Blood vs Saliva vs Urine: Choosing Optimal Hormone Sampling for Circadian Rhythm Research and Drug Development

Abstract

This article provides a comprehensive analysis of blood, saliva, and urine sampling methodologies for circadian hormone assessment, tailored for researchers and drug development professionals. It explores the foundational biology of key circadian biomarkers like cortisol and melatonin, detailing the distinct clinical information each matrix provides—from bioavailable hormone levels in saliva to metabolic pathways in urine. The content delivers practical guidance on protocol optimization, analytical technique selection, and troubleshooting common pitfalls. A critical comparative framework is presented to validate method selection for specific research intents, including chronotherapy and biomarker discovery, ultimately supporting robust experimental design and precise diagnostic development in circadian medicine.

Circadian Rhythms and Hormonal Biomarkers: Foundations for Biomedical Research

The human circadian system is a hierarchical network of biological clocks that orchestrates near-24-hour oscillations in physiology and behavior. This system is composed of a central master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral oscillators found in virtually every organ and tissue [1] [2] [3]. The synchronization between central and peripheral clocks is fundamental to health, while their disruption is implicated in various pathologies, including metabolic disorders, cardiovascular diseases, and cancer [4] [3]. A critical area of circadian research involves the accurate assessment of circadian phase, for which hormone measurements in different biological matrices—blood, saliva, and urine—are essential. This article provides application notes and detailed protocols for studying these oscillators, framed within the context of comparative hormone sampling for circadian research.

Molecular Architecture of the Circadian Clock

At the molecular level, the circadian clock operates through autoregulatory transcription-translation feedback loops (TTFLs) comprising core clock genes and their protein products [1] [3].

• Core Feedback Loop: The heterodimeric complex of CLOCK and BMAL1 (also known as ARNTL1) acts as the primary transcriptional activator. This complex binds to E-box elements in the promoters of target genes, including the Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2) genes [3].
• Negative Feedback: PER and CRY proteins accumulate, form complexes in the cytoplasm, and translocate back to the nucleus to inhibit CLOCK-BMAL1-mediated transcription, thereby repressing their own expression [1] [3].
• Stabilizing Loop: CLOCK-BMAL1 also drives the expression of nuclear receptors Rev-Erbα/β (repressors) and RORα/β/γ (activators), which compete for ROR elements (ROREs) in the Bmal1 promoter, creating an additional interlocking loop that stabilizes the core oscillator [2] [3].

Post-translational modifications (e.g., phosphorylation by CK1δ/ε) regulate protein stability and nuclear translocation, fine-tuning the period of the rhythm to approximately 24 hours [1] [2]. The following diagram illustrates these core molecular interactions:

Diagram 1: Core Molecular Feedback Loops of the Circadian Clock. The core loop (center) involves CLOCK-BMAL1 activation and PER-CRY repression. The stabilizing loop (sides) shows ROR activation and REV-ERB repression of BMAL1 transcription.

Central and Peripheral Clocks: A Hierarchical System

The mammalian circadian system is organized hierarchically.

• The Central Pacemaker: The Suprachiasmatic Nucleus (SCN) serves as the master clock. It is entrained primarily by light signals received via the retinohypothalamic tract and is responsible for synchronizing peripheral oscillators throughout the body [2] [3].
• Peripheral Clocks: Clocks in peripheral tissues (e.g., liver, heart, salivary glands) are synchronized by the SCN through a combination of neural, endocrine (e.g., glucocorticoids), and behavioral (e.g., feeding-fasting cycles) cues [5] [3]. While they can be entrained independently of the SCN by non-photic zeitgebers like food intake, they generally remain subordinated to the central pacemaker [1] [5].
• Synchronization Across Tissues: Importantly, molecular studies have demonstrated phase synchronization of core clock genes (e.g., ARNTL1, PER2) across diverse peripheral tissues, validating the use of accessible tissues like saliva as a proxy for assessing the state of the peripheral circadian system [6].

Hormones as Circadian Biomarkers: A Comparison of Sampling Matrices

The hormones melatonin and cortisol are robust biomarkers for assessing the phase and amplitude of circadian rhythms in humans [7] [8]. The choice of biological matrix for their measurement is a critical consideration in research design, balancing analytical requirements, practicality, and participant burden.

Table 1: Comparison of Biological Matrices for Circadian Hormone Sampling

Parameter	Saliva	Blood (Plasma/Serum)	Urine
Key Hormones Measured	Cortisol, Melatonin (free fraction)	Cortisol (total/free), Melatonin (total/free)	Cortisol (free), Melatonin metabolites (6-Sulfatoxymelatonin)
Invasiveness	Non-invasive	Invasive (venipuncture or catheter)	Non-invasive
Home/Ambulatory Sampling	Excellent; suitable for frequent, time-series sampling	Poor; typically restricted to clinical settings	Good; for pooled or sequential collections
Analytical Challenges	Low analyte concentrations; potential for contamination	Higher analyte levels; requires clinical expertise for collection	Measures metabolites; requires volume/creatinine correction
Primary Circadian Metrics	DLMO (Melatonin), CAR (Cortisol)	DLMO, Cortisol Acrophase	24-hour total production, Diurnal rhythm patterns
Major Strengths	Ideal for dense circadian phase assessment (e.g., DLMO) in real-world settings	Gold standard for total hormone levels and pulsatility analysis	Provides integrated measure of hormone secretion over time
Major Limitations	Low hormone levels demand high-sensitivity assays (e.g., LC-MS/MS) [7]	Impractical for high-frequency sampling outside the lab; disrupts sleep	Low temporal resolution; not suitable for precise phase markers like DLMO

Detailed Experimental Protocols

This section provides standardized protocols for assessing circadian rhythms using saliva, the preferred matrix for non-invasive, high-density time-series sampling in ambulatory settings.

Protocol: Saliva Collection for Circadian Hormone and Gene Expression Analysis

This protocol is optimized for the simultaneous analysis of cortisol and melatonin rhythms, as well as circadian gene expression from saliva [6] [7].

5.1.1. Pre-Collection Considerations

• Participant Screening: Exclude individuals with recent shift work, transmeridian travel (within 2 weeks), extreme chronotypes, or uncontrolled sleep disorders. Document sleep-wake patterns and medication use (e.g., beta-blockers, NSAIDs) as they can affect hormone levels [9] [7].
• Timing: For a full phase assessment, collect samples at 3-4 time points per day over 2-3 consecutive days. For DLMO determination, a 4-6 hour sampling window before and after habitual bedtime is sufficient [7].
• Lighting Control: For melatonin sampling, enforce dim light conditions (<10-30 lux) starting at least 1 hour before the first sample and throughout the collection period [7].

5.1.2. Materials and Reagents

Table 2: Research Reagent Solutions for Salivary Circadian Analysis

Item	Function/Description	Example/Note
Saliva Collection Aid	Facilitates hygienic saliva collection into a tube	Salivette (cotton or polyester swab)
RNA Stabilizer	Preserves RNA integrity for gene expression analysis	RNAprotect Saliva Reagent [6]
Hormone Assay Kits	Quantification of cortisol and melatonin	Highly sensitive ELISA or LC-MS/MS kits [7]
RNA Extraction Kit	Isolation of high-quality total RNA from saliva	Commercially available kits for biofluids
qPCR Reagents	Quantification of core clock gene expression	Primers/Probes for ARNTL1, PER2, NR1D1 [6]

5.1.3. Step-by-Step Procedure

• Sample Collection:

  • Instruct participants not to eat, drink (except water), or brush teeth for at least 30 minutes before sampling.
  • For hormone-only studies, have participants passively drool into a pre-chilled tube or use a dedicated saliva collection aid.
  • For combined gene expression and hormone analysis, collect 1.5 mL of saliva and immediately mix with 1.5 mL of RNA stabilization reagent (1:1 ratio) to preserve nucleic acids [6].

• Sample Processing and Storage:

  • Centrifuge saliva samples to precipitate cellular debris.
  • Aliquot the supernatant for hormone analysis and store at -80°C until assayed.
  • For RNA extraction, process the cell pellet or stabilized saliva according to the manufacturer's protocol.

• Hormone Analysis:

  • Use LC-MS/MS where possible for its superior specificity and sensitivity for salivary melatonin and cortisol [7].
  • If using immunoassays, validate for minimal cross-reactivity with salivary matrix components.

• Data Analysis:

  • DLMO Calculation: Plot melatonin concentration against clock time. Calculate the time at which levels consistently exceed a predetermined threshold (e.g., 3 pg/mL or 2 standard deviations above the mean of three baseline values) [7].
  • Cortisol Acrophase: Fit a cosine curve to the time-series data to determine the peak time (acrophase) of the rhythm.
  • Gene Expression Rhythms: Analyze the expression of core clock genes (e.g., ARNTL1, PER2) using algorithms like TimeTeller to determine the circadian phase of the peripheral oscillator [6].

The following workflow diagram summarizes this integrated protocol:

Diagram 2: Integrated Workflow for Salivary Circadian Analysis. The protocol outlines the parallel processing of saliva for concurrent gene expression and hormone analysis.

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Essential Research Toolkit for Circadian Rhythm Studies

Category	Item	Specific Application/Function
Participant Characterization	Morningness-Eveningness Questionnaire (MEQ) [10]	Determines chronotype (morning, intermediate, evening type).
	Sleep Diaries [10]	Tracks sleep-wake patterns and timing in the natural environment.
Sample Collection & Stabilization	RNAprotect Saliva Reagent [6]	Stabilizes RNA in saliva samples for reliable gene expression analysis.
	Salivette or similar aids	Standardizes and simplifies the collection of clean saliva samples.
	Cryogenic Tubes (-80°C)	For long-term storage of sample aliquots to preserve analyte integrity.
Hormone Analysis	LC-MS/MS Platform [7]	Gold-standard for specific, sensitive quantification of salivary melatonin and cortisol.
	High-Sensitivity Salivary Melatonin ELISA	Immunoassay alternative for melatonin, requires careful validation.
	High-Sensitivity Salivary Cortisol ELISA	Widely used for cortisol measurement, including the Cortisol Awakening Response (CAR).
Molecular Biology	RNA Extraction Kit (for biofluids)	Isolves high-quality total RNA from saliva samples.
	Reverse Transcription Kit	Synthesizes cDNA from extracted RNA for downstream qPCR.
	TaqMan qPCR Assays	For precise quantification of core clock gene expression (e.g., ARNTL1, PER2, NR1D1) [6].
Data Analysis	TimeTeller or Similar Algorithm [6]	Computes circadian phase and rhythm strength from time-series gene expression data.
	Cosinor Analysis Software	Fits rhythmic data (e.g., hormone levels) to determine acrophase, amplitude, and mesor.

Melatonin and Cortisol as Primary Circadian Endocrine Markers

Circadian rhythms are endogenous, near-24-hour cycles that orchestrate a wide range of physiological processes in humans, including the sleep-wake cycle, hormone secretion, metabolism, and behavior [11] [12]. These intrinsic rhythms persist even in the absence of external cues, reflecting the activity of an internal biological clock centered in the suprachiasmatic nucleus (SCN) of the hypothalamus [7]. The hormones melatonin and cortisol represent crucial biochemical markers of circadian phase, providing valuable proxies for assessing the timing of the central circadian clock in humans [11] [12] [7]. Within the context of comparative biofluid sampling research, this review systematically evaluates contemporary methodologies for quantifying these endocrine markers across different biological matrices, with particular emphasis on blood, saliva, and urine specimens.

Circadian Endocrinology: Core Hormonal Rhythms

Melatonin Rhythm and Dim Light Melatonin Onset (DLMO)

Melatonin is produced by the pineal gland with secretion following a distinct daily rhythm, reaching nadir during daytime and peaking in the early night [12] [7]. Its production is exquisitely sensitive to light exposure, with secretion inhibited by light and promoted in darkness [13]. The Dim Light Melatonin Onset (DLMO), defined as the time when melatonin concentrations begin to rise under dim light conditions, is widely considered the most reliable marker of internal circadian timing [11] [12] [7]. DLMO typically occurs 2-3 hours before habitual sleep time and serves as a critical phase marker for the biological night [12].

Cortisol Rhythm and Cortisol Awakening Response (CAR)

Cortisol, a glucocorticoid hormone produced by the adrenal cortex, exhibits a circadian rhythm roughly opposite to melatonin, with levels peaking early in the morning and reaching their nadir around midnight [12] [7]. The Cortisol Awakening Response (CAR) - a sharp rise in cortisol levels occurring within 30-45 minutes after waking - serves as an index of hypothalamic-pituitary-adrenal (HPA) axis activity and is influenced by circadian timing, sleep quality, and psychological stress [11] [12] [7]. Although cortisol-based methods offer less precision for circadian phase assessment compared to melatonin (standard deviation of ~40 minutes versus 14-21 minutes), it remains a valuable marker when melatonin assessment is confounded by medications or other factors [12] [7].

Comparative Analysis of Biological Matrices

The selection of appropriate biological matrices is crucial for circadian endocrine research, with each offering distinct advantages and limitations for hormone assessment.

Table 1: Comparison of Biological Matrices for Circadian Hormone Assessment

Matrix	Melatonin Measurement	Cortisol Measurement	Key Advantages	Major Limitations
Blood	Gold standard for plasma/serum melatonin; DLMO reference method [12]	Reliable measurement of total and free cortisol [14]	High analyte levels, better reliability [12]; Direct measurement of circulating hormones	Invasive, logistically demanding; Not suitable for frequent sampling [12]
Saliva	Salivary DLMO with thresholds of 3-4 pg/mL [12] [7]; Non-invasive collection	CAR assessment; Non-invasive ambulatory collection [12] [13]	Non-invasive, suitable for repeated ambulatory measurements [12] [6]; Correlates well with free hormone levels	Low hormone concentrations challenge analytical sensitivity [12]
Urine	6-sulfatoxymelatonin as primary metabolite [11]	Urinary free cortisol measurement; Integrated hormone exposure assessment	Non-invasive; Provides integrated hormone exposure over time [11]	Does not capture rapid ultradian patterns; Time-lagged metabolite measurements
Novel Matrices	Passive perspiration (wearable sensors) [15]	Passive perspiration (wearable sensors) [15]	Continuous, real-time monitoring [15]; Minimal subject burden	Emerging technology requiring further validation [15]

Table 2: Analytical Platforms for Hormone Quantification

Analytical Method	Sensitivity	Specificity	Throughput	Cost	Best Applications
LC-MS/MS	High (ideal for low-abundance analytes like melatonin) [12]	Excellent (minimal cross-reactivity) [11] [12]	Moderate	High	Gold standard for salivary and serum hormones; Research requiring high precision [12]
Immunoassays (ELISA, RIA)	Moderate to high	Moderate (potential cross-reactivity with metabolites) [12]	High	Moderate to low	High-throughput screening; Clinical settings where highest specificity not required [12]
Wearable Biosensors	Developing	Developing	Continuous	Variable (development vs. production)	Ambulatory monitoring; Longitudinal studies [15]

Detailed Experimental Protocols

Protocol for Dim Light Melatonin Onset (DLMO) Assessment in Saliva

Principle: Determine the time of melatonin onset under dim light conditions using salivary samples collected in the evening hours [12] [16].

Materials:

• Salivette collection devices or similar saliva collection tubes
• Low-light environment (<10-50 lux) verified by lux meter
• Freezer (-20°C or -80°C) for sample storage
• LC-MS/MS system or validated immunoassay for melatonin quantification

Procedure:

• Participant Preparation: Participants should avoid non-steroidal anti-inflammatory drugs (suppress melatonin), beta-blockers (suppress melatonin), antidepressants or melatonin supplements (elevate melatonin), and alcohol for 24-48 hours prior to testing [12] [16]. Caffeine should be avoided on the testing day [16].
• Light Control: Implement dim light conditions (<10-50 lux) beginning at least 2 hours before the first sample collection and maintain throughout sampling [12] [16]. Use red light if illumination is necessary as it minimally suppresses melatonin.
• Sampling Schedule: Collect samples every 30-60 minutes for 4-6 hours, typically from 5 hours before to 1 hour after habitual bedtime [12]. Exact timing may be adjusted based on suspected circadian rhythm disorder and age of participant [12].
• Sample Collection: Participants should refrain from eating, drinking (except water), brushing teeth, or using mouthwash for at least 30 minutes before each sample [16]. Note exact collection time for each sample.
• Sample Processing: Centrifuge saliva collection devices if required by manufacturer instructions. Store samples at -20°C or -80°C until analysis.
• DLMO Calculation: Determine DLMO using either:
  • Fixed threshold method: Time when interpolated melatonin concentration reaches 3-4 pg/mL in saliva (or 10 pg/mL in serum) [12] [7]
  • Dynamic threshold method: Time when melatonin exceeds 2 standard deviations above the mean of 3 or more baseline values [12]
  • Hockey-stick algorithm: Objective, automated assessment of change from baseline to rise [12]

Protocol for Cortisol Awakening Response (CAR) Assessment

Principle: Measure the rapid increase in cortisol that occurs within the first 30-45 minutes after waking [12].

Materials:

• Salivette collection devices or similar saliva collection tubes
• Home freezer for sample storage or cooler with ice packs for transport
• Timer or alarm clock
• LC-MS/MS system or validated immunoassay for cortisol quantification

Procedure:

• Participant Preparation: Participants should avoid smoking, eating, drinking (except water), and brushing teeth for at least 30 minutes before each sample. Document wake time, sleep quality, and any medications [12] [13].
• Sampling Schedule:
  • Sample 1: Immediately upon waking (0 minutes)
  • Sample 2: 30 minutes after waking
  • Sample 3: 45 minutes after waking
  • Additional samples may be collected at 60 minutes for extended assessment
• Sample Collection: Participants record exact collection time for each sample. If multiple days are assessed, maintain consistent procedure across days.
• Sample Processing: Centrifuge if required and freeze samples at -20°C or lower until analysis.
• CAR Calculation: Calculate area under the curve (AUC) or peak increase from waking to 30-45 minutes post-awakening.

Protocol for Simultaneous Assessment of Melatonin and Cortisol

Principle: Leverage the analytical capability of LC-MS/MS to simultaneously quantify both melatonin and cortisol from the same sample, providing a comprehensive circadian assessment [12].

Materials:

• Appropriate collection devices for chosen matrix (saliva, blood, urine)
• LC-MS/MS system with validated method for simultaneous quantification
• Standard protocols for sample collection and storage as above

Procedure:

• Follow relevant sampling procedures from protocols 4.1 and 4.2 based on research question.
• Process samples according to laboratory standard operating procedures.
• Analyze using LC-MS/MS method validated for simultaneous quantification of melatonin and cortisol.
• Interpret results considering the differential rhythmicity and phase relationship between these hormones [15].

Signaling Pathways and Regulatory Mechanisms

The circadian system operates through a complex hierarchy of transcriptional-translational feedback loops that regulate hormonal output.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Research Reagent Solutions for Circadian Endocrine Studies

Item	Function	Application Notes
Salivette Collection Devices	Saliva sample collection	Preferred for consistency; various types available (cotton, polyester) [12]
LC-MS/MS System	Gold standard hormone quantification	Provides high sensitivity and specificity for simultaneous melatonin and cortisol measurement [12]
Validated Immunoassay Kits	Hormone quantification	ELISA kits available for melatonin and cortisol; check cross-reactivity [12]
Lux Meter	Verify dim light conditions	Critical for DLMO assessment (<10-50 lux) [16]
Portable Freezers	Sample transport and storage	Maintain cold chain for hormone stability [13]
Actigraphy Devices	Objective sleep-wake monitoring	Correlate hormone measures with rest-activity patterns [17]
RNAprotect Solution	RNA preservation for gene expression	For studies integrating transcriptomics with hormone analysis [6]

Data Analysis and Interpretation

Analytical Considerations for Cross-Matrix Comparisons

When comparing hormone measurements across different biological matrices, several analytical factors must be considered:

• Temporal resolution: Blood provides real-time assessment, saliva shows slight delays (15-20 minutes), while urine provides integrated measures over several hours [11] [12].
• Correlation between matrices: Strong correlations exist between saliva and blood for cortisol (r=0.92) and melatonin (r=0.90) when using appropriate collection and analytical methods [15].
• Absolute concentrations: Vary significantly between matrices, requiring matrix-specific reference ranges and thresholds for interpretation.

Cosinor Analysis and Rhythm Assessment

For comprehensive circadian analysis, cosinor methods can be applied to characterize multiple rhythm parameters:

• Acrophase: Time of peak hormone concentration
• Amplitude: Half the difference between peak and trough levels
• Mesor: Rhythm-adjusted mean
• Period: Duration of one complete cycle (typically ~24 hours)

Tools such as CircaCompare enable differential rhythmicity analysis between hormones and across experimental conditions [15].

Melatonin and cortisol serve as fundamental endocrine markers of circadian phase, with applications spanning basic circadian research, clinical diagnostics, and therapeutic monitoring. The selection of appropriate biological matrices - blood, saliva, or urine - depends on specific research questions, required temporal resolution, and practical considerations regarding participant burden and analytical capabilities. Saliva has emerged as an optimal matrix for many applications due to its non-invasive nature and good correlation with unbound, biologically active hormone fractions. LC-MS/MS methodology provides the highest quality data for simultaneous assessment of both hormones, while rigorous protocol standardization is essential for reliable circadian phase assessment. As circadian medicine continues to evolve, precise measurement of these endocrine markers will remain crucial for understanding circadian disruption in disease and optimizing chronotherapeutic interventions.

Dim Light Melatonin Onset (DLMO) is the most reliable and biologically accurate marker of central circadian phase in humans [18] [19]. It represents the time in the evening when endogenous melatonin secretion from the pineal gland begins to rise, marking the transition into the biological night [7]. The quantification of DLMO provides a critical objective measure for diagnosing circadian rhythm sleep-wake disorders, evaluating circadian phase shifts, and optimizing chronotherapy in drug development [18]. Within the broader research context comparing blood, saliva, and urine for hormone circadian sampling, salivary DLMO has emerged as the preferred method in modern research due to its non-invasive nature, high participant compliance, and strong correlation with plasma melatonin levels [18] [19]. This protocol outlines standardized procedures for salivary DLMO assessment, emphasizing its advantages over blood and urine sampling for circadian phase determination.

Comparative Analysis of Circadian Sampling Matrices

Table 1: Comparison of Biological Matrices for Circadian Melatonin Assessment

Parameter	Saliva	Blood (Plasma/Serum)	Urine
Sample Collection	Non-invasive, suitable for home/remote collection [18] [20]	Invasive, requires venipuncture or cannulation [18]	Non-invasive, but cumbersome for timed collections
Participant Burden	Low, enables high compliance and frequent sampling [18] [19]	High, especially for serial sampling overnight [18]	Moderate, requires complete timed urine voids
Melatonin Correlation	Highly correlated with blood levels [18]	Gold standard reference	Correlates with plasma but reflects clearance rather than real-time secretion
DLMO Protocol Feasibility	Excellent for home-based protocols (7-13 samples over 6-8 hours) [18] [19]	Limited to laboratory settings due to invasiveness [18]	Less suitable for precise DLMO determination
Analytical Challenges	Low concentrations requiring sensitive assays [7]	Higher concentrations, but sample processing required	Requires creatinine correction and volume measurement
Ideal Application	Routine DLMO assessment, field studies, pediatric and special populations [19] [20]	Method validation research, cases requiring highest precision	6-sulfatoxymelatonin for overall melatonin production assessment

Salivary DLMO Assessment Protocol

Pre-Assessment Requirements

• Participant Preparation: Participants should avoid the following for 24 hours prior to testing: melatonin supplements, beta-blockers, non-steroidal anti-inflammatory drugs (NSAIDs), antidepressants, and contraceptive medications as they can interfere with natural melatonin production [7]. They should also avoid alcohol and caffeine during the testing day [19].
• Lighting Control: Maintain dim light conditions (<50 lux) during the entire collection period, verified using a digital lux meter [18] [20]. Participants should avoid direct light exposure, particularly blue wavelength light (~460-480 nm) which strongly suppresses melatonin secretion [8] [7].
• Timing Considerations: Schedule the DLMO assessment based on the participant's habitual sleep schedule. Collection typically begins 5-6 hours before and continues until 1-2 hours after habitual bedtime [18] [19].

Sample Collection Workflow

Diagram 1: Salivary DLMO Collection and Analysis Workflow

Detailed Collection Procedure

• Sample Timing: Collect 7-13 saliva samples at regular intervals (typically hourly or half-hourly) beginning 5 hours before habitual bedtime through 1 hour after bedtime [18]. For populations with suspected severe phase shifts or irregular rhythms, extend the collection window [18].
• Collection Method: Use passive drool or salivette collection systems. For passive drool, participants should drool through a straw into appropriate collection tubes, with 0.5 mL sufficient for duplicate assays [18].
• Sample Handling: Immediately freeze samples at -20°C after collection. Maintain cold chain during transport to the analytical laboratory [20].
• Compliance Monitoring: Utilize objective compliance measures such as medication event monitoring system (MEMS) caps to record exact sampling times, actigraphy watches to monitor activity, and light sensors to verify dim light conditions [20].

DLMO Calculation Methodologies

Analytical Methods

Table 2: Comparison of Melatonin Detection Methods

Method	Sensitivity	Sample Volume	Throughput	Advantages	Limitations
ELISA	1.35 pg/mL [18]	100 μL/well [18]	38 samples in duplicate in 3.5 hours [18]	Cost-effective, no extraction needed, established protocols	Potential cross-reactivity with metabolites [7]
LC-MS/MS	Higher than ELISA [7]	Similar to ELISA [7]	Lower throughput but multiplexing capability [7]	Gold standard specificity, can simultaneously analyze multiple hormones [7]	Higher equipment costs, requires technical expertise [7]

DLMO Calculation Algorithms

Diagram 2: DLMO Calculation Method Decision Pathway

• Fixed Threshold Method: DLMO is defined as the time when interpolated melatonin concentrations cross a predetermined absolute threshold, typically 3 pg/mL or 4 pg/mL for saliva [18] [19]. This method works well for normal melatonin producers but may miss DLMO in low producers or those with high baseline levels [18].

• Variable Threshold Method (3k Method): Calculate the mean and standard deviation of the first three low daytime samples. DLMO is defined as the time when melatonin levels rise and remain above 2 standard deviations from this baseline mean [18]. This method accommodates individual differences in baseline melatonin production and is particularly suitable for low melatonin producers, such as older adults [18].

• Hockey-Stick Algorithm: An objective, automated method that estimates the point of change from baseline to the rising phase of melatonin secretion using curve-fitting algorithms. This method shows strong agreement with expert visual assessment and reduces subjective interpretation [7].

The Scientist's Toolkit: Essential Research Reagents and Equipment

Table 3: Essential Materials for Salivary DLMO Assessment

Item	Specification	Application	Protocol Notes
Saliva Collection Devices	Untreated Salivettes or passive drool tubes [20]	Sample collection	Avoid treated devices that may interfere with assays
Light Meter	Digital lux meter (e.g., VWR LXM001) [20]	Verify dim light conditions (<50 lux)	Calibrate regularly; use throughout collection
Blue Light-Blocking Glasses	Orange/amber lenses blocking ~460-480 nm [20]	Prevent melatonin suppression if light exposure necessary	Required if participants use electronic devices
Actigraphy Device	(e.g., ActTrust 2) [20]	Monitor activity and sleep-wake patterns	Wear for 1-2 weeks before DLMO to establish habitual sleep time
Time-Stamping Cap	Medication Event Monitoring System (MEMS) [20]	Objective compliance monitoring	Records exact sample collection times
Cold Chain Supplies	Freezer bags, ice packs, -20°C freezer [20]	Sample preservation	Maintain freezing until analysis
Melatonin Assay	Validated ELISA or LC-MS/MS [18] [7]	Melatonin quantification	Verify sensitivity for salivary concentrations (≤1.35 pg/mL)

Protocol Validation and Technical Considerations

Recent studies have validated home-based DLMO protocols across diverse populations, including individuals with obesity [19] and pediatric patients with chronic pain [20]. These protocols demonstrate high success rates, with DLMO detection in 98.2% of participants using individualized thresholds and 89.6% using standardized thresholds [19]. The feasibility of entirely self-directed, remote DLMO collections has been established with appropriate compliance monitoring [20].

When implementing salivary DLMO assessment, consider that phase angles (the relationship between DLMO and sleep timing) show consistent patterns across populations, with DLMO typically occurring 1-2 hours before sleep onset [20]. This relationship provides a biological framework for interpreting DLMO results in clinical and research contexts.

The simultaneous measurement of cortisol may provide complementary circadian information, as LC-MS/MS methods can analyze both hormones without additional costs [7]. However, cortisol's circadian rhythm shows greater variability than melatonin, with a standard deviation of approximately 40 minutes compared to 14-21 minutes for melatonin [7].

Cortisol Awakening Response (CAR) as an HPA Axis Indicator

The Cortisol Awakening Response (CAR) is a distinct neuroendocrine phenomenon characterized by a rapid increase in cortisol secretion during the first 30-45 minutes after awakening [12] [21]. This dynamic response is theorized to provide an "allostatic boost" that prepares the individual for anticipated energy demands and stressors of the forthcoming day [22] [23]. As a biomarker, CAR offers valuable insights into the integrity of the hypothalamic-pituitary-adrenal (HPA) axis and its interaction with circadian regulatory systems [12]. Within the broader context of comparative hormone sampling research, CAR measurement presents distinct methodological considerations across blood, saliva, and urine matrices, each offering unique advantages for circadian rhythm assessment [21].

Recent research utilizing innovative continuous sampling methodologies has revealed that the rate of cortisol increase following awakening may not differ significantly from the pre-awakening period, challenging the concept of CAR as a purely awakening-dependent phenomenon [22]. Instead, evidence suggests CAR reflects a continuation of underlying circadian rhythms influenced by multiple factors including sleep duration and wake time consistency [22]. Despite these complexities, CAR remains a valuable indicator of HPA axis reactivity with demonstrated relevance for emotional processing, stress-related disorders, and overall circadian health [12] [23].

Biological Basis and Significance

Circadian Regulation and HPA Axis Function

Cortisol secretion follows a robust diurnal rhythm characterized by a morning peak, gradual decline throughout the day, and nadir during the early sleep phase [12] [21]. This rhythm is regulated by the central circadian pacemaker in the suprachiasmatic nucleus (SCN), which coordinates peripheral clocks throughout the body [6] [21]. The HPA axis represents the primary neuroendocrine system governing cortisol release, integrating neural signals from the SCN with hormonal outputs from the adrenal cortex [21].

The CAR is superimposed on this circadian pattern, representing a discrete period of enhanced HPA axis reactivity upon awakening. Recent evidence suggests this response may be more tightly regulated by intrinsic cortisol rhythmicity than previously thought, with the pre-awakening cortisol level being the strongest predictor of post-awakening increases [22]. The CAR appears to be most pronounced when awakening is anticipated, as evidenced by blunted responses when participants are forcibly awoken during the night [22].

Functional Significance

CAR serves as a proactive neuroendocrine mechanism that modulates brain function to meet anticipated daily demands. Pharmacological suppression of CAR leads to impaired discrimination of negative facial expressions and altered functional connectivity between the amygdala and prefrontal cortex during emotional processing [23]. This supports the concept that CAR prepares limbic circuitry for upcoming emotional challenges by setting a "tonic tone" for the day [23].

Disturbances in CAR patterns are associated with various pathological conditions. Both elevated and blunted CAR profiles have been reported in stress-related disorders including depression, post-traumatic stress disorder, and burnout [22] [12]. These alterations reflect potential maladaptation of the HPA axis and may serve as biomarkers for disease risk, progression, and treatment response [24] [12].

Comparative Analysis of Sampling Matrices

The selection of appropriate biological matrices is crucial for accurate CAR assessment, with each medium offering distinct advantages and limitations for circadian research.

Table 1: Comparison of Biological Matrices for CAR Assessment

Matrix	Cortisol Form Measured	Sampling Frequency	Key Advantages	Major Limitations
Saliva	Free (biologically active)	High-frequency (multiple samples over 30-45 min)	Non-invasive; suitable for home collection; reflects free cortisol [12] [21]	Low concentrations challenge analytical sensitivity; potential lag in hormone appearance [22] [12]
Blood	Total (free + protein-bound)	Limited by invasiveness	Higher analyte concentrations; better reliability; gold standard reference [12]	Invasive; impractical for frequent sampling; requires clinical setting [22]
Interstitial Fluid (ISF)	Free (biologically active)	Continuous (20-min intervals)	Continuous ambulatory monitoring; minimal disruption to daily activities [22]	Technological complexity; potential lag behind plasma/saliva; research-use only [22]
Urine	Free cortisol	Cumulative (overnight/morning collection)	Non-invasive; integrated measure over time [21]	Cannot capture rapid dynamics of CAR; timing resolution insufficient for precise CAR calculation [21]

Table 2: Analytical Methods for Cortisol Detection

Method	Sensitivity	Specificity	Throughput	Suitable Matrices	Key Considerations
LC-MS/MS	High (pg/mL range)	Excellent (minimal cross-reactivity)	Moderate to High	Saliva, serum, urine, ISF [12]	Gold standard; requires specialized equipment and expertise [12]
Immunoassays (ELISA)	Moderate to High	Moderate (potential cross-reactivity)	High	Saliva, serum [12] [21]	Cost-effective; accessible; validation against LC-MS/MS recommended [12]
Microdialysis + LC-MS/MS	High	Excellent	Low	ISF [22]	Research technique; enables continuous monitoring; technically complex [22]

Methodological Protocols

Salivary CAR Assessment Protocol

Sample Collection

• Participants should provide 4-5 saliva samples at scheduled intervals: immediately upon awakening (S1), then 15, 30, and 45 minutes post-awakening (S2-S4) [12]. An additional late-evening sample may be collected to establish diurnal baseline.
• Use validated saliva collection devices (e.g., Salivettes) that do not interfere with immunoassays or LC-MS/MS analysis.
• Participants should refrain from eating, drinking, brushing teeth, or smoking during the 45-minute sampling period to avoid contamination.
• Record exact sampling times using electronic monitoring devices (e.g., TrackCaps) to ensure protocol compliance and temporal accuracy [12].

Sample Processing and Storage

• Centrifuge saliva samples at 3000×g for 10 minutes to separate clear supernatant from mucins and cellular debris.
• Aliquot supernatant into cryovials and store at -80°C until analysis to prevent degradation.
• Avoid multiple freeze-thaw cycles by creating single-use aliquots.

Analytical Procedure (LC-MS/MS Recommended)

• Extract cortisol using supported liquid extraction (SLE) or protein precipitation.
• Use deuterated cortisol-d4 as internal standard for quantification.
• Separate analytes using reverse-phase C18 column with methanol/water gradient.
• Employ multiple reaction monitoring (MRM) transitions m/z 363.2→121.2 (quantifier) and 363.2→309.2 (qualifier) for cortisol.
• Establish calibration curve using matrix-matched standards (0.5-50 ng/mL) [12].

CAR Calculation

• Calculate CAR using area under the curve with respect to increase (AUCi) or as the difference between peak (usually 30-45 min) and awakening concentrations [12].

Plasma CAR Assessment Protocol

Sample Collection

• Insert intravenous catheter prior to anticipated awakening time to minimize sleep disruption.
• Collect blood samples at identical timepoints as salivary protocol: pre-awakening (if possible), immediately upon awakening, and 15, 30, 45 minutes post-awakening.
• Collect 2-3 mL blood into EDTA or heparin tubes at each timepoint, invert gently, and place on ice.

Sample Processing

• Centrifuge blood samples at 1500×g for 10 minutes at 4°C within 30 minutes of collection.
• Transfer plasma to polypropylene tubes and store at -80°C until analysis.
• For total cortisol measurement, analyze without extraction; for free cortisol, implement equilibrium dialysis or ultrafiltration prior to analysis.

Analytical Considerations

• Plasma cortisol concentrations are approximately 2-3 times higher than in saliva.
• LC-MS/MS methods should utilize serum-based calibrators and quality controls with appropriate matrix-matching procedures.

Continuous Interstitial Fluid Monitoring Protocol

Microdialysis System Setup

• Insert linear microdialysis probe (e.g., 30 kDa molecular weight cut-off) into abdominal subcutaneous tissue.
• Perfuse with sterile isotonic solution at 0.3-1.0 μL/min flow rate using portable micropump system.
• Collect dialysate fractions at 20-minute intervals over 24-hour period [22].
• Participants record sleep and wake times using sleep diaries or actigraphy monitoring.

Sample Analysis

• Analyze dialysate cortisol using ultrasensitive LC-MS/MS methods with minimal sample dilution.
• Concentrations will be significantly lower than saliva or plasma, requiring optimized sensitivity.
• Normalize recovery rates using reference compounds (e.g., dexamethasone) when possible.

Data Interpretation

• Align cortisol profiles relative to individual wake times.
• Compare rate of cortisol increase before and after awakening to determine awakening-specific effects [22].

Visualization of CAR Assessment Pathways

HPA Axis Regulation and CAR Measurement

Experimental Workflow for Multi-Matrix CAR Assessment

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Research Reagents and Materials for CAR Assessment

Category	Specific Items	Application/Function	Key Considerations
Sample Collection	Salivette cortisol collection devices	Non-invasive saliva sampling	Choose synthetic fiber swabs; avoid cotton which can interfere with assays [12]
	EDTA or Heparin blood collection tubes	Plasma preparation for total cortisol	Maintain consistent anticoagulant across study [21]
	Microdialysis systems with 30 kDa cut-off probes	Continuous ISF sampling	Requires specialized training; validate recovery rates [22]
	Electronic monitoring devices (TrackCaps)	Verify sampling compliance	Critical for protocol adherence in home collection [12]
Sample Processing	Refrigerated centrifuges	Cellular separation	Maintain 4°C for plasma processing [21]
	Cryogenic vials	Sample storage	Use low-protein binding polypropylene [12]
	-80°C freezers	Long-term sample preservation	Monitor temperature stability [12]
Analytical Standards	Certified cortisol reference standard	Quantification calibration	Source from accredited reference material providers [12]
	Deuterated cortisol-d4 internal standard	LC-MS/MS quantification	Essential for mass spectrometry-based methods [12]
	Matrix-matched calibrators	Standard curve preparation	Match biological matrix of samples for accurate quantification [12]
Analytical Instruments	LC-MS/MS system with C18 column	Gold-standard quantification	Requires MRM capability for optimal sensitivity [12]
	Automated immunoassay platform	High-throughput screening	Validate against LC-MS/MS for salivary cortisol [12]

Methodological Considerations and Best Practices

Controlling for Confounding Factors

Accurate CAR assessment requires strict control of several confounding variables:

• Light exposure: Maintain dim light conditions before and during sampling, as light can suppress melatonin and potentially affect cortisol rhythms [12].
• Posture: Ensure consistent semi-recumbent or seated position during sampling, as posture changes can affect cortisol concentrations [12].
• Medications: Document and consider excluding participants using medications affecting HPA axis function (e.g., corticosteroids, psychotropic drugs) [12].
• Wake time consistency: Account for sleep duration and variability in wake times, as short sleep and misaligned wake times significantly impact CAR dynamics [22].
• Food intake: Implement fasting during the sampling period, as eating can stimulate cortisol secretion independently of the CAR [12].

Data Interpretation and Normalization

Between-subject variability in CAR is substantial, with individual differences influenced by sleep patterns, psychological factors, and genetic predispositions [22]. Normalization strategies include:

• Expressing values as percentage change from awakening concentration
• Calculating area under the curve with respect to ground (AUCg) and increase (AUCi)
• Using multiple assessment days to establish stable CAR estimates
• Incorporating covariance analysis for known moderators (age, sex, chronotype)

Recent research indicates CAR variability is partly explained by sleep duration and wake time consistency, with short sleepers showing maximal cortisol increase after waking, while long sleepers peak before waking [22]. These findings highlight the importance of considering sleep-wake patterns when interpreting CAR data.

The Cortisol Awakening Response represents a valuable window into HPA axis dynamics and circadian regulation. When properly assessed using standardized protocols across appropriate biological matrices, CAR provides unique insights into neuroendocrine function relevant to both basic research and clinical applications. The comparative analysis of blood, saliva, and urine sampling presented herein highlights the particular utility of salivary cortisol for CAR assessment due to its balance of methodological practicality, non-invasive nature, and measurement of biologically active hormone fractions. As research methodologies continue to advance, particularly with the development of continuous monitoring technologies, our understanding of CAR as an HPA axis indicator will further refine its utility in circadian medicine and stress-related pathology.

Comparative Suitability of Blood, Saliva, and Urine for Biomarker Access

The choice of biofluid is a critical determinant of success in clinical research and diagnostics, particularly for investigating dynamic physiological processes like circadian rhythms. Traditional blood-based sampling, while considered the gold standard, presents significant limitations for intensive time-course studies. This application note provides a structured comparison of blood, saliva, and urine, framing their technical and practical suitability within the context of advanced hormone circadian rhythm research. The objective is to equip researchers and drug development professionals with the data and protocols necessary to select the optimal biofluid for specific biomarker applications, with a special emphasis on emerging, non-invasive methodologies that facilitate dense temporal sampling.

Comparative Analysis of Biofluids

The selection of a biofluid involves balancing analytical performance, practical feasibility, and biological relevance. The following tables provide a quantitative and qualitative summary of key considerations.

Table 1: Quantitative Diagnostic Performance of Key Biomarkers Across Biofluids

Biomarker	Biofluid	Correlation with Serum	Key Performance Metrics	References
C-Reactive Protein (CRP)	Serum	Gold Standard	N/A	[25]
	Saliva	r~sp~=0.709; P<.001	Significantly elevated in inflammation group (P=.001)	[25]
	Urine	r~sp~=0.886; P<.001	Significantly elevated in inflammation group (P=.001)	[25]
Creatinine (for CKD)	Serum	Gold Standard	N/A	[26]
	Saliva	Strong Correlation	AUC up to 1.00; Sensitivity & Specificity >85%	[26]
Urea (for CKD)	Serum	Gold Standard	N/A	[26]
	Saliva	Strong Correlation	AUC up to 1.00; Sensitivity & Specificity >85%	[26]

Table 2: Practical and Analytical Characteristics for Circadian Research

Characteristic	Blood (Serum/Plasma)	Saliva	Urine
Invasiveness	High (venipuncture)	Low (non-invasive)	Low (non-invasive)
Patient Preference	Least Preferred	Favored	Favored	[25]
Collection Logistics	Requires trained phlebotomist; clinic visit	Simple self-collection; suitable for home/remote use	Simple self-collection; suitable for home/remote use	[26] [27] [25]
Biomarker Representativeness	Total hormone levels (bound + free)	Free, bioavailable hormone levels	Hormone metabolites; integrated over time	[27] [28]
Ideal for Circadian Assessment	Challenging due to invasiveness	Excellent for dense sampling (e.g., Cortisol Awakening Response)	Good for 24-hour integrated patterns	[27] [6]
Major Advantages	Gold standard for many analytes; comprehensive panel availability	Reflects biologically active fraction; ideal for stress axis (HPA) mapping	Provides metabolic pathway information (e.g., estrogen metabolism)	[27] [28]
Key Limitations/Considerations	Stress of collection can influence biomarker levels (e.g., cortisol); not suitable for frequent sampling	Susceptible to contamination (food, drink); requires specific protocols (e.g., fasting)	Concentration varies; requires normalization (e.g., to creatinine)	[28] [25]

Experimental Protocols for Non-Invasive Biomarker Analysis

Protocol: Salivary CRP and Circadian Hormone Assessment

This protocol is adapted for the simultaneous assessment of inflammatory markers and circadian hormonal rhythms, such as cortisol.

A. Pre-Collection Requirements:

• Fasting: Participants should fast for at least 2 hours prior to sample collection, consuming only water [25].
• Oral Hygiene Restrictions: Avoid brushing teeth, using mouthwash, or chewing gum for at least 30 minutes prior to saliva collection [25].
• Timing: For circadian or Cortisol Awakening Response (CAR) studies, collect samples at precise, pre-defined times (e.g., immediately upon waking, 30 minutes post-waking, 60 minutes post-waking, and throughout the day) [27].

B. Sample Collection:

• Use standardized, inert collection tools such as the Salimetrics SalivaBio Oral Swab [25].
• Place the swab under the tongue for 2-3 minutes to ensure sufficient saturation.
• Transfer the swab directly into a pre-labeled collection tube without touching the swab.
• For RNA-based circadian rhythm analysis (e.g., core clock genes), collect 1.5 mL of saliva and immediately mix with 1.5 mL of RNAprotect reagent to preserve nucleic acid integrity [6].

C. Sample Storage and Transport:

• Store samples in a refrigerator or with cold packs immediately after collection.
• Transport to the laboratory as soon as possible. For long-term storage, freeze at -80°C.

D. Analytical Methods:

• CRP Quantification: Immunoassays (ELISA) are commonly used. Recent studies show strong correlation with serum CRP [25].
• Hormone Quantification: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold standard for its high specificity and accuracy in measuring steroids like cortisol, cortisone, estrogen, and progesterone [27].
• Gene Expression Analysis: For circadian clock genes (e.g., ARNTL1, PER2), perform RNA extraction followed by reverse transcription and quantitative PCR (qPCR) [6].

Protocol: Urinary Hormone Metabolite and CRP Assessment

This protocol is suited for comprehensive hormone metabolism profiling and inflammation monitoring.

A. Sample Collection:

• For a spot urine sample, collect a midstream urine sample into a sterile cup.
• For a 24-hour urinary hormone metabolite profile, collect all urine over a 24-hour period into a single container provided by the laboratory, keeping it cool throughout the collection period [28].
• Perform a dipstick test (e.g., Roche Combur Test Strip) immediately after collection to screen for urinary tract infections, which can confound CRP results [25].

B. Sample Processing:

• Aliquot the urine sample for different analyses.
• For metabolomic/hormone analysis: Use dried urine spots (DUTCH test) or freeze liquid urine at -20°C or below [28].
• For CRP analysis: Freeze aliquots at -80°C prior to analysis.

C. Analytical Methods:

• Hormone Metabolites: LC-MS/MS is used to profile a wide array of sex hormone and cortisol metabolites, providing insights into metabolic pathways [28].
• CRP Quantification: Utilize sensitive immunoassays validated for urine matrix. Note that values are typically normalized to urinary creatinine to account for dilution [25].

Workflow Visualization

The following diagram illustrates the logical decision-making process for selecting the most appropriate biofluid based on research objectives.

Biofluid Selection Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Non-Invasive Biomarker Research

Item	Function/Application	Example Product / Note
Saliva Collection Swab	Absorbs unstimulated whole saliva for hormone, protein, or DNA/RNA analysis. Inert material prevents analyte binding.	Salimetrics SalivaBio Oral Swab [25]
Saliva Collection Kit (Multi-Timepoint)	Facilitates at-home self-collection for circadian/CAR studies. Includes tubes, labels, and cold storage solutions.	Vibrant Wellness Salivary Hormones Test Kit [27]
RNA Stabilization Reagent	Preserves RNA integrity in saliva immediately upon collection for downstream gene expression analysis of circadian clock genes.	RNAprotect [6]
Dried Urine Kit	Enables convenient collection and stabilization of urine for comprehensive hormone metabolite profiling via LC-MS/MS.	DUTCH Plus Test Kit [28]
LC-MS/MS Instrumentation	Gold-standard method for high-specificity quantification of steroid hormones and their metabolites in saliva, urine, and blood.	Essential for validating biomarker assays [27] [28]
High-Sensitivity CRP Immunoassay	Quantifies low levels of CRP in non-invasive biofluids like saliva and urine.	Requires validation for the specific matrix [25]
Core Body Temperature Sensor	Provides a physiological correlate of circadian rhythm. Can be used alongside salivary and urinary biomarkers.	greenTEG CaleraResearch CORE [25]

Methodological Protocols and Research Applications for Different Sampling Matrices

The accurate assessment of hormone levels is a cornerstone of endocrinology and circadian biology research. While blood sampling has been the traditional methodology, saliva sampling has emerged as a robust, non-invasive alternative for measuring the bioavailable fraction of hormones, which is the portion that is biologically active and available to target tissues [29]. This protocol details the application of saliva sampling for the assessment of free, bioavailable hormones, placing specific emphasis on its critical role in circadian rhythm research. The diurnal patterns of hormones like cortisol and melatonin are central markers of the body's internal clock [8] [7]. Saliva collection is uniquely suited for high-density time-series sampling required to characterize these rhythms accurately, offering a practical advantage over more invasive blood collection and less specific urine collection [6] [30]. This document provides a standardized protocol for researchers and drug development professionals to implement reliable saliva sampling for hormonal and circadian applications.

Saliva as a Biological Matrix for Hormone Assessment

Saliva is an ultrafiltrate of blood, containing unbound, free steroid hormones that diffuse passively from the plasma into salivary glands [29] [30]. In circulation, 95-99% of steroid hormones are bound to carrier proteins (e.g., sex hormone-binding globulin, albumin) and are not biologically active. Saliva testing measures the bioavailable hormone fraction, which better correlates with clinical symptoms and tissue-specific hormone activity than total hormone levels measured in serum [29]. This makes saliva an ideal matrix for assessing hormonally driven physiological processes.

The non-invasive nature of saliva collection facilitates frequent sampling, which is imperative for capturing dynamic hormonal fluctuations, such as the cortisol awakening response (CAR) or the dim light melatonin onset (DLMO) [8] [7]. From a practical standpoint, saliva collection is cost-effective, reduces patient burden, and enables sampling in remote or non-clinical settings, including the patient's home [31] [30]. This is particularly advantageous for large-scale epidemiological studies and long-term monitoring of hormonal circadian rhythms.

Table 1: Comparison of Hormone Sampling Matrices

Feature	Saliva	Blood (Serum/Plasma)	Urine
Biomarker Measured	Free, bioavailable hormone fraction [29]	Total hormone (free + protein-bound) [29]	Metabolized hormone (integrated over time)
Invasiveness	Non-invasive	Invasive	Non-invasive
Collection Feasibility	High (self-collection at home) [30]	Low (requires phlebotomist)	High (self-collection)
Ideal for Circadian Profiling	Excellent (allows high-density time-series) [6]	Good (limited by invasiveness)	Poor (coarse temporal resolution)
Key Circadian Applications	Cortisol Awakening Response (CAR), Dim Light Melatonin Onset (DLMO) [8] [7]	Full 24-h hormonal profiles	24-hour integrated hormone output

Pre-Sampling Considerations and Protocol Design

Selection of Collection Device

The choice of collection device is critical and must be validated for the specific analyte of interest.

• Passive Drool: The gold standard for most steroid hormones. Participants drool through a short straw into a polypropylene tube [30] [32].
• Salivette-type Devices: Use a synthetic swab (e.g., polyester), not cotton. Cotton swabs contain plant sterols that can cross-react in immunoassays, leading to spuriously high results for hormones like testosterone, progesterone, and estradiol [30] [32]. Cotton swabs are validated only for cortisol.
• Tube Material: Use polypropylene tubes. Polyethylene tubes can adsorb steroids, reducing recovery [30] [32].

Protocol Design for Circadian Studies

Capturing an accurate circadian rhythm requires careful planning of the sampling schedule.

• Cortisol: The rhythm peaks around 30-45 minutes after waking and declines throughout the day [8]. A robust protocol includes samples immediately upon waking, 30 minutes post-waking, 45 minutes post-waking, and at strategic times throughout the day (e.g., 1200 h, 1700 h, 2000 h) [8].
• Melatonin: To assess DLMO, sampling should begin 5 hours before and continue until 1 hour after habitual bedtime, typically every 30-60 minutes under dim light conditions [7].

All sampling protocols must account for potential confounders. Participants should be instructed to avoid the following before providing a sample:

• Food and Drink: Avoid eating, drinking (especially caffeinated beverages), or brushing teeth for at least 30-60 minutes prior to sampling [30].
• Substances: Avoid alcohol and tobacco use.
• Blood Contamination: Vigorous tooth brushing can cause blood contamination, which skews results [30].

Step-by-Step Sample Collection and Handling Protocol

Materials and Reagents

Table 2: Research Reagent Solutions and Essential Materials

Item	Function/Description	Key Considerations
Polypropylene Tube	Sample collection and storage receptacle.	Prefer over polyethylene to minimize steroid adsorption [30].
DNA/RNA Shield or TNA Buffer	Preservative for nucleic acid and/or hormone integrity.	Protects biomarkers from degradation; added to tube pre-collection [31].
Zirconium Beads	Homogenization of viscous saliva samples.	Enables efficient processing for high-throughput workflows [31].
Barcode Labels	Sample tracking and de-identification.	Essential for maintaining chain of custody in large studies.
Cooling Pack & Insulated Mailer	Sample transport at stable temperature.	Maintains sample integrity from home collection to lab.

Detailed Collection Procedure

• Preparation: Provide participants with pre-labeled collection kits containing the appropriate tubes, detailed instructions, and a pre-filled sample log sheet.
• Rinsing: Instruct participants to rinse their mouth with water 10 minutes before sample collection.
• Collection:
  • For passive drool: Participants should tilt their heads forward and allow saliva to pool in the mouth before drooling through a straw into the collection tube. The process is repeated until the required volume (e.g., 1.5 mL) is collected [6].
  • For swab-based collection: Participants place the swab in their mouth and chew gently for 1-2 minutes until the swab is saturated, then place it back into the storage tube.
• Stabilization: If the assay requires it, a preservative (e.g., RNAprotect for gene expression, or specific hormone stabilizers) should be added to the tube at a predetermined ratio (e.g., 1:1 saliva-to-preservative) [6].
• Storage: Participants should immediately place the sample in their home freezer (-20°C) after collection. Studies show steroid hormones are stable in frozen saliva for at least a year at -20°C [30].
• Transport: Samples should be transported to the laboratory on dry ice or with cooling packs to maintain a frozen state.

Sample Processing Workflow in the Laboratory

The following diagram illustrates the core workflow for processing saliva samples upon receipt in the laboratory.

Diagram 1: Saliva Sample Processing Workflow

• Visual Inspection: Check for blood contamination or unusual viscosity/color [31].
• Homogenization: For viscous samples, bead-beating with zirconium beads (e.g., 45 s at 6 m/s) ensures even liquification and homogenization, which is critical for automated liquid handling [31].
• Aliquoting: Samples are aliquoted for different downstream analyses (e.g., hormone quantification, RNA extraction for gene expression).
• Downstream Analysis: Proceed with specific protocols for RNA extraction and gene expression analysis (e.g., of core clock genes like ARNTL1, PER2) [6] or hormone quantification.

Analytical Methods for Hormone Quantification

The low concentration of steroid hormones in saliva necessitates highly sensitive analytical methods. The two primary platforms are immunoassays and mass spectrometry.

• Enzyme-Linked Immunosorbent Assay (ELISA): A widely used and accessible method. Saliva-based ELISA tests must be highly sensitive and standardized. Performance metrics should include an inter-assay coefficient of variation (CV) <15% and an intra-assay CV <10% [30]. While practical for high-throughput analysis, ELISA can be susceptible to cross-reactivity with similar molecules.
• Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS): Considered the gold standard for hormone quantification due to its superior specificity, sensitivity, and ability to multiplex (measure multiple hormones simultaneously) [7]. It is particularly valuable for measuring low-concentration hormones like estradiol and for creating reference ranges [29].

Table 3: Comparison of Hormone Quantification Methods

Parameter	ELISA	LC-MS/MS
Sensitivity	Good (must be validated for saliva)	Excellent (high sensitivity for low-concentration analytes) [29]
Specificity	Moderate (potential for cross-reactivity)	High (distinguishes between structurally similar molecules) [7]
Throughput	High	Moderate to High
Cost	Lower	Higher (capital equipment)
Multiplexing	Limited (typically single-plex)	High (can measure a panel of hormones) [29]

Data Analysis and Interpretation in Circadian Context

Analyzing data from circadian sampling requires specialized approaches to determine key rhythm parameters.

• Acrophase: The time at which the peak of a rhythm occurs.
• Amplitude: The magnitude of the peak relative to the mean.
• Mesor: The rhythm-adjusted mean.

For cortisol, the Cortisol Awakening Response (CAR) is calculated as the area under the curve (AUC) or the increase from the waking sample to the 30-45 minute post-waking sample [8]. For melatonin, the Dim Light Melatonin Onset (DLMO) is often calculated using a fixed threshold (e.g., 3-4 pg/mL in saliva) or a variable threshold based on baseline values [7].

The molecular machinery of the circadian clock, which drives these hormonal rhythms, is based on interlocking transcription-translation feedback loops. The following diagram illustrates this core mechanism.

Diagram 2: Core Circadian Clock Feedback Loop

The CLOCK:BMAL1 heterodimer activates the transcription of Per and Cry genes. After translation, PER and CRY proteins form a complex that translocates to the nucleus to repress CLOCK:BMAL1 activity, forming the core negative feedback loop [33]. A stabilizing loop involves the transcriptional regulation of BMAL1 by REV-ERB (repressor) and ROR (activator). This molecular oscillator regulates the expression of clock-controlled genes, including those involved in hormone secretion [33].

Application Notes: Integration in Drug Development and Research

The reliability of saliva sampling opens doors for specific applications in pharmaceutical and clinical research.

• Chronotherapy Trials: Saliva sampling is ideal for personalizing drug administration times based on a patient's circadian phase to maximize efficacy and minimize side effects [6] [7].
• Adrenal and Gonadal Function Assessment: The non-invasive nature allows for easy monitoring of HPA axis function (via cortisol) and sex hormone profiles in response to therapeutic interventions.
• Longitudinal Studies: The feasibility of home collection enables robust long-term studies on the impact of diseases, lifestyle, or treatments on circadian hormonal rhythms.

In conclusion, this protocol provides a comprehensive framework for implementing saliva sampling to assess bioavailable hormones in circadian rhythm research. Adherence to standardized collection, processing, and analytical methods is critical for generating reliable, reproducible data that can advance our understanding of circadian biology and inform drug development.

Within the field of human chronobiology and endocrine research, the choice of biological matrix is critical for capturing accurate hormonal data. While blood serum analysis provides a snapshot of circulating hormone levels and saliva testing measures the bioavailable fraction, urine sampling offers a unique and non-invasive window into hormone metabolism and clearance pathways over time [34] [35]. This protocol focuses on the comprehensive assessment of hormone metabolites through urinary analysis, a methodology that reveals the functional endpoints of steroid hormone processing, conjugation, and elimination that are not accessible through other matrices.

The fundamental advantage of urine hormone metabolite profiling lies in its capacity to provide an integrated view of endocrine activity across circadian rhythms, capturing diurnal variations in hormone production and metabolism through controlled collection protocols [34]. Unlike single-timepoint serum measurements, which represent a momentary glimpse of hormonal status, urine collections spanning multiple time points or 24-hour periods reflect the dynamic interplay between hormone secretion, metabolic transformation, and clearance mechanisms [34] [10]. This approach is particularly valuable for investigating the circadian nature of endocrine function and its disruption in various pathological states.

Scientific Rationale and Circadian Context

Hormone Metabolism and Clearance Pathways

Steroid hormones undergo complex metabolic transformations that modulate their biological activity and clearance rates. These processes follow circadian patterns influenced by both central clocks in the suprachiasmatic nucleus and peripheral clocks in metabolic tissues [6] [10]. Urine hormone metabolite analysis captures the outputs of these pathways, providing functional readouts of enzymatic activity that are often obscured in blood or saliva testing.

The major metabolic pathways detectable in urine include hydroxylation, reduction, and methylation of parent steroid hormones [34]. For estrogens, the balance between 2-, 4-, and 16-hydroxylation pathways has clinical significance, with the 2:16 hydroxyestrone ratio serving as a marker of estrogenic balance [34]. Similarly, the metabolism of cortisol to tetrahydrocortisol (THF) and tetrahydrocortisone (THE) via 5α- and 5β-reductase pathways provides insights into glucocorticoid clearance rates that are relevant to metabolic health [36]. These metabolite patterns reflect the integrated activity of hepatic and peripheral tissue enzymes over the collection period.

Table 1: Key Hormone Metabolite Ratios and Their Clinical Significance in Urine Analysis

Metabolite Ratio	Pathway Assessed	Clinical Significance
2-OHE1:16α-OHE1	Estrogen hydroxylation	<1.5 suggests estrogen dominance; >2.0 considered protective [34]
2-MeOE1:2-OHE1	COMT methylation activity	Low ratio suggests need for methylation cofactors (SAMe, B vitamins) [34]
Androsterone:Etiocholanolone	5α- vs. 5β-reductase activity	Indicates androgen metabolism bias; high ratio may drive androgenic symptoms [34]
(THF+THE):(Free Cortisol+Free Cortisone)	Cortisol clearance rate	Fast clearance associated with obesity, hyperthyroidism; slow clearance with hypothyroidism, liver issues [36]
5α-Androstanediol:Testosterone	Tissue-level 5α-reductase activity	Marker of intracellular DHT metabolism; elevated in hirsutism, acne, alopecia [36]

Comparative Analysis of Biological Matrices

Each biological matrix used in hormone assessment offers distinct advantages and limitations that must be considered within research design. Blood serum provides total hormone concentrations with high analytical sensitivity but captures only a momentary snapshot and requires invasive collection. Saliva measures the bioavailable, unbound fraction of hormones and allows convenient circadian sampling but may not reflect comprehensive metabolic pathways [37] [35]. Urine integrates hormone production and metabolism over time, captures metabolite patterns essential for understanding clearance pathways, and enables non-invasive collection, though it requires creatinine normalization and careful timing of collections [34].

The circadian dimension is particularly relevant when comparing matrices. While saliva can track diurnal cortisol rhythms through multiple collections, urine provides additional data on metabolic clearance through the quantification of cortisol metabolites (THF, THE) that reflect overall production and elimination [10] [36]. This comprehensive view of both rhythmic secretion and metabolic processing makes urine particularly valuable for investigating endocrine disruptions in conditions with altered circadian function, such as shift work disorders or metabolic diseases.

Materials and Equipment

Research Reagent Solutions

Table 2: Essential Materials for Urine Hormone Metabolite Analysis

Item	Function/Application
LC-MS/MS System	Gold-standard analytical platform for simultaneous quantification of multiple steroid hormones and metabolites with high specificity and sensitivity [34] [38]
Dried Urine Collection Cards	Matrix for timed spot collections (typically 4-5 time points) to capture diurnal hormone variations; enables stable transport at ambient temperature [34] [36]
24-Hour Urine Collection Container	Chilled container for total 24-hour collection when complete hormone output assessment is required [34] [39]
Solid-Phase Extraction (SPE) Cartridges	Sample preparation to isolate and concentrate steroids from urine matrix prior to LC-MS/MS analysis [38]
Enzymatic Deconjugation Reagents	Hydrolysis of phase II conjugates (glucuronides/sulfates) to measure total hormone output [34]
Creatinine Standard	Reference compound for normalization of hormone values to account for urine concentration variability [34]
Acidification Reagents	pH stabilization for certain hormone metabolites during collection and storage [34]

Experimental Protocol

Sample Collection Procedures

Pre-Collection Guidelines

• Participant Preparation: Instruct participants to avoid alcohol, nutritional supplements, and vigorous exercise for 48 hours prior to and during collection, as these factors may influence hormone metabolism [34].
• Timing Considerations: For circadian rhythm studies, establish collection times that capture critical phases of hormone secretion (e.g., waking, mid-morning, afternoon, bedtime) [34] [10].
• Collection Materials: Provide appropriate collection kits containing either dried urine cards for spot collections or pre-chilled containers for 24-hour collections, along with detailed instructions and cold packs if required for shipping [39].

Dried Urine Spot Collection (4-Time Point Method)

• First Morning Void: Collect immediately upon waking. Record exact collection time. This sample typically reflects overnight hormone production and metabolism.
• Mid-Morning Collection: Collect approximately 2-3 hours after waking, before lunch. Record exact collection time.
• Afternoon Collection: Collect between 2-4 PM. Record exact collection time.
• Bedtime Collection: Collect immediately before going to sleep. Record exact collection time.
• Sample Processing: Apply 2-3 drops of each urine sample to the designated circles on the dried urine card. Allow cards to air dry completely before sealing in the provided barrier pouch with desiccant [34] [36].

24-Hour Urine Collection

• Collection Initiation: Begin collection after discarding the first morning void. Note the exact start time.
• Total Collection: Collect all urine throughout the 24-hour period in the provided chilled container.
• Final Collection: Include the first morning void of the following day to complete the 24-hour cycle.
• Volume Measurement and Aliquotting: After mixing the total collection, measure and record total volume. Remove a 10-15 mL aliquot for analysis and store immediately at -20°C or colder if shipping will be delayed [34] [39].

Sample Processing and Storage

• Preservation: For liquid urine collections, acidification may be required for certain hormone metabolites (e.g., catechol estrogens). Follow specific assay requirements [34].
• Short-Term Storage: Refrigerate samples at 2-8°C if processing within 72 hours of collection completion.
• Long-Term Storage: Freeze samples at -20°C or colder for extended storage. Avoid repeated freeze-thaw cycles.
• Shipping: Ship frozen samples on dry ice or dried urine cards at ambient temperature with adequate desiccant [39].

Analytical Methodology: LC-MS/MS

Sample Preparation

• Aliquoting: Divide 24-hour liquid collections into four aliquots representing waking, mid-morning, afternoon, and bedtime periods, or process dried urine card punches.
• Solid-Phase Extraction: Isolate free and conjugated steroids using optimized SPE protocols [38].
• Enzymatic Deconjugation: Treat samples with glucuronidase/sulfatase enzymes to hydrolyze conjugates and measure total hormone output [34].
• Creatinine Measurement: Analyze creatinine concentration in each sample to normalize hormone values for urine concentration [34].

LC-MS/MS Analysis

• Chromatographic Separation: Utilize reverse-phase liquid chromatography with optimized gradients to separate isobaric compounds (e.g., cortisol vs. cortisone) [34] [38].
• Mass Spectrometric Detection: Employ tandem mass spectrometry with multiple reaction monitoring for specific quantification of target analytes at picogram sensitivity [34] [38].
• Quality Control: Include calibration standards, quality control samples, and blanks in each analytical run. Acceptable precision should demonstrate <15% coefficient of variation [38].

Diagram 1: Experimental Workflow for Urine Hormone Metabolite Analysis

Data Analysis and Interpretation

Data Normalization and Quality Assessment

• Creatinine Indexing: Normalize all hormone metabolite values to urine creatinine concentration (ng/mg creatinine) to correct for variations in urine concentration and ensure valid comparisons [34].
• Recovery Assessment: Monitor internal standard recovery rates during LC-MS/MS analysis to ensure extraction efficiency.
• Reference Ranges: Compare results to sex- and age-specific reference ranges established for each metabolite and ratio [34] [36].

Key Analytical Outputs

Table 3: Comprehensive Urine Hormone Metabolite Panel

Analyte Category	Specific Biomarkers	Biological Significance
Estrogen Metabolites	Estrone (E1), Estradiol (E2), 2-OHE1, 4-OHE1, 16α-OHE1, 2-MeOE1, 4-MeOE1	Assess estrogen balance, hydroxylation pathways, and methylation capacity [34] [39]
Androgen Metabolites	Testosterone, 5α-DHT, Androsterone, Etiocholanolone, 5α-Androstanediol	Evaluate androgen production, 5α-reductase activity, and tissue-level androgenic impact [34] [36]
Glucocorticoid Metabolites	Free cortisol (x4-5 time points), Free cortisone, THF, α-THF, THE	Map diurnal cortisol rhythm, cortisol-cortisone shuttle, and glucocorticoid clearance [34] [36]
Progesterone Metabolites	Pregnanediol, Allopregnanolone, 5α-Pregnanediol, 5β-Pregnanediol	Assess progesterone production and metabolism to neuroactive metabolites [34]
Oxidative Stress Marker	8-hydroxy-2-deoxyguanosine (8-OHdG)	Evaluate systemic oxidative DNA damage [39]

Interpretation Framework

• Pattern Analysis: Identify consistent patterns across multiple metabolites rather than focusing on individual values in isolation.
• Pathway Assessment: Evaluate the balance between competing metabolic pathways (e.g., 2- vs. 16-hydroxylation of estrogens; 5α- vs. 5β-reduction of androgens).
• Circadian Alignment: Assess the timing and amplitude of cortisol secretion across collection time points relative to established norms.
• Clinical Correlation: Integrate laboratory findings with clinical presentation to develop targeted interventions.

Diagram 2: Hormone Metabolism and Clearance Pathways Detectable in Urine

Applications in Research and Drug Development

Urine hormone metabolite profiling provides valuable insights across multiple research domains:

• Circadian Rhythm Research: The non-invasive nature of urine collection enables detailed mapping of hormone rhythms across the 24-hour cycle without disrupting sleep or normal activities [6] [10].
• Drug Development: Assessment of drug effects on specific metabolic pathways (e.g., 5α-reductase inhibitors, aromatase modulators, COMT inhibitors) through changes in metabolite patterns [34] [36].
• Personalized Medicine: Identification of individual metabolic phenotypes that may predict treatment response or susceptibility to adverse effects [34] [40].
• Intervention Studies: Monitoring changes in hormone metabolism in response to nutritional, lifestyle, or pharmaceutical interventions through pre- and post-testing [40].
• Toxicology Assessment: Evaluation of endocrine disruptor effects on hormone metabolic pathways through altered metabolite profiles.

Limitations and Considerations

While urine hormone metabolite testing offers substantial advantages, researchers should consider several methodological factors:

• Collection Compliance: Incomplete or improperly timed collections can significantly impact results, particularly for circadian studies.
• Analytical Specificity: While LC-MS/MS provides high specificity, isobaric compounds may require sophisticated separation techniques.
• Phase III Metabolism: Urine analysis primarily captures glucuronidated and sulfated metabolites; additional genetic or functional testing may be needed for comprehensive assessment of elimination pathways [34].
• Circadian Alignment: Collection times should be aligned with individual sleep-wake cycles rather than fixed clock times for accurate circadian assessment.
• Medication Interference: Certain medications may alter hormone metabolism or analytical interference; careful medication history is essential.

This protocol provides a standardized approach for implementing urine hormone metabolite analysis in research settings, with particular relevance for studies investigating circadian endocrine function, metabolic clearance pathways, and personalized therapeutic interventions.

Blood sampling remains a cornerstone in clinical diagnostics and research for quantifying total hormone concentrations and executing broad-panel analyses. An estimated 60-70% of clinical decisions are based on laboratory test results, with the pre-analytical phase accounting for 46-68% of total testing errors [41]. This application note provides a standardized protocol for blood collection specifically optimized for comprehensive hormone profiling, with consideration of the comparative advantages and limitations of saliva and urine matrices within circadian rhythm research contexts.

Pre-Analytical Considerations

Patient Preparation and Pre-Sampling Controls

Table 1: Key Patient Preparation Factors Influencing Hormone Measurements

Factor	Impact on Hormone Measurements	Protocol Recommendation
Posture	Transitioning from supine to upright reduces circulating blood volume by up to 10%, affecting renin, aldosterone, and catecholamines [41].	Maintain supine position for 30 minutes prior to sampling for catecholamine/metanephrine tests; document posture for aldosterone/renin testing [41].
Fasting Status	Glucose and bone turnover markers fluctuate postprandially; prolonged fasting (>16 hours) may cause false positives in glucose tolerance tests [41].	Implement 10-12 hour fast for specific metabolic panels; avoid excessive water restriction; fasting not routinely required for lipid panels [41].
Circadian Variation	Cortisol, growth hormone, testosterone, renin, and aldosterone exhibit strong diurnal rhythms [41] [42].	Collect cortisol in morning; mid-morning collection for aldosterone-renin ratio; document collection time for all samples [41].
Medications & Supplements	Biotin supplements (>1 week washout) interfere with immunoassays; antihypertensives affect aldosterone-renin ratios; trimethoprim elevates creatinine [41].	Document all medications/supplements; withhold high-dose biotin ≥1 week before testing; consult laboratory for critical tests [41].

Sample Matrix Selection: Serum vs. Plasma

Table 2: Quantitative Comparison of 17β-estradiol and Progesterone in Plasma vs. Serum

Parameter	Plasma (EDTA) vs. Serum	Statistical Significance	Clinical/Research Implication
17β-estradiol	44.2% higher in plasma (40.75 pg/mL vs. 28.25 pg/mL) [43]	P < 0.001 [43]	Use matrix-specific reference ranges; critical for participant classification [43].
Progesterone	78.9% higher in plasma (1.70 ng/mL vs. 0.95 ng/mL) [43]	P < 0.001 [43]	Plasma may better tolerate processing delays; serum preferred for cleaner matrix [43].
Correlation	Strong positive correlation (17β-estradiol: r=0.72; progesterone: r=0.89) [43]	P < 0.001 for both [43]	Both matrices suitable for trend analysis despite concentration differences [43].

Experimental Protocol: Blood Collection for Hormone Panels

Materials and Equipment

• Tourniquet: Single-use, non-latex
• Evacuated Blood Collection System: Needles (21G or 23G), safety holder [41]
• Collection Tubes: Serum separator tubes (SST), K₂EDTA tubes, lithium heparin tubes [41] [44]
• Sample Transport Supplies: Biohazard bags, temperature-maintaining containers, secure packaging
• Laboratory Equipment: Centrifuge capable of 3500g, calibrated pipettes, -80°C freezer [43]

Step-by-Step Collection Procedure

• Patient Identification: Verify identity using at least two permanent identifiers (e.g., name, date of birth) [41]. Confirm test requisition form is complete and signed.

• Venipuncture Site Preparation: Apply tourniquet 3-4 inches above antecubital site. Disinfect with alcohol and allow to dry completely to prevent in vitro haemolysis [41].

• Blood Collection with Order of Draw: Adhere strictly to recommended sequence to prevent cross-contamination [41]:

  1. Blood culture bottles
  2. Sodium citrate tubes
  3. Serum separator tubes (SST)
  4. Lithium heparin tubes
  5. EDTA tubes (for transfusion)
  6. EDTA tubes (for hematology)
  7. EDTA tubes with gel separator
  8. Sodium fluoride/EDTA tubes

• Sample Handling Post-Collection:

  • Gently invert tubes 5-10 times (do not shake) [41]
  • Process serum tubes within 30-60 minutes: clot for 15 minutes at room temperature, then centrifuge at 3500g for 10 minutes [43]
  • Process EDTA plasma tubes: centrifuge at 3500g for 10 minutes at 4°C [43]
  • Aliquot supernatant into cryovials, avoiding disturbance of buffy coat or bottom layers

• Sample Storage: Freeze immediately at -80°C if analysis cannot be performed within 24 hours [44]. Avoid repeated freeze-thaw cycles.

Circadian Rhythm Sampling Considerations

For circadian studies, document precise collection times relative to the patient's wake time. The cortisol awakening response (CAR) requires sampling at wake, +30, and +60 minutes, as minutes matter for accuracy [42]. For melatonin rhythm assessment, saliva samples collected every 30-60 minutes under dim light conditions (DLMO protocol) are the reference standard, though sweat-based wearable sensors are emerging as alternatives [15] [42].

Comparative Biofluid Analysis in Circadian Research

Table 3: Comparison of Biofluids for Hormone Circadian Rhythm Assessment

Parameter	Blood (Serum/Plasma)	Saliva	Urine (24-hour)
Analytes Measured	Total hormone concentration (bound + free) [45]	Free, biologically active hormones [45]	Hormone metabolites, total output [34]
Invasiveness	High (venipuncture) [45]	Low (non-invasive) [45] [6]	Low (non-invasive) [34]
Temporal Resolution	Single time-point snapshot [34]	Multiple time points feasible for dense mapping [45]	24-hour integrated profile [34]
Circadian Applications	Gold standard for most clinical assays; confirms menstrual cycle status [43]	Ideal for cortisol awakening response, dim light melatonin onset [42]	Comprehensive metabolite profiling; estrogen metabolism ratios [34]
Key Advantages	Wide assay availability; established reference ranges [43]	Correlates with free serum fraction; home collection possible [45]	Captures hormone production, biotransformation, and elimination [34]
Key Limitations	Does not distinguish free from bound hormone; stressful collection may affect levels [45]	Affected by oral contamination, flow rate; limited analyte spectrum [46]	Requires creatinine normalization; complex collection [34]

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Materials for Blood-Based Hormone Analysis

Item	Function	Application Note
K₂EDTA Tubes	Chelates calcium to prevent coagulation; preserves labile hormones [43]	Yields higher 17β-estradiol and progesterone vs. serum; check assay compatibility [43]
Serum Separator Tubes (SST)	Contains clot activator and gel separator [41]	Provides cleaner matrix for immunoassays; process within 30-60 minutes [43]
Competitive Immunoenzymatic Assays	Quantify specific hormones (e.g., 17β-estradiol, progesterone) [43]	Document intra-assay CV (target <5%); know cross-reactivity profiles [43]
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)	Gold standard for metabolite profiling; detects isobaric compounds [34]	Required for urinary hormone metabolites (e.g., 2-OHE1, 16α-OHE1); superior specificity [34]
Enzyme-Linked Immunosorbent Assay (ELISA)	Alternative for saliva hormone testing [45]	Precise, cost-effective for salivary steroids which reflect free hormone levels [45]

Analytical Workflow and Decision Pathways

Diagram 1: Biofluid Selection and Processing Workflow for Hormone Analysis. This workflow outlines the decision pathway for selecting appropriate biofluids and processing methodologies based on research objectives in hormone profiling and circadian rhythm studies.

Optimal blood sampling for total hormone concentration and broad panels requires meticulous attention to pre-analytical variables, particularly sample matrix selection, circadian timing, and patient preparation. While blood remains the gold standard for total hormone quantification, integrating saliva and urine matrices provides complementary data on free hormone dynamics and metabolic clearance patterns essential for comprehensive circadian rhythm assessment. Adherence to standardized protocols minimizes pre-analytical errors and ensures data reliability across longitudinal studies and multi-center trials.

Within circadian rhythm research, the accurate quantification of hormones such as cortisol and melatonin in biological matrices like blood, saliva, and urine is paramount. The choice between immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) hinges on the specific demands of sensitivity, specificity, and practicality for high-frequency sampling protocols common in circadian studies. Immunoassays offer accessibility and high throughput, whereas LC-MS/MS provides superior specificity and precision, especially at the low hormone concentrations found in saliva. This application note provides a comparative analysis of these techniques and detailed protocols to guide researchers in selecting and implementing the optimal method for their circadian research.

Comparative Performance Analysis

The following tables summarize key performance characteristics for each analytical technique across different hormonal biomarkers, drawing on recent comparative studies.

Table 1: Method Comparison for Cortisol Measurement in Urine (Cushing's Syndrome Diagnosis) [47]

Method	Correlation with LC-MS/MS (Spearman r)	Sensitivity (%)	Specificity (%)	Diagnostic AUC
Autobio CLIA	0.950	89.7	96.7	0.953
Mindray CLIA	0.998	93.1	93.3	0.969
Snibe CLIA	0.967	92.0	94.7	0.963
Roche ECLLA	0.951	89.7	95.3	0.958
LC-MS/MS	(Reference)	-	-	-

Table 2: General Analytical Characteristics for Hormone Assays [48] [49] [12]

Parameter	Immunoassay	LC-MS/MS
Principle	Antibody-antigen binding with colorimetric, chemiluminescent, or electrochemiluminescent detection [47]	Physical separation and mass-to-charge ratio detection [47]
Specificity	Susceptible to cross-reactivity with structurally similar compounds and metabolites; can be affected by matrix interferents like CRP [49] [12]	High specificity; minimizes cross-reactivity through chromatographic separation and unique mass transitions [48] [12]
Sensitivity	Generally sufficient for cortisol in most matrices; can struggle with low salivary melatonin and estradiol [12]	Superior sensitivity and precision, especially for low-concentration analytes (e.g., salivary estradiol, melatonin) [48] [12]
Sample Throughput	High	Moderate to High
Multiplexing Capability	Limited (typically single-plex or low-plex on specialized platforms)	High (can simultaneously quantify multiple hormones in a single run)
Cost & Accessibility	Lower instrument cost, widely available	Higher instrument cost, requires specialized expertise

Experimental Protocols for Circadian Hormone Assessment

Protocol A: Urinary Free Cortisol (UFC) Analysis by Immunoassay

Urinary free cortisol is a key integrated measure of cortisol exposure and is used as an initial diagnostic test for Cushing's syndrome [47].

• Sample Collection: Collect a complete 24-hour urine sample in a container without preservatives. Aliquot and store at -20°C or -80°C until analysis [47].
• Sample Pretreatment: For certain platforms (e.g., Autobio, Mindray), an organic solvent extraction (e.g., ethyl acetate) may be used to improve specificity, but newer direct (extraction-free) methods are available [47]. If required, dilute the sample according to the manufacturer's instructions using the specified diluent (e.g., sample diluent, phosphate-buffered saline) [47].
• Analysis:
  • Calibrate the automated immunoassay analyzer (e.g., Roche Cobas e801, Mindray CL-1200i) according to the manufacturer's protocol using the provided calibrators [47].
  • Process quality control samples at two levels to ensure assay performance.
  • Load samples, calibrators, and controls onto the instrument.
  • The assay typically employs a competitive principle using chemiluminescent or electrochemiluminescent detection. Cortisol in the sample competes with a labeled cortisol derivative for binding sites on specific antibodies [47].
  • The instrument calculates cortisol concentration in nmol/24h or μg/24h based on the generated calibration curve.

Protocol B: Salivary Melatonin and Cortisol Analysis by LC-MS/MS

Saliva provides a non-invasive matrix for high-frequency sampling to determine circadian phase markers like Dim Light Melatonin Onset (DLMO) and the Cortisol Awakening Response (CAR) [12].

• Sample Collection: Collect saliva using specialized collection devices (e.g., Salivette). For DLMO assessment, collect samples under dim light every 30-60 minutes for 4-6 hours before and after habitual bedtime. For CAR, collect samples immediately upon waking and at 15, 30, and 45 minutes post-awakening [12]. Centrifuge samples to obtain clear saliva and store at ≤ -20°C.
• Sample Preparation:
  • Thaw samples on ice or at room temperature and vortex.
  • Dilute an aliquot (e.g., 200 μL) with pure water (e.g., 20-fold dilution) [47].
  • Add a known concentration of internal standard (e.g., deuterated cortisol-d4 and melatonin-d4) to correct for sample preparation and ionization variability [47].
  • Centrifuge the mixture to pellet any particulates.
• LC-MS/MS Analysis:
  • Chromatography: Inject the supernatant (e.g., 10 μL) onto a UPLC system. Use a suitable column (e.g., ACQUITY UPLC BEH C8, 2.1 × 100 mm, 1.7 μm) and a binary mobile phase gradient (e.g., water and methanol with modifiers) to achieve chromatographic separation [47].
  • Mass Spectrometry: Operate the mass spectrometer in positive electrospray ionization (ESI+) mode. Use Multiple Reaction Monitoring (MRM) to detect specific transitions for each analyte and its internal standard. Example transitions include cortisol: 363.2 → 121.0, melatonin: 233.2 → 174.2 [47] [12].
  • Quantification: Construct a calibration curve with known standards. The analyte-to-internal standard response ratio is used to interpolate the concentration of unknowns.

Figure 1: LC-MS/MS Workflow for Salivary Hormones

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for Hormone Assays [47] [12]

Item	Function/Description	Example Use Cases
Specific Antibodies	High-affinity, monoclonal or polyclonal antibodies for target hormone (e.g., cortisol, melatonin). Critical for immunoassay specificity and sensitivity [47].	Immunoassay development on automated or plate-based platforms.
Deuterated Internal Standards	Stable isotope-labeled analogs of the target analytes (e.g., cortisol-d4). Used for normalization in LC-MS/MS to correct for matrix effects and recovery losses [47].	LC-MS/MS method for salivary melatonin and cortisol; urinary free cortisol.
Certified Reference Materials	Highly purified and characterized hormone standards of known concentration and purity. Essential for calibrating both immunoassays and LC-MS/MS instruments [47].	Preparation of calibration curves in all quantitative methods.
Chromatography Columns	UPLC/HPLC columns (e.g., C8, C18) for separating analytes from matrix components prior to mass spectrometric detection [47].	Reversed-phase separation of hormones in saliva or urine for LC-MS/MS.
Sample Collection Kits	Specialized kits for specific matrices (e.g., Salivettes for saliva, 24-hr urine containers). Ensure sample integrity and compatibility with downstream assays [12].	Circadian rhythm studies requiring frequent or timed sampling.

The choice between immunoassay and LC-MS/MS in circadian research is context-dependent. For high-throughput urinary cortisol analysis where extreme sensitivity is not the primary concern, modern direct immunoassays demonstrate excellent diagnostic agreement with LC-MS/MS [47]. However, for the precise quantification of low-level hormones in saliva, such as melatonin for DLMO or estradiol, LC-MS/MS is the superior technique due to its enhanced specificity and sensitivity [48] [12]. Researchers must weigh factors including required specificity, target analytes, sample volume, throughput, and budget. As circadian medicine advances, LC-MS/MS is poised to become the gold standard for validating biomarkers and generating highly reliable data in complex, multi-hormone studies.

Circadian rhythms, the endogenous ~24-hour oscillations in physiology and behavior, are increasingly recognized as critical determinants of drug efficacy and safety. The emerging field of chronotherapy—timing drug administration to align with the body's internal circadian biology—leverages these rhythms to optimize therapeutic outcomes [50]. This approach is grounded in the understanding that the suprachiasmatic nucleus (SCN), the master circadian clock, regulates endocrine activity and peripheral clocks found in most cells and tissues, driving daily fluctuations in hormone levels, metabolic processes, and immune function [6] [50]. These rhythms profoundly influence pharmacokinetics (drug absorption, distribution, metabolism, excretion) and pharmacodynamics (drug activity and duration), making dosing timing a crucial variable in drug development [50].

Accurate assessment of an individual's circadian phase is therefore essential for personalizing chronotherapy regimens. This application note details protocols for quantifying circadian rhythms using hormone sampling from blood, saliva, and urine, providing a methodological framework for informing dosing schedules in clinical drug development.

Circadian Biomarkers and Sampling Matrices: A Comparative Analysis

The hormones melatonin and cortisol serve as robust, phase-defining circadian biomarkers. Melatonin, secreted by the pineal gland, signals the onset of the biological night, while cortisol, produced by the adrenal cortex, peaks around waking and promotes alertness [8] [7]. Their complementary rhythms provide a comprehensive view of circadian phase, which can be assessed through various biological matrices.

Table 1: Comparison of Biofluids for Circadian Hormone Sampling

Parameter	Blood (Serum/Plasma)	Saliva	Urine
Primary Analytes	Total melatonin & cortisol (free + protein-bound) [8] [7]	Free, biologically active melatonin & cortisol [8] [7]	Cortisol metabolites (e.g., 24-hour free cortisol) [8]
Key Circadian Metrics	Dim Light Melatonin Onset (DLMO), Cortisol Awakening Response (CAR) [7]	DLMO, CAR [7]	24-hour integrated cortisol secretion pattern [8]
Invasiveness & Practicality	High; requires clinical supervision, unsuitable for frequent home sampling [7]	Low; non-invasive, ideal for frequent, ambulatory, and home sampling [6] [7]	Moderate; non-invasive but requires 24-hour collection, cumbersome for participants [8]
Stability & Key Advantages	High analyte concentration, considered highly reliable [7]	Good stability with preservatives; reflects biologically active hormone fraction [8] [7]	Suitable for identifying chronic changes and prolonged cortisol elevation [8]
Major Limitations	Logistically demanding, stressful sampling may alter hormone levels [7]	Low hormone concentrations demand highly sensitive assays (e.g., LC-MS/MS) [7]	Does not capture rapid ultradian pulsatility or provide precise phase markers like DLMO [8]

Saliva presents a particularly compelling matrix for circadian research in drug development due to its non-invasive nature, allowing for dense, ecologically valid sampling schedules in outpatient settings [6]. This is critical for capturing the dynamic, pulsatile nature of hormone secretion without disrupting natural sleep-wake cycles.

Experimental Protocols for Circadian Phase Assessment

This section provides detailed methodologies for determining circadian phase using gold-standard hormonal markers.

Protocol for Salivary Dim Light Melatonin Onset (DLMO) Assessment

The DLMO is the gold standard for assessing the timing of the central circadian pacemaker [7].

• Primary Objective: To determine the clock time when endogenous melatonin secretion onset occurs under dim light conditions.
• Sample Collection:
  • Timing: Samples are collected every 30-60 minutes over a 4-6 hour window, typically starting 5 hours before and ending 1 hour after habitual bedtime [7].
  • Conditions: Participants must remain in dim light (<10-30 lux) for the duration of sampling to prevent melatonin suppression. They should refrain from eating, drinking (except water), smoking, or brushing teeth 10-30 minutes before each sample [7].
  • Materials: Salivettes or similar collection devices containing stabilizing agents (e.g., citric acid) are used to preserve sample integrity.
• Hormone Quantification:
  • Recommended Method: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). This method offers superior specificity, sensitivity, and reproducibility for low-abundance salivary analytes compared to immunoassays, which can suffer from cross-reactivity [7].
  • Alternative Method: High-sensitivity Enzyme-Linked Immunosorbent Assay (ELISA).
• Data Analysis (DLMO Calculation):
  • Fixed Threshold Method: DLMO is interpolated as the time when melatonin concentration crosses a predetermined threshold (e.g., 3-4 pg/mL for saliva) [7].
  • Variable Threshold Method: The threshold is set as two standard deviations above the mean of at least three baseline (pre-rise) values. This method may be more accurate for individuals with low melatonin production [7].

Protocol for Cortisol Awakening Response (CAR) and Diurnal Profile

This protocol assesses the integrity of the hypothalamic-pituitary-adrenal (HPA) axis, which exhibits a strong circadian rhythm.

• Primary Objective: To characterize the diurnal pattern of cortisol secretion, including the sharp rise that occurs 30-45 minutes after awakening.
• Sample Collection:
  • CAR: Collect saliva immediately upon waking, and again at 30, 45, and 60 minutes post-awakening. Participants should record exact wake and sampling times.
  • Diurnal Profile: Collect additional samples at 2-3 hour intervals throughout the day (e.g., 10:00, 13:00, 16:00, 19:00, before bed) [8].
• Hormone Quantification: LC-MS/MS or a validated, high-sensitivity immunoassay [8] [7].
• Data Analysis:
  • Calculate the area under the curve (AUC) for the CAR.
  • Determine the acrophase (time of peak concentration) and nadir (time of lowest concentration) for the diurnal profile. The acrophase of cortisol has been shown to correlate with the acrophase of core clock gene expression in saliva, such as ARNTL1, providing a molecular link to the peripheral clock [6].

Table 2: Essential Research Reagent Solutions for Circadian Hormone Sampling

Reagent/Material	Function & Application	Key Considerations
Salivette Collection Devices	Non-invasive collection of whole saliva; often contain cotton swabs and neutral/ citric acid-treated tubes [7].	Citric acid-treated versions stimulate saliva flow but may dilute analyte concentration; neutral is standard for hormone assays.
RNAprotect Solution	Preservative for RNA in saliva samples for concurrent gene expression analysis [6].	A 1:1 ratio with 1.5 mL saliva was optimized for high RNA yield and quality for transcriptomic studies [6].
LC-MS/MS Kits	Gold-standard for quantifying low-concentration hormones (melatonin, cortisol) in saliva [7].	Offers high specificity and sensitivity, overcoming cross-reactivity issues of immunoassays.
High-Sensitivity ELISA Kits	Immunoassay-based alternative for hormone quantification.	Lower cost but potential for cross-reactivity; requires rigorous validation for salivary matrices [7].
TimeTeller Kits	Commercial solution for assessing circadian phase via core clock gene expression (ARNTL1, PER2, NR1D1) in saliva [6].	Provides a molecular method to complement hormonal phase assessment.

Workflow Visualization for Chronotherapy Protocol Development

The following diagram illustrates the integrated workflow for developing a chronotherapy dosing schedule based on circadian phase assessment.

Application in Drug Development: Informing Dosing Schedules

Integrating circadian phase assessment into clinical trials can rationally guide the timing of drug administration. The resulting circadian phase data can directly inform dosing schedules to enhance target engagement and minimize off-target effects.

• Glucocorticoid Therapy: For patients with adrenal insufficiency, administering hydrocortisone in the early morning supports the physiological circadian rise in cortisol. Conversely, modified-release hydrocortisone taken at night can suppress the early-morning adrenocorticotropic hormone surge in congenital adrenal hyperplasia, demonstrating how timing achieves different therapeutic goals [50].
• Thyroid Hormone Therapy: While levothyroxine is typically taken in the morning, bedtime administration may be equally effective and improve adherence for some patients, provided meals and bedtime are sufficiently spaced [50].
• Chronotherapy in Oncology: Research indicates that the circadian timing of anti-cancer drug administration can significantly influence both efficacy and toxicity profiles. For instance, one study cited a doubling of survival rates in cancer patients when treatment was aligned with peak tumor temperature, a circadian rhythm [51].

The integration of circadian biomarker assessment—particularly using practical matrices like saliva—into drug development pipelines represents a paradigm shift towards more precise and personalized medicine. By moving beyond fixed-clock dosing to biology-guided scheduling, developers can significantly enhance therapeutic indices. Future efforts should focus on standardizing protocols, validating salivary biomarkers against clinical endpoints, and prospectively testing chronotherapy regimens in Phase II and III trials to fully realize the potential of timing as a critical variable in medicinal efficacy.

The accurate measurement of hormonal circadian rhythms has become a cornerstone in the diagnosis and management of endocrine and sleep disorders. The hypothalamic-pituitary-adrenal (HPA) axis, a critical regulator of circadian rhythm in humans, produces cortisol following a distinct 24-hour pattern that is fundamentally disrupted in conditions like Cushing's syndrome and circadian rhythm sleep disorders (CRSDs). The choice of biological matrix—blood, saliva, or urine—profoundly influences the diagnostic information obtained, each offering distinct advantages for capturing different aspects of hormonal secretion. Saliva provides a non-invasive means for assessing free, biologically active hormone levels, particularly useful for capturing diurnal patterns. Blood measurements reflect total hormone levels (both free and protein-bound), while 24-hour urine collection measures integrated hormone production over time. Understanding these distinctions is essential for developing targeted diagnostic protocols for conditions ranging from Cushing's syndrome to shift work disorder, advanced sleep phase disorder (ASPD), and delayed sleep phase disorder (DSPD).

Comparative Analysis of Sampling Matrices

Table 1: Comparison of Biological Matrices for Circadian Hormone Assessment

Parameter	Saliva	Blood (Serum/Plasma)	Urine
Analytes	Free cortisol, melatonin [6] [8]	Total cortisol, ACTH, melatonin [7]	Urine free cortisol (UFC), cortisol metabolites [8]
Key Circadian Metrics	Dim Light Melatonin Onset (DLMO), Cortisol Awakening Response (CAR), late-night salivary cortisol [7]	DLMO, CAR, ACTH-cortisol paired measurements [7]	24-hour integrated cortisol production [52]
Primary Clinical Applications	Cushing's syndrome screening, CRSD diagnosis, chronotype assessment [6] [52]	Differential diagnosis of Cushing's syndrome, ACTH measurement [52]	Cushing's syndrome screening, monitoring hypercortisolism [52]
Key Advantages	Non-invasive, suitable for home collection, reflects biologically active free hormone, excellent for time-series sampling [6] [8]	Gold standard for some analytes (e.g., ACTH), higher analyte concentrations, well-established protocols [7]	Integrated measure over time, not affected by pulsatile secretion, no circadian variation in sample [8]
Key Limitations	Lower analyte concentrations requiring sensitive assays, potential for oral contamination, influenced by collection method [6] [7]	Invasive, stressful (may affect cortisol), requires clinical setting, single time-point snapshot [8]	Cumbersome collection, requires patient adherence, affected by renal function, normal values not excluding Cushing's in ~20-25% of cases [52]
Stability & Handling	Stable at room temperature for mailing; use of RNAprotect for gene expression studies [6] [52]	Requires rapid processing and freezing; strict temperature control [7]	Requires refrigeration during collection; preservatives needed; volume measurement critical [52]

Diagnostic Applications for Cushing's Syndrome

Cushing's syndrome represents a paradigmatic condition of circadian disruption characterized by loss of normal cortisol rhythmicity and sustained hypercortisolism. The Endocrine Society guidelines recommend three primary diagnostic studies, each utilizing different biological matrices: late-night salivary cortisol, low-dose dexamethasone suppression test (blood), and 24-hour urine free cortisol [52].

Experimental Protocols for Cushing's Syndrome Diagnosis

Protocol 1: Late-Night Salivary Cortisol Collection

• Principle: Loss of circadian rhythm is an early marker of Cushing's; late-night samples detect elevated cortisol when levels should be nadir [52].
• Materials: Salivette or similar saliva collection device; freezer for storage.
• Procedure:
  • Collect sample between 11:00 p.m. and midnight.
  • Avoid food, coffee, tea, or brushing teeth for at least 30 minutes prior.
  • Place saliva collection device in mouth until saturated (typically 2-3 minutes).
  • Close device securely and freeze immediately if not shipping same day.
  • Mail to reference laboratory at room temperature (cortisol is stable) [52].
• Interpretation: Normal ranges vary by lab; most US labs report normal <3.0-4.0 nmol/L or 0.10-0.15 μg/dL. Elevated values suggest Cushing's with 93-100% sensitivity [52].

Protocol 2: 24-Hour Urine Free Cortisol (UFC)

• Principle: Measures integrated cortisol production over 24 hours, bypassing pulsatile secretion.
• Materials: Large collection jug (3-5 L) with preservative (e.g., boric acid); cooler with ice packs.
• Procedure:
  • Discard first morning void.
  • Collect all urine for next 24 hours, including first morning void of following day.
  • Keep collection jug refrigerated or on ice throughout.
  • Record total volume after completion; mix well and aliquot sample for laboratory.
  • Transport to lab on ice [52].
• Interpretation: Normal typically <40-50 μg/day; elevated suggests Cushing's. Note: 20-25% of Cushing's patients may have normal UFC [52].

Protocol 3: Overnight 1 mg Dexamethasone Suppression Test

• Principle: Tests HPA axis negative feedback; dexamethasone should suppress cortisol in normal individuals.
• Materials: 1 mg dexamethasone tablets; materials for blood draw.
• Procedure:
  • Administer 1 mg dexamethasone orally at 11:00 p.m.
  • Obtain serum cortisol between 8:00-9:00 a.m. the following morning.
  • Process serum promptly and freeze if not testing immediately [52].
• Interpretation: Normal suppression to <1.8 μg/dL; lack of suppression suggests Cushing's with 95-97% sensitivity [52].

Signaling Pathway in Cushing's Syndrome

Diagram Title: HPA Axis Dysregulation in Cushing's Syndrome

Diagnostic Applications for Circadian Rhythm Sleep Disorders

Circadian Rhythm Sleep Disorders (CRSDs) encompass conditions where the internal circadian timing system misaligns with the external environment. The American Academy of Sleep Medicine (AASM) practice parameters outline assessment methods, with melatonin and cortisol as key circadian phase markers [53].

Experimental Protocols for CRSD Assessment

Protocol 1: Dim Light Melatonin Onset (DLMO) Assessment

• Principle: DLMO is the gold standard for determining endogenous circadian phase, marking the onset of biological night [7].
• Materials: Salivette tubes; dim red light (<10 lux); freezer (-20°C); sensitive melatonin assay (LC-MS/MS preferred).
• Procedure:
  • Conduct in dim light (<10 lux) from 5 hours before until 1 hour after habitual bedtime.
  • Collect saliva samples every 30-60 minutes.
  • Maintain seated posture; prohibit exercise, food, and caffeine during sampling.
  • Freeze samples immediately after collection.
  • Analyze using LC-MS/MS for optimal sensitivity and specificity [7].
• Interpretation: DLMO typically defined as time when melatonin exceeds 3-4 pg/mL (saliva) or 10 pg/mL (serum) threshold. Phase delays in DSPD; phase advances in ASPD [7].

Protocol 2: Cortisol Awakening Response (CAR) Profile

• Principle: CAR reflects HPA axis reactivity and shows characteristic circadian pattern.
• Materials: Salivette tubes; home collection kit; freezer; cortisol immunoassay.
• Procedure:
  • Collect saliva immediately upon awakening (S1).
  • Collect subsequent samples at 30, 45, and 60 minutes post-awakening.
  • Record exact sampling times and wake time.
  • Avoid eating, drinking, or brushing teeth before completion.
  • Freeze samples until analysis [7].
• Interpretation: Normal pattern shows 40-60% increase within 30-45 minutes post-awakening. Altered patterns may indicate circadian misalignment or HPA axis dysfunction [7].

Protocol 3: Actigraphy with Sleep Diaries

• Principle: Objective measurement of sleep-wake patterns over extended periods.
• Materials: Actigraph device; sleep diary (Consensus Sleep Diary recommended).
• Procedure:
  • Wear actigraph on non-dominant wrist for 7-14 days minimum.
  • Complete sleep diary daily: bedtime, sleep onset latency, awakenings, rise time.
  • Maintain normal routine.
  • Download data and analyze with validated algorithms [17].
• Interpretation: Provides sleep parameters (timing, duration, regularity). AASM recommends as guideline for CRSD evaluation [53].

Molecular Circadian Clock Machinery

Diagram Title: Molecular Circadian Clock Feedback Loop

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Research Reagents for Circadian Hormone Studies

Reagent/Material	Function/Application	Key Considerations
Salivette Tubes (Sarstedt)	Standardized saliva collection for cortisol/melatonin	Different versions (cotton, polyester); choose based on analyte compatibility [6]
RNAprotect Saliva Reagent (Qiagen)	Preserves RNA for gene expression studies from saliva	Enables transcriptomic analysis of core clock genes (ARNTL1, PER2, NR1D1) [6]
LC-MS/MS Kits for melatonin/cortisol	Gold standard analytical method for hormone quantification	Superior specificity/sensitivity vs. immunoassays; crucial for low salivary concentrations [7]
Dexamethasone Tablets (1 mg)	HPA axis suppression testing for Cushing's diagnosis	Ensure proper formulation; timing critical (11 p.m. administration) [52]
Actigraphy Devices (e.g., Actiwatch)	Objective sleep-wake monitoring for CRSD assessment	AASM guideline recommended; minimum 7 days for reliable rhythm assessment [53] [17]
CRH/DDAVP	Stimulating agents for inferior petrosal sinus sampling (IPSS)	Used in differential diagnosis of ACTH-dependent Cushing's; CRH currently scarce in US [52]

Integrated Diagnostic Workflow

Diagram Title: Integrated Diagnostic Pathway for Circadian Disorders

The strategic selection of biological matrices for hormone circadian sampling provides complementary insights essential for modern clinical diagnostics. Saliva has emerged as a particularly valuable matrix for ambulatory circadian monitoring, enabling sensitive detection of rhythm disturbances in both Cushing's syndrome and CRSDs through measurements of late-night salivary cortisol and DLMO. Blood remains indispensable for analytical precision and certain specialized tests, while urine offers unique value for integrated hormone production assessment. As circadian medicine advances, optimized protocols leveraging the distinct advantages of each matrix will continue to enhance diagnostic accuracy, therapeutic monitoring, and personalized treatment approaches for patients with circadian rhythm disorders.

Troubleshooting Sampling Protocols and Optimizing Data Reliability

Accurate assessment of hormonal circadian rhythms is critical for research and drug development. The pre-analytical phase—encompassing all steps from participant preparation to sample processing—is a significant source of variability, with errors accounting for over 60% of laboratory testing errors [54]. Factors such as light exposure, patient posture, and precise time stamping are not mere procedural details; they are fundamental determinants of data integrity, as they can directly influence hormone concentration and interpretation of the circadian phase [55] [10]. This document provides detailed application notes and protocols for controlling these variables in blood, saliva, and urine-based circadian hormone sampling, framed within a comparative research context.

The following table summarizes the critical pre-analytical variables and their specific impacts on different biological matrices used for circadian hormone assessment.

Table 1: Influence of Pre-Analytical Variables on Different Sample Matrices for Circadian Research

Pre-Analytical Variable	Impact on Blood	Impact on Saliva	Impact on Urine	Supporting Evidence
Light Exposure	Direct light degrades bilirubin (~2.3%/hour) and can affect photosensitive hormones [54].	Specific light sensitivity for saliva analytes is less documented; general protection is recommended.	Light can degrade specific metabolites; protection recommended for certain assays.	Clinical laboratory case studies [54].
Posture	Posture shifts can cause hemodilution/hemoconcentration, significantly affecting protein-bound hormones and electrolytes. Postural changes during phlebotomy can alter renin and aldosterone levels.	Considered minimal impact due to non-invasive collection.	Not applicable.	Documented effects on electrolytes and proteins [55].
Tourniquet Time	>60 sec can increase K+ by 2.5%; 60-120 sec can increase total cholesterol by 5% [54].	Not applicable.	Not applicable.	Analysis of pre-analytical errors [54].
Sample Processing Delays	Delays affect stability: glucose decreases 5-7%/hour; bilirubin decreases ~2.3%/hour in unprocessed samples [54].	Requires protocol optimization (e.g., use of RNAprotect) for transcriptomic analysis [6].	Typically stable for longer periods at room temperature for many hormones; requires preservation for specific analytes.	Stability data for analytes in blood [54]; saliva protocol optimization [6].
Time Stamp Accuracy	Critical for defining acrophase of cortisol, melatonin, and other circadian hormones.	Critical for correlating gene expression (e.g., ARNTL1) with hormone rhythms [6].	Essential for calculating fractional excretion or total output over timed intervals.	Correlation of saliva gene expression acrophase with cortisol and bedtime [6].

Experimental Protocols for Circadian Sampling

Protocol for Controlled Light Exposure and Postural Assessment

Aim: To standardize light conditions and posture prior to and during sample collection for accurate circadian hormone measurement. Materials: Dim light source (<50 lux), light meter, phlebotomy chair or bed, timer, sample collection kits. Methodology:

• Light Control: For melatonin assessment, samples must be collected under dim light conditions (<10-50 lux) to prevent suppression of melatonin secretion. Verify light levels at the participant's eye level using a calibrated light meter [10].
• Postural Control: For blood collection, the participant should rest in a semi-recumbent or seated position for at least 15 minutes prior to venipuncture. The use of an indwelling catheter is preferred for serial sampling to avoid repeated postural changes and tourniquet application [55] [54].
• Sampling: Perform phlebotomy or saliva collection without a tourniquet or with minimal tourniquet time (<60 seconds). Note the exact posture and time of collection.
• Documentation: Record light levels, posture, and precise collection time for every sample.

Protocol for High-Density Time-Course Sampling in Saliva

Aim: To characterize the molecular circadian rhythm using saliva samples for gene expression and hormone analysis. Materials: Saliva collection kits (e.g., Salivettes), RNA stabilizer (e.g., RNAprotect), cold storage, labeled cryovials, timer. Methodology:

• Study Design: Collect saliva samples at 3-4 time points per day over at least 2 consecutive days [6].
• Sample Collection: Use a standardized volume of saliva (e.g., 1.5 mL) mixed with a stabilizer at a predefined ratio (e.g., 1:1 saliva:RNAprotect) to ensure RNA integrity for gene expression analysis [6].
• Time Stamping: Record the exact clock time of each sample. The bedtime and wake time of the participant on the sampling day should also be recorded, as these behavioral cues correlate with acrophases of gene expression and cortisol [6].
• Processing: Centrifuge samples as required and store at -80°C until analysis for hormones (cortisol, melatonin via ELISA) or RNA extraction for core-clock gene expression (e.g., ARNTL1, PER2, NR1D1) [6].

Graphviz DOT script for the circadian sampling workflow:

Diagram 1: Workflow for controlled circadian sampling.

Protocol for Enzyme-Linked Immunosorbent Assay (ELISA)

Aim: To accurately quantify hormone levels (e.g., cortisol, melatonin) in biological samples. Materials: 96-well microplate, coating buffer (e.g., PBS or carbonate), capture antibody, analyte standard, sample, blocking buffer (e.g., BSA), detection antibody, enzyme conjugate, substrate, stop solution, ELISA plate reader [56] [57]. Methodology:

• Coating: Adsorb the capture antibody to the wells by incubating in coating buffer. The optimal concentration must be determined via checkerboard titration during assay optimization to form a monolayer [57].
• Blocking: Saturate remaining protein-binding sites with a blocking buffer (e.g., 1-5% BSA) to minimize non-specific binding [57].
• Sample & Analyte Incubation: Add samples and a dilution series of the standard to respective wells. The sample diluent should mimic the sample matrix to avoid interference [57].
• Detection: Add a detection antibody specific to the analyte, followed by an enzyme-conjugated secondary antibody (for indirect detection) [56].
• Signal Development & Readout: Add an enzyme substrate (e.g., TMB for HRP) to produce a colored product. Stop the reaction with acid and measure the absorbance with a plate reader [56].
• Validation: Perform spike-and-recovery and linearity-of-dilution experiments to validate the assay in the specific sample matrix (serum, saliva, urine) [57].

Graphviz DOT script for the core circadian clock mechanism:

Diagram 2: Core circadian clock transcriptional-translational feedback loop.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Circadian Hormone Sampling and Analysis

Item	Function/Application
RNAprotect / Other Stabilizers	Preserves RNA integrity in saliva and other tissues immediately upon collection, preventing degradation prior to RNA extraction and gene expression analysis [6].
ELISA Kits	Enable quantitative measurement of specific hormones (e.g., cortisol, melatonin) and peptides in biological fluids like serum, plasma, saliva, and urine [56].
Dim Light Source (<50 lux)	Critical for melatonin sampling protocols to prevent light-induced suppression of the hormone, which would distort the true circadian rhythm measurement [10].
Portible Light Meter	Verifies that ambient light levels at the participant's eye level are within the required dim light range for valid melatonin phase assessment [10].
Actigraphy Monitors	Provides objective, continuous measurement of sleep-wake cycles and activity rhythms, used to correlate with hormonal data and assess behavioral entrainment [10].
Saliva Collection Kits (e.g., Salivettes)	Provide a standardized, non-invasive method for collecting sterile saliva samples for subsequent hormone or RNA analysis [6].
Blocking Buffers (BSA, Non-Fat Milk)	Used in ELISA to saturate unused binding sites on the microplate well surface, thereby reducing nonspecific background signal and improving assay specificity [57].
Microtiter Plates	Specialized plates with a high-binding surface (e.g., charged polystyrene) for immobilizing proteins (antigens or antibodies) in the ELISA procedure [56] [57].

In circadian rhythm research, the choice of biological matrix—blood, saliva, or urine—profoundly impacts data quality and interpretation. Each matrix presents unique advantages and specific vulnerabilities to pre-analytical errors, contamination, and confounding variables that can compromise hormone measurement accuracy. This application note synthesizes current methodologies and protocols to address matrix-specific challenges in circadian sampling for hormones such as cortisol and melatonin, enabling researchers to optimize data reliability in chronobiological studies.

Matrix-Specific Challenges and Quantitative Comparisons

The table below summarizes the primary contamination sources and collection errors associated with each biological matrix used in circadian hormone assessment.

Table 1: Matrix-Specific Challenges in Circadian Hormone Sampling

Matrix	Primary Contamination Sources	Common Collection Errors	Key Circadian Analytes	Major Interference Factors
Blood	Hemolysis, improper anticoagulants [7]	Incorrect tube filling, prolonged tourniquet time, improper processing delay [9]	Total cortisol, melatonin	Shift work, sleep deprivation, drug interactions [9]
Saliva	Food particles, blood from gingivitis, topical creams [6] [7]	Stimulated saliva production, variable sample volume, contamination during expectoration [6]	Free cortisol, melatonin (DLMO) [7]	Light exposure (melatonin), smoking, alcohol consumption [9]
Urine	Microbial degradation, oxidizing agents [58]	Incomplete voiding, inaccurate timing/volume recording, improper preservation [58]	Cortisol metabolites, 6-sulfatoxymelatonin	Hydration status, renal function, paraben exposure [58]

Experimental Protocols for Robust Circadian Assessment

Salivary Hormone Collection Protocol

Saliva provides a non-invasive means for assessing the free, biologically active fraction of hormones and is particularly useful for dim light melatonin onset (DLMO) and cortisol awakening response (CAR) measurements [7].

Detailed Methodology:

• Pre-collection Instructions: Participants should refrain from eating, drinking (except water), brushing teeth, or smoking for at least 30 minutes before sampling. These activities can alter saliva composition and introduce contaminants [7].
• Sample Collection: Use specialized saliva collection aids (e.g., Salivettes). For passive drool, participants should drip saliva directly into a collection vial. For swab-based methods, the swab is placed in the mouth until saturated. Avoid stimulating saliva flow, as this can dilute analyte concentration [6].
• Preservation and Storage: Immediately upon collection, stabilize samples using a preservative like RNAprotect (for gene expression) at a 1:1 ratio if also analyzing transcriptomics. For hormone-only analysis, freeze samples at ≤ -20°C immediately after collection [6] [7].
• Circadian Protocol: For DLMO assessment, collect samples in dim light (<10 lux) every 30-60 minutes for 4-6 hours before habitual bedtime. For CAR, collect immediately upon waking, and at 30, 45, and 60 minutes post-awakening [7].

Urinary Hormone Collection and Clean-up Protocol

Urine analysis reflects integrated hormone secretion over time and is suitable for assessing metabolite excretion patterns.

Detailed Methodology:

• Sample Collection: Participants should provide first-morning voids or timed collections (e.g., 24-hour). Record exact start/stop times and total volume. Use pre-cleaned containers to avoid chemical contamination [58].
• Sample Preparation (QuEChERS Approach):
  • Volume & Dilution: Optimize sample and final dilution volumes to balance matrix interference and sensitivity. Tested combinations include 2 mL or 5 mL urine diluted to 100 µL, 500 µL, or 1000 µL [58].
  • Extraction: Employ a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) salt mixture (e.g., SALT-Kit-AC2) for clean-up. This step removes proteins and other matrix interferents [58].
  • Analysis: Analyze extracts using HPLC-QTOF (High-Performance Liquid Chromatography coupled to a Quadrupole Time-of-Flight mass spectrometer). Chromatographic separation can be achieved within 16 minutes, providing high sensitivity for endocrine-disrupting chemicals and endogenous hormones [58].

Workflow Visualization

The following diagram illustrates the logical workflow for selecting and handling biological matrices in circadian research, highlighting critical control points to mitigate contamination and collection errors.

The Scientist's Toolkit: Essential Research Reagents and Materials

The table below lists key reagents and materials essential for mitigating contamination and ensuring sample integrity in circadian hormone studies.

Table 2: Research Reagent Solutions for Circadian Sampling

Item	Function	Application Notes
RNAprotect Saliva Reagent	Stabilizes RNA in saliva for transcriptomic analysis of core clock genes (e.g., ARNTL1, PER2) [6].	Use at a 1:1 ratio with saliva; enables concurrent analysis of hormonal and gene expression rhythms [6].
QuEChERS Salt Kits	Provides a quick, effective clean-up step for complex urine matrices, removing interfering compounds prior to analysis [58].	Critical for reducing matrix effects in LC-HRMS analysis; allows simultaneous assessment of multiple endocrine disruptors [58].
LC-HRMS/MS Systems	High-resolution mass spectrometry for highly specific and sensitive detection of hormones and contaminants [58] [59].	Superior to immunoassays by minimizing cross-reactivity; essential for accurate low-concentration salivary melatonin measurement [7].
Certified Reference Materials (CRMs)	Provides quality assurance and quality control (QA/QC) for method validation and calibration [60].	Mitigates data heterogeneity; especially important for biomonitoring studies in blood and urine [60].
Dim Light Melatonin Onset (DLMO) Protocol	Standardized procedure for assessing circadian phase using salivary melatonin [7].	Requires strict control of ambient light (<10 lux) and timing; the gold standard for non-invasive phase assessment [7] [9].

Within circadian rhythm research and chronotherapy development, the strategic selection of a biological sampling matrix is paramount for accurately assessing the pharmacokinetic and pharmacodynamic profiles of therapeutic interventions. The rising prominence of non-invasive sampling and the need to align biomarker measurement with a drug's route of administration present significant methodological considerations [6] [11]. This protocol is framed within an extensive thesis exploring the comparative advantages of blood, saliva, and urine for hormone circadian sampling. Each matrix offers a unique window into the body's circadian phase and response to therapy, influenced by the supplementation type—whether topical or oral [11] [7]. The following application notes provide a structured guide for researchers and drug development professionals to match the sampling matrix to the supplementation type, ensuring robust and clinically relevant data on circadian entrainment and therapy efficacy.

Matrix Characteristics and Comparative Analysis

Table 1: Comparative Analysis of Biological Sampling Matrices for Circadian Research

Matrix	Key Advantages	Primary Limitations	Ideal for Supplementation Type	Key Circadian Biomarkers
Saliva	Non-invasive, suitable for frequent/home collection, reflects bioavailable hormone levels [6] [11].	Lower analyte concentrations, sensitivity to collection protocol (e.g., food, contamination), requires sensitive analytical methods (e.g., LC-MS/MS) [11] [7].	Topical & Oral	Melatonin (DLMO), Cortisol (CAR), Core Clock Gene Expression (e.g., ARNTL1, PER2) [6] [7].
Blood (Serum/Plasma)	High analyte concentration, rich in metabolites & proteins, gold standard for many hormonal assays [11].	Invasive, requires clinical setting, unsuitable for high-frequency sampling, influenced by stress from venipuncture itself [11].	Oral & Systemic	Full melatonin & cortisol profile, inflammatory markers, drug metabolites [11] [7].
Urine	Integrated measure over time (e.g., overnight), non-invasive, suitable for metabolite ratio analysis [11].	Does not provide precise phase timing, time-lagged concentration, influenced by renal function and hydration status.	Oral	6-sulfatoxymelatonin (aMT6s), Cortisol metabolites [11].

The choice of matrix directly impacts the reliability of circadian phase assessment. For instance, Dim Light Melatonin Onset (DLMO), the gold standard for circadian phase mapping, can be measured in saliva or plasma, with salivary offering a practical advantage for ambulatory studies [11] [7]. Conversely, the Cortisol Awakening Response (CAR) is most accurately captured through serial saliva samples due to the stress-induced perturbation of cortisol levels from blood draws [11]. When assessing the impact of a therapy on the peripheral molecular clock, saliva provides direct access to clock gene expression in oral mucosal cells, a valid proxy for the systemic circadian rhythm [6].

Experimental Protocols for Matrix-Specific Sampling

Saliva Sampling Protocol for Topical and Oral Therapy Monitoring

Application: This protocol is optimized for assessing circadian phase shifts in response to both topical and oral therapies, focusing on hormones like melatonin and cortisol, as well as gene expression analysis [6] [7].

Workflow Overview:

Materials:

• Saliva Collection Device: Salivettes or similar passive drool kits.
• Preservative: RNAprotect for gene expression; no preservative needed for hormone assays if frozen promptly [6].
• Cooling: Portable cooler or frozen gel packs.
• Time-Keeping: Dedicated digital timer and collection log sheet.

Detailed Procedure:

• Participant Preparation: Instruct participants to avoid the following for 60 minutes before each sample: eating, drinking (except water), brushing teeth, and applying topical products to the face or lips [11]. For DLMO assessment, samples must be collected under dim light (<10 lux) [7].
• Sample Collection: Collect 1.5 mL of unstimulated saliva via passive drool into a provided tube. For RNA studies, immediately mix with an equal volume (1.5 mL) of RNAprotect to stabilize nucleic acids [6].
• Sampling Timecourse: For a foundational circadian profile, collect samples at 3-4 timepoints per day (e.g., upon waking, +30 minutes for CAR, afternoon, before bed) over at least two consecutive days [6]. For precise DLMO, collect samples every 30-60 minutes in the 4-6 hours before habitual bedtime [7].
• Recording Metadata: Participants must record the exact time of sample collection, time of therapy application (for topical) or ingestion (for oral), and any deviations from the protocol.
• Storage and Transport: Freeze samples at -20°C or lower immediately after collection. Transport to the lab on dry ice to maintain sample integrity [6].

Protocol for Blood and Urine Sampling in Oral Therapy Pharmacokinetics

Application: Blood and urine are best suited for oral therapy development to characterize systemic exposure, metabolism, and full 24-hour hormone profiles [11].

Workflow Overview:

Blood Sampling:

• Procedure: In a clinical setting, collect serial blood samples via venipuncture or an indwelling catheter. A comprehensive 24-hour profile may require 4-6 samples, while a focused CAR assessment requires waking and +30 minute samples.
• Analysis: Serum/plasma is analyzed using high-sensitivity platforms like LC-MS/MS for melatonin, cortisol, and drug concentrations, providing a systemic measure [11] [7].

Urine Sampling:

• Procedure: Participants collect all urine over a defined period (e.g., 24-hour or overnight). The total volume is recorded, and an aliquot is taken for analysis.
• Analysis: The primary circadian marker is 6-sulfatoxymelatonin (aMT6s), a stable melatonin metabolite. Measurement provides an integrated output of melatonin production over the collection period [11].

The Scientist's Toolkit: Key Reagents and Materials

Table 2: Essential Research Reagent Solutions for Circadian Sampling

Item	Function/Application	Key Considerations
RNAprotect Solution	Stabilizes RNA in saliva samples immediately upon collection, preventing degradation for subsequent gene expression analysis of clock genes [6].	A 1:1 ratio with saliva is optimal for yield and quality [6].
Salivette / Passive Drool Kits	Provides a standardized, hygienic method for saliva collection.	Choose polyester/polypropylene swabs; avoid cotton for hormone assays due to interference.
LC-MS/MS Kits	Gold-standard analytical platform for quantifying low-abundance hormones (melatonin, cortisol) in saliva and blood with high specificity and sensitivity [11] [7].	Overcomes cross-reactivity issues of immunoassays; essential for reliable salivary melatonin measurement [7].
Melatonin Immunoassay Kits	Alternative to LC-MS/MS for hormone quantification; can be used for urine aMT6s or serum melatonin.	Potential for cross-reactivity with metabolites; verify analytical validation for the intended matrix [11].
Consensus Sleep Diary	Subjective tool to record sleep-wake timing, therapy intake, and potential confounders.	Critical for correlating biomarker data with behavior and identifying social jetlag [10] [17].
Dim Light Melatonin Onset (DLMO) Protocol	A standardized procedure for determining the circadian phase by measuring the evening rise in melatonin.	Requires strict control of ambient light (<10 lux) and precise timing of saliva or blood samples [11] [7].

Data Interpretation and Integration

Interpreting data from multi-matrix studies requires an understanding of the temporal and physiological relationships between them. Salivary cortisol CAR reflects the acute HPA axis response upon waking, while urinary cortisol metabolites represent a longer-term, integrated output. For oral therapies, blood sampling provides the peak concentration (Cmax) and pharmacokinetic curve, whereas saliva may reflect the bioavailable, unbound fraction of the drug [11].

Gene expression data from saliva, such as the acrophase of ARNTL1 expression, can be directly correlated with the timing of hormonal rhythms (e.g., cortisol) and behavioral data like self-reported bedtime to build a comprehensive picture of an individual's circadian state and its modulation by therapy [6]. This integrated approach is the cornerstone of personalized chronotherapy.

Optimizing Sampling Frequency for Diurnal and Ultradian Rhythm Capture

The accurate capture of diurnal (24-hour) and ultradian (<24-hour) rhythmicity is fundamental to advancing the field of circadian medicine. The choice of biological matrix—blood, saliva, or urine—and its associated sampling protocol directly influences the resolution, accuracy, and practicality of circadian research and clinical application. This document provides detailed application notes and protocols for optimizing sampling frequency across these biofluids, framed within a broader research context comparing their respective advantages and limitations for hormone circadian sampling. Aimed at researchers, scientists, and drug development professionals, these guidelines are designed to enhance the quality and reliability of data collected in both controlled laboratory and real-world settings.

Theoretical Foundations of Sampling Frequency

The core principle guiding sampling frequency is the Nyquist-Shannon theorem, which states that to accurately reconstruct a signal, the sampling frequency must be at least twice the highest frequency component of the signal of interest.

• For Diurnal Rhythms (≈24-hour period): A minimum of 12 samples over 24 hours (i.e., every 2 hours) is theoretically sufficient to capture the fundamental rhythm. However, higher-frequency sampling is often required to define the precise shape and timing (phase) of the waveform, especially for hormones with sharp peaks, such as cortisol [8].
• For Ultradian Rhythms (e.g., cortisol pulses with 60-90 minute cycles): A much higher sampling frequency is necessary. Capturing these rhythms may require sampling as often as every 10-30 minutes over a significant portion of the day to resolve individual pulses without aliasing [8] [61].

In practice, the optimal design must balance statistical power with practical constraints such as participant burden, cost, and sample volume. For a rhythm of known period (e.g., 24 hours), equispaced sampling is statistically the most powerful design [61]. However, when investigating rhythms with unknown or variable periods, or when equispaced sampling is logistically infeasible, optimized irregular sampling schedules can be developed using computational tools like PowerCHORD to maximize detection power across a range of frequencies [61].

Biofluid-Specific Sampling Protocols and Analytical Methods

The selection of biofluid dictates not only the sampling protocol but also the analytical approach, as each matrix reflects different physiological compartments and hormone fractions.

Saliva Sampling

Saliva offers a non-invasive means to measure the biologically active, free fraction of hormones, which is crucial for assessing physiological activity.

• Detailed Protocol for Salivary Circadian Assessment [6]:
  • Collection: Use Salivette collection devices (sarstedt). Participants should chew the synthetic swab for at least 1 minute. Avoid consuming food, caffeine, or dairy products at least 30 minutes prior to collection. Ensure proper oral hygiene before sampling.
  • Sampling Frequency: For a robust diurnal profile, collect samples at a minimum of 3-4 time points per day over 2 consecutive days. Key time points should capture the circadian dynamics, such as upon awakening, 30 minutes post-awakening, in the early afternoon, and before sleep [6] [62].
  • Sample Handling: Following collection, swabs can be temporarily stored at -20°C. For long-term storage, centrifuge the samples and store the supernatant at -80°C. For RNA analysis from saliva, stabilize with RNAprotect at a 1:1 ratio [6].
  • Analysis: Hormones like cortisol and melatonin can be quantified using enzyme-linked immunosorbent assays (ELISA) or radioimmunoassay (RIA). For gene expression analysis of core-clock genes (e.g., ARNTL1, PER2, NR1D1), RT-qPCR is used following RNA extraction [6].

Urine Sampling

Urine analysis is particularly valuable for measuring hormone metabolites and providing an integrated view of hormone production over time.

• Detailed Protocol for Urinary Metabolite Analysis [63]:
  • Collection: Collect urine as spot samples (e.g., first morning void, daytime, nighttime) or over timed intervals (e.g., 24-hour collection). Record the exact time and volume of each collection.
  • Sampling Frequency: Spot samples at key circadian phases (e.g., morning, afternoon, night) can reveal diurnal variation. For a comprehensive profile of ultradian and diurnal patterns, fractionated urine collection every 2-4 hours over a 24-48 hour period is recommended.
  • Sample Handling: Freeze urine samples at -20°C or lower immediately after collection to prevent degradation.
  • Analysis: A state-of-the-art method involves Low-Density Solvent-Dispersive Liquid-Liquid Microextraction coupled with Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (LDS-DLLME-UPLC-MS/MS). This green chemistry approach allows for the simultaneous quantification of cortisol, melatonin, and their major metabolites (e.g., 6-sulfatoxymelatonin, 6-hydroxycortisol) with high sensitivity and specificity [63].

Blood Sampling

Blood provides a comprehensive matrix, reflecting both free and protein-bound hormones, and allows for analysis of cellular transcriptomes.

• Detailed Protocol for Blood-Based Circadian Biomarkers [64]:
  • Collection: Blood can be drawn via venipuncture at discrete time points or through an indwelling catheter for frequent sampling. For gene expression studies, collect blood into PAXgene RNA tubes for immediate stabilization.
  • Sampling Frequency: The frequency is highly dependent on the analyte. For transcriptomic biomarker development, sampling every 4 hours over a 24-hour cycle is common. To resolve ultradian cortisol pulses, sampling every 15-30 minutes for 4-8 hours may be necessary [64] [8].
  • Sample Handling: Process plasma or serum within 2 hours of collection. For RNA studies, follow the manufacturer's protocol for PAXgene tubes.
  • Analysis: Hormones are typically measured with immunoassays or LC-MS/MS. For transcriptomic analysis, microarrays or RNA-Seq are used on whole blood or isolated peripheral blood mononuclear cells (PBMCs). Machine learning algorithms (e.g., ZeitZeiger, Elastic Net) can then be applied to transcriptomic data to build multivariate predictors of circadian phase [64].

Table 1: Comparative Analysis of Biofluids for Circadian Hormone Sampling

Parameter	Saliva	Urine	Blood
Measured Fraction	Free (bioactive)	Integrated metabolites & free hormone	Total (free + protein-bound)
Key Hormones	Cortisol, Melatonin [62] [8]	Cortisol, Melatonin, 6-Sulfatoxymelatonin [63]	Cortisol, Melatonin, Transcriptomic panels [64]
Invasiveness	Low (non-invasive)	Low (non-invasive)	High (invasive)
Sampling Burden	Low (self-collection)	Low to Moderate	High (requires clinical setting)
Ideal Sampling Frequency for Diurnal Rhythm	3-4 times/day for 2 days [6]	Timed voids (e.g., 2-4 hour intervals) [63]	Every 4 hours for 24h (transcriptomics) [64]
Ideal Sampling Frequency for Ultradian Rhythm	Every 30-60 minutes	Every 1-2 hours (limited by bladder filling)	Every 15-30 minutes [8]
Primary Analytical Methods	ELISA, RIA, RT-qPCR	LC-MS/MS, UPLC-MS/MS [63]	ELISA, LC-MS/MS, Microarrays, RNA-Seq [64]

Experimental Workflow for Sampling Protocol Design

The following diagram outlines the logical decision-making process for designing an optimized sampling protocol based on research objectives and practical constraints.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Research Reagent Solutions for Circadian Sampling

Item	Function/Application	Example/Notes
Salivette Collection Device	Non-invasive collection of whole saliva.	Synthetic swab in a centrifuge tube; allows for clean saliva recovery post-centrifugation [62].
RNAprotect Saliva Reagent	Stabilizes RNA in saliva samples at point of collection.	Prevents degradation of RNA for subsequent gene expression analysis of circadian genes [6].
PAXgene Blood RNA Tubes	Stabilizes intracellular RNA in whole blood at point of draw.	Essential for transcriptomic studies to generate accurate gene expression profiles from blood [64].
C18 Solid-Phase Extraction (SPE) Cartridges	Purification and concentration of analytes from complex biological matrices.	Used in sample preparation for LC-MS/MS analysis of hormones in urine and saliva [63].
LC-MS/MS System	High-sensitivity, high-specificity quantification of hormones and their metabolites.	Gold-standard for multiplexed hormone analysis; used for simultaneous measurement of cortisol, melatonin, and metabolites in urine [63].
ELISA Kits	Immunoassay-based quantification of specific hormones.	Widely used for measuring cortisol and melatonin in saliva and blood serum/plasma.
PowerCHORD Software	Computational tool for optimizing sampling time points for rhythm detection.	Used to design non-equispaced sampling schedules that maximize statistical power when period is uncertain [61].

The optimization of sampling frequency is a critical determinant of success in circadian research. While theoretical principles provide a foundation, the practical execution must be tailored to the specific biofluid, analyte, and research question. Saliva offers an optimal balance of non-invasiveness and biological relevance for free hormone assessment, blood remains indispensable for transcriptomic biomarker discovery, and urine is powerful for metabolic profiling. By employing the detailed protocols, comparative data, and specialized tools outlined in this document, researchers can design rigorous and efficient studies that accurately capture the complex temporal dynamics of diurnal and ultradian rhythms, thereby advancing the translation of circadian biology into clinical and therapeutic applications.

Standardization Strategies for Multi-Center and Longitudinal Studies

In the context of research comparing blood, saliva, and urine for hormone circadian sampling, standardization is not merely a methodological preference but a fundamental requirement for scientific validity. Multi-center longitudinal studies investigating circadian rhythms face unique challenges in maintaining data consistency across different locations and over time. The lack of standardized data collection procedures poses significant threats to the internal validity of such research, potentially compromising the reliability of findings related to hormonal oscillations in different biological matrices [65]. This article establishes comprehensive standardization protocols specifically designed for circadian hormone research across multiple centers, addressing critical phases from sample collection to data analysis to ensure the cross-site comparability and longitudinal consistency required for robust circadian science.

Methodological Standardization Strategies

Sample Collection and Handling Protocols

Standardization must begin at the pre-analytical phase, as variations in sample collection can significantly alter analytical results. For circadian rhythm assessment, consistency in timing, matrix handling, and participant instructions is paramount.

• Temporal Standardization: Circadian studies require strict synchronization of sampling times across centers. For cortisol assessment, collection should target the cortisol awakening response (CAR), requiring samples immediately upon waking and at 30-45 minute intervals thereafter [12]. For melatonin, the dim light melatonin onset (DLMO) assessment typically requires a 4-6 hour sampling window before habitual bedtime [12].
• Matrix-Specific Handling: For saliva collection, which offers a non-invasive advantage for dense temporal sampling, protocols should specify exact saliva volumes (e.g., 1.5 mL) and preservative ratios (e.g., 1:1 with RNAprotect for transcriptomic analyses) [6]. Blood collection protocols should standardize anticoagulants, centrifugation conditions, and plasma/serum separation parameters. Urine collection should specify complete voiding vs. spot samples and standardization approaches for concentration variations.
• Environmental Controls: Melatonin sampling requires strict dim light conditions (<50 lux) as light exposure suppresses secretion [12]. Protocols should standardize body posture, as recumbency affects hormone levels, and document potential confounders like ambient light, sleep deprivation, and medication use [12].

Analytical Standardization Across Centers

Consistent analytical methodologies are crucial for reliable multi-center hormone data.

Table 1: Analytical Method Comparison for Circadian Hormone Assessment

Method	Sensitivity	Specificity	Feasibility	Best Application
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)	High (detects pg/mL range)	Excellent (minimal cross-reactivity)	Moderate (specialized equipment required)	Gold standard for melatonin/cortisol in all matrices [12]
Immunoassays (ELISA, RIA)	Moderate to High	Moderate (cross-reactivity concerns)	High (widely available)	High-throughput screening when cross-reactivity is characterized [12]
RNA Expression Analysis	High (for transcriptomic rhythms)	Target-specific	Moderate (requires RNA stabilization)	Core clock gene expression in saliva [6]

Laboratories should implement identical analytical platforms where possible, or establish cross-validation procedures using shared reference materials. For hormone assays, LC-MS/MS offers superior specificity over immunoassays, particularly for low-concentration analytes like salivary melatonin [12]. For gene expression studies of peripheral clocks, quantitative PCR protocols should be standardized across sites using reference genes validated for circadian research.

Data Processing and Statistical Standardization

Longitudinal circadian data presents unique statistical challenges that require standardized approaches.

• Circadian Parameter Calculation: Standardize algorithms for determining key circadian parameters: DLMO (using fixed or variable thresholds), acrophase (peak time), mesor (24h mean), and amplitude (peak-trough difference) [12]. For cortisol, standardize CAR calculation methods.
• Avoid Statistical Pitfalls: In longitudinal analyses, avoid full z-score standardization across time points as this obfuscates true mean-level changes over time [66]. Instead, use alternative transformations like Proportion of Maximum Scaling (POMS) or Percent of Maximum Possible (POMP) which maintain the covariance metric essential for longitudinal models [66].
• Covariate Harmonization: Standardize the collection and coding of critical covariates known to affect circadian rhythms: age, sex, chronotype (using standardized questionnaires like MEQ), sleep quality, medication use, and light exposure history [6] [10].

Experimental Protocols for Circadian Hormone Sampling

Multi-Center Protocol for Salivary Circadian Assessment

Saliva provides a non-invasive matrix ideal for high-density circadian sampling in ambulatory settings. The following protocol standardizes salivary collection for simultaneous hormonal and molecular circadian profiling:

Table 2: Standardized Saliva Collection Protocol for Multi-Center Circadian Studies

Protocol Component	Standardization Specification	Rationale
Sample Collection	Passive drool into polypropylene tubes; target volume 1.5 mL; 1:1 ratio with RNAprotect for transcriptomics	Standardizes RNA quality and analyte preservation [6]
Temporal Schedule	3-4 timepoints/day over ≥2 consecutive days; exact clock times synchronized across sites	Captures circadian waveform with minimal participant burden [6]
Pre-collection Conditions	No food, caffeine, or tooth brushing 60 minutes before collection; document medication timing	Minimizes confounding effects on hormone levels and gene expression
Immediate Processing	Centrifuge at 4°C within 30 minutes of collection; aliquot into cryovials; freeze at -80°C	Preserves analyte integrity for both hormonal and molecular analyses
Documentation	Standardized forms for exact collection time, light exposure, sleep timing, and participant compliance	Essential for interpreting potential phase shifts or rhythm disruptions

This protocol enables simultaneous assessment of hormonal rhythms (cortisol/melatonin) and peripheral clock gene expression (e.g., ARNTL1, PER2) from the same biological matrix, providing complementary circadian information [6].

Cross-Matrix Validation Protocol

For method comparison studies, implement a standardized protocol for simultaneous collection of different matrices:

Procedure: Recruit participants representing expected variability in chronotypes. Collect matched blood, saliva, and urine samples at 3-4 hour intervals across a 24-hour period under controlled conditions. For blood, standardize serum vs. plasma collection tubes across sites. For saliva, use the protocol above. For urine, collect total output per interval with volume recording and aliquot for analysis.

Analysis: Process all matrices following standardized protocols in Table 2. Assay all samples for target hormones (melatonin, cortisol) using the preferred LC-MS/MS method [12]. Calculate phase relationships between matrices and within-individual phase stability across matrices.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Research Toolkit for Multi-Center Circadian Hormone Studies

Item	Function	Specification Considerations
Saliva Collection System	Non-invasive sample collection for hormonal and molecular analyses	Polypropylene tubes; RNA stabilizers for transcriptomics; volume measurement [6]
LC-MS/MS System	Gold-standard quantification of low-abundance hormones	Standardized across sites or cross-validated with reference materials [12]
Portible Actigraphs	Objective sleep-wake cycle and activity rhythm monitoring	Standardized models across sites; synchronized timekeeping
Chronotype Questionnaires	Subject classification for sampling protocol adaptation	Morningness-Eveningness Questionnaire (MEQ) or Munich Chronotype Questionnaire [10]
Dim Light Compliance Verification	Ensures valid melatonin assessment	Lux meters with standardized thresholds (<50 lux) [12]
Standardized Reference Materials	Cross-site assay calibration	Pooled saliva/plasma/urine samples with characterized analyte concentrations
Temperature-Controlled Centrifuges	Sample processing consistency	Standardized g-force, duration, and temperature across sites
Cryogenic Storage Systems	Sample preservation for longitudinal analysis	Standardized -80°C freezers with temperature monitoring and backup

Workflow Visualization

Figure 1: Multi-center circadian study standardization workflow.

Figure 2: Multi-matrix approach to comprehensive circadian profiling.

Validating Methods and Comparative Analysis of Sampling Matrices

Circadian rhythms are endogenous, roughly 24-hour cycles that govern a wide spectrum of physiological processes, from sleep-wake cycles to hormone secretion and metabolism [11] [7]. The hormones melatonin and cortisol are established as crucial biochemical markers for assessing the phase and integrity of this internal timing system [11] [8]. Melatonin, produced by the pineal gland, signals the onset of the biological night, while cortisol, a glucocorticoid from the adrenal cortex, peaks in the morning and supports alertness and energy mobilization [8] [7].

A critical challenge in circadian research and clinical diagnostics is the accurate and reliable measurement of these hormones across different biological matrices. The choice of matrix—whether blood, saliva, or urine—directly influences the methodological approach, the interpretation of results, and the clinical insights that can be gleaned. This application note provides a detailed comparison of these matrices and protocols for robust hormone analysis, supporting research within a broader thesis on circadian sampling methodologies.

Matrix Comparison: Blood, Saliva, and Urine

The selection of a biological matrix is a fundamental decision that depends on the research question, required analytical precision, and practical considerations of participant burden. The table below summarizes the key characteristics of blood, saliva, and urine for measuring melatonin and cortisol.

Table 1: Comparison of Biological Matrices for Circadian Hormone Assessment

Matrix	Hormone Fraction Measured	Key Circadian Metrics	Key Advantages	Key Limitations
Blood (Serum/Plasma)	Total hormone (free + protein-bound) [8]	Full 24-hour profile; DLMO (serum threshold: ~10 pg/mL) [7]	High analyte concentration; good reliability; gold standard for reference methods [7]	Invasive collection; not suitable for frequent, ambulatory sampling; reflects total, not just bioavailable, hormone [8] [7]
Saliva	Free (bioactive) hormone [8]	DLMO (saliva threshold: 3-4 pg/mL) [7]; Cortisol Awakening Response (CAR) [11]	Non-invasive; ideal for frequent, home-based collection; measures biologically active fraction [6] [7]	Low hormone concentration demands high analytical sensitivity; potential for contamination; flow rate and composition can vary [6] [7]
Urine	Hormone metabolites (e.g., 6-sulfatoxymelatonin, free cortisol) [8]	24-hour hormone output or timed collections (e.g., overnight) [8]	Non-invasive; integrates hormone secretion over a period (e.g., 24 hours) [8]	Does not provide precise phase timing (e.g., DLMO); results influenced by renal function; cumbersome collection protocol [8]
Hair	Cortisol incorporated over time [8]	N/A for circadian phase; identifies chronic, prolonged elevations in cortisol levels [8]	Provides a long-term retrospective measure (weeks to months) of HPA axis activity [8]	Cannot capture diurnal or ultradian rhythms [8]

Analytical Methodologies

Accurate quantification is paramount, as hormone concentrations in saliva, particularly for melatonin, can be very low [7].

Immunoassays vs. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Two primary analytical platforms are used for hormone detection:

• Immunoassays (e.g., ELISA, Chemiluminescence Immunoassays): Traditionally used due to their wide availability and high throughput. However, they can suffer from cross-reactivity with structurally similar molecules, leading to potentially reduced specificity and overestimation of hormone levels [11] [38].
• Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): This technology has emerged as the superior alternative, offering enhanced specificity, sensitivity, and reproducibility [11] [7]. It allows for the simultaneous analysis of multiple steroids in a single run and is less susceptible to cross-reactivity issues [38]. A recent 2026 study validated an LC-MS/MS method for 19 steroids, demonstrating strong linearity and excellent precision, outperforming immunoassays, especially at lower concentrations [38].

Chemical derivatization is a powerful technique often employed in LC-MS/MS analysis to improve the ionization efficiency and stability of small molecules like melatonin and cortisol, thereby enhancing detection sensitivity and throughput in clinical laboratories [67].

Determining Circadian Phase

The analytical method must be matched with an appropriate sampling protocol to define key circadian parameters:

• Dim Light Melatonin Onset (DLMO): The gold standard for assessing circadian phase. It is typically determined from serial saliva or plasma samples collected in the evening (e.g., 4-6 hour window before habitual bedtime) under dim light conditions [11] [7]. DLMO can be calculated using a fixed threshold (e.g., 3-4 pg/mL in saliva) or a variable threshold based on baseline values [7].
• Cortisol Awakening Response (CAR): A sharp rise in cortisol levels peaking 30-45 minutes after waking. It requires saliva or blood samples collected immediately upon waking and at subsequent intervals (e.g., 15, 30, 45 minutes) [11] [7]. While more variable than DLMO, CAR provides a valuable index of HPA axis health and is influenced by circadian timing [7].

Detailed Experimental Protocols

Protocol A: Salivary Dim Light Melatonin Onset (DLMO) Assessment

This protocol is designed for the determination of DLMO using saliva samples, suitable for at-home collection by participants.

1. Materials and Reagents

• Saliva Collection Kit: Salivettes or similar collection devices.
• Portable Freezer: -20°C freezer for sample storage post-collection.
• Light Meter: To verify dim light conditions (<10-30 lux).
• LC-MS/MS System: For hormone analysis [7].
• Solid-Phase Extraction (SPE) Plates: For sample preparation and analyte purification [38].
• Chemical Derivatization Reagents: If required for sensitivity enhancement in LC-MS/MS (e.g., for melatonin) [67].

2. Pre-Collection Participant Guidelines (Critical)

• Light Exposure: Participants must remain in dim light (<10-30 lux) from the start of sampling until its completion. Avoid screen use (phones, laptops) as blue light suppresses melatonin [68] [9].
• Posture & Diet: Maintain a seated or semi-recumbent position. Avoid vigorous physical activity. Refrain from eating, drinking (except water), brushing teeth, or smoking during the 60 minutes before each sample to prevent contamination [9].
• Substances to Avoid: Abstain from alcohol, caffeine, and non-steroidal anti-inflammatory drugs (NSAIDs) for 24 hours prior, as they can suppress or alter melatonin secretion [7].

3. Sampling Procedure

• Timing: Begin sampling 5 hours before and continue until 1 hour after habitual bedtime [7]. A typical schedule: 18:00, 19:00, 20:00, 21:00, 22:00, 23:00, 00:00.
• Collection at Each Timepoint: Provide at least 1 mL of saliva per sample. For RNA co-analysis, 1.5 mL of saliva mixed 1:1 with RNAprotect is recommended [6].
• Handling: Clearly label each sample with time and date. Freeze immediately at –20°C or lower after collection. Transport frozen to the analytical laboratory.

4. Data Analysis

• Analyze samples using a validated LC-MS/MS method [38] [7].
• Plot melatonin concentration against time.
• Calculate DLMO using an established method, such as the fixed threshold of 3-4 pg/mL or the "hockey-stick" algorithm [7].

Protocol B: Salivary Cortisol Awakening Response (CAR)

This protocol outlines the procedure for assessing the CAR, a key marker of HPA axis dynamics.

1. Materials and Reagents

• Same as Protocol A.

2. Pre-Collection Participant Guidelines

• Participants should set an alarm for their usual wake time. Upon waking, they must note the exact wake-up time and collect the first sample immediately while remaining in bed.

3. Sampling Procedure

• Timing: Collect samples at awakening (S1), and then at 15 (S2), 30 (S3), and 45 (S4) minutes post-awakening.
• Collection at Each Timepoint: Record the exact clock time for each sample. Provide at least 0.5 mL of saliva per sample.
• Handling: Freeze samples immediately after each collection.

4. Data Analysis

• Analyze cortisol levels for all four samples.
• The CAR can be expressed as the area under the curve (AUC) with respect to ground (AUCg) or as the peak cortisol level observed in the 45-minute window.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Research Reagent Solutions for Circadian Hormone Analysis

Item	Function/Application	Key Considerations
LC-MS/MS System	Gold-standard quantification of melatonin and cortisol with high specificity and sensitivity [38] [7].	Allows for multiplexing (measuring multiple steroids simultaneously). Requires significant capital investment and technical expertise.
Chemical Derivatization Reagents	Enhances ionization efficiency and detection sensitivity of hormones (like melatonin) in LC-MS/MS [67].	Critical for achieving low limits of detection in challenging matrices like saliva.
Solid-Phase Extraction (SPE) Plates	Purifies and concentrates analytes from complex biological matrices (saliva, serum, urine) prior to LC-MS/MS analysis [38].	Improves assay sensitivity and protects the LC-MS/MS instrumentation from matrix effects.
Validated Saliva Collection Kits (e.g., Salivettes)	Standardized, non-invasive collection of saliva for hormone analysis.	Minimizes interference; some kits contain cotton which can interfere with some assays—polyethylene-based kits are often preferred.
RNAprotect Saliva Reagent	Preserves RNA in saliva for concurrent transcriptomic analysis of circadian gene expression (e.g., ARNTL1, PER2) [6].	Enables multi-omics approaches (e.g., integrating hormone and gene expression data from the same sample).
Certified Reference Standards	Calibrators and internal standards (preferably stable isotope-labeled) for LC-MS/MS quantification [38].	Essential for achieving accurate and reproducible results; corrects for sample preparation losses and matrix effects.

Visualization of an Integrated Circadian Workflow

The following diagram illustrates a comprehensive workflow that integrates sampling from different matrices with multi-omics analysis to provide a holistic view of circadian system function.

In the field of circadian biology research, the accurate assessment of hormonal rhythms is paramount for both scientific discovery and clinical application. The choice of biological matrix—blood, saliva, or urine—fundamentally influences the analytical validity of circadian phase measurements, directly impacting sensitivity, specificity, and reproducibility. Hormones such as melatonin and cortisol serve as crucial peripheral markers of the central circadian pacemaker located in the suprachiasmatic nucleus (SCN) [7] [69]. Each matrix offers distinct advantages and limitations for capturing these endocrine oscillations, requiring researchers to make informed decisions based on their specific experimental questions and constraints. This application note provides a comprehensive comparison of these matrices, detailing standardized protocols for their implementation in circadian rhythm studies, with particular relevance for researchers and drug development professionals working in chronobiology and related fields.

The mammalian circadian system is governed by a master pacemaker in the SCN, which synchronizes peripheral clocks throughout the body via neural, hormonal, and behavioral pathways [7] [69]. Two hormones, melatonin and cortisol, have emerged as the most reliable peripheral biomarkers for assessing the phase and amplitude of the central circadian clock in humans.

• Melatonin: Produced by the pineal gland, melatonin secretion is tightly regulated by the light-dark cycle. Its levels rise in the evening, peak during the night, and decline in the early morning, serving as a primary signal for the biological night [7] [69]. The Dim Light Melatonin Onset (DLMO), the time at which melatonin concentrations begin to rise under dim light conditions, is considered the gold standard marker for circadian phase [70] [7].
• Cortisol: This glucocorticoid, produced by the adrenal cortex, exhibits a diurnal rhythm roughly opposite to that of melatonin. It peaks shortly after awakening—a phenomenon known as the Cortisol Awakening Response (CAR)—and declines throughout the day to reach a nadir around midnight [7] [8]. While its rhythm can be more susceptible to stress and other non-photic stimuli compared to melatonin, its stability and reproducibility over time make it a valuable circadian marker [8].

The following diagram illustrates the complementary diurnal rhythms of these two key hormones and the standard sampling points for DLMO and CAR.

Comparative Analytical Validity by Matrix

The selection of an appropriate biological matrix is a critical methodological decision that directly influences the sensitivity, specificity, and reproducibility of circadian hormone measurements. The table below provides a quantitative and qualitative comparison of blood (serum/plasma), saliva, and urine for the analysis of melatonin and cortisol.

Table 1: Comparative Analytical Validity of Blood, Saliva, and Urine for Circadian Hormone Assessment

Parameter	Blood (Serum/Plasma)	Saliva	Urine
Invasiveness & Participant Burden	High (venipuncture) [7]	Low (non-invasive, self-collection possible) [6] [7]	Low (non-invasive) [8]
Suitability for Frequent/Dense Sampling	Low (clinically intensive)	High (ideal for ambulatory, at-home studies) [6] [7]	Medium (typically cumulative, less frequent)
Approximate Melatonin Concentration	~10-60 pg/mL (peak) [7]	~3-4 pg/mL (DLMO threshold) [7]	6-Sulfatoxymelatonin (aMT6s) metabolite
Approximate Cortisol Concentration	High (total cortisol) [8]	Low (free, biologically active cortisol) [8]	Free cortisol (24h collection) [8]
Sensitivity Considerations	High absolute concentration; gold standard reference [7]	Lower absolute concentration requires highly sensitive assays (e.g., LC-MS/MS) [7] [8]	Measures metabolite; reflects hormone production over time
Specificity & Key Analytical Challenges	Measures total hormone (free + protein-bound); high specificity with LC-MS/MS [7]	Measures free hormone; immunoassays susceptible to cross-reactivity; LC-MS/MS recommended for superior specificity [7]	Subject to renal function and hydration status; excellent specificity for metabolites via LC-MS/MS
Reproducibility & Key Confounding Factors	High intra-assay precision; confounded by stress of venipuncture [8]	High reproducibility for CAR & DLMO; critically dependent on strict adherence to pre-sampling protocols (e.g., no eating/drinking) [7] [9]	High reproducibility for 24h output; timing and completeness of collection are major factors [8]
Primary Circadian Applications	DLMO (in lab); pharmacokinetic studies [7]	DLMO (at-home); Cortisol Awakening Response (CAR) [7] [8]	24h hormone production rhythm (e.g., aMT6s for melatonin rhythm) [8]

Key Insights from Comparative Data

• Sensitivity and Specificity: While blood offers the highest analyte concentrations, saliva provides a direct measurement of the biologically active, free fraction of hormones, which is often more physiologically relevant [8]. The adoption of liquid chromatography–tandem mass spectrometry (LC–MS/MS) has become the method of choice for salivary hormone analysis due to its enhanced sensitivity and specificity, overcoming the limitations of traditional immunoassays which can suffer from cross-reactivity [7].
• Reproducibility: Salivary DLMO and CAR have been validated as highly stable and reproducible measures over time in individuals, making them suitable for longitudinal studies [70] [8]. This reproducibility, however, is contingent upon strict protocol adherence, including controlled lighting (dim light for DLMO), posture, and avoidance of contaminants before saliva sampling [9].
• Matrix Choice in Context: For high-frequency phase assessment (e.g., DLMO), saliva is often the optimal matrix due to its non-invasiveness. For measuring total 24-hour hormone output, urine collection is highly effective. Blood remains the reference material for validating assays and establishing absolute hormone concentrations [7] [8].

Detailed Experimental Protocols

Protocol for Salivary Dim Light Melatonin Onset (DLMO) Assessment

Salivary DLMO is the preferred method for determining circadian phase in ambulatory and field-based studies due to its non-invasive nature and strong correlation with the gold-standard plasma DLMO [7].

Workflow Overview:

Step-by-Step Protocol:

• Pre-Study Screening & Preparation

  • Participant Selection: Screen participants for exclusion criteria, including recent shift work, transmeridian travel (>2 time zones in past 2 weeks), substance use (alcohol, caffeine, nicotine during protocol), and use of medications affecting melatonin secretion (e.g., beta-blockers, NSAIDs) [9].
  • Protocol Familiarization: Provide participants with detailed instructions and a sampling kit. Emphasize the critical requirement for dim light conditions (<10-30 lux) from 2-3 hours before the first sample until the end of collection. Use a lux meter for verification [9].

• Sample Collection (At-Home/Clinic)

  • Timing: Begin sampling 5-6 hours before and continue until 1 hour after habitual bedtime. A typical schedule involves collecting samples every 30-60 minutes (e.g., 18:00, 18:30, 19:00... 00:00) [7].
  • Procedure: Use standardized, low-binding saliva collection tubes (e.g., Salivettes). Participants should not eat, drink (except water), brush their teeth, or smoke for at least 30 minutes before each sample. They should rinse their mouth with water 10 minutes before collection [9].
  • Documentation: Record the exact clock time of each sample.

• Sample Processing & Storage

  • Centrifuge samples upon return to the lab (if using Salivettes) to separate saliva from the cotton roll.
  • Aliquot supernatant into cryovials and immediately freeze at -20°C or -80°C until analysis to prevent degradation.

• Hormone Analysis via LC-MS/MS

  • This protocol step utilizes LC-MS/MS for its superior sensitivity and specificity for low-concentration salivary melatonin [7].
  • Extraction: Thaw samples on ice. Perform a liquid-liquid or solid-phase extraction to purify and concentrate melatonin from the saliva matrix.
  • LC-MS/MS Analysis:
    • Chromatography: Use a reverse-phase C18 column. Mobile phase: water and methanol, both with 0.1% formic acid, with a gradient elution.
    • Mass Spectrometry: Operate in positive electrospray ionization (ESI+) mode. Monitor multiple reaction monitoring (MRM) transitions for melatonin (e.g., 233.2 → 174.2 for quantification; 233.2 → 159.0 for qualification) and a stable isotope-labeled internal standard (e.g., d4-melatonin, 237.2 → 178.2).

• Data Analysis & DLMO Calculation

  • Generate a standard curve from known melatonin concentrations to interpolate sample concentrations.
  • Plot melatonin concentration against sample clock time.
  • Calculate DLMO using a fixed threshold (commonly 3 pg/mL or 4 pg/mL in saliva) or a variable threshold (2 standard deviations above the mean of three baseline values) [7]. The fixed threshold of 3 pg/mL is widely used and provides a robust estimate.

Protocol for Salivary Cortisol Awakening Response (CAR)

The CAR is a distinct and robust marker of HPA axis activity, influenced by the circadian clock [8].

Step-by-Step Protocol:

• Participant Preparation: Provide participants with labeled salivettes and a detailed timeline. Instruct them to avoid eating, drinking, brushing teeth, or smoking before completing the series. The first sample must be taken immediately upon waking.

• Sample Collection:

  • Collect samples at awakening (0 min), 30 min, and 45 min post-awakening. Record the exact time of each sample.
  • Maintain this protocol for multiple consecutive days (e.g., 2-3 days) to establish a reliable baseline and account for day-to-day variability.

• Sample Processing & Analysis:

  • Process and store samples as described in the DLMO protocol (Step 3).
  • Analyze cortisol concentrations using a highly sensitive and specific salivary cortisol immunoassay or LC-MS/MS.

• Data Analysis:

  • The CAR is typically calculated as the area under the curve (AUC) with respect to ground or the absolute increase from the waking value to the peak value (usually at 30-45 min).

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 2: Key Research Reagent Solutions for Circadian Hormone Sampling

Item	Function/Application	Example & Notes
Saliva Collection Device	Non-invasive collection of whole saliva for hormone analysis.	Salivette (Sarstedt). Choose cotton or polyester synthetic roll based on analyte of interest to minimize interference.
Sample Preservation Tube	Stabilizes RNA in saliva for concurrent gene expression analysis of circadian clock genes.	RNAprotect (Qiagen), used at a 1:1 ratio with saliva to preserve transcript integrity [6].
LC-MS/MS System	Gold-standard analytical platform for sensitive and specific quantification of low-abundance hormones in saliva.	Systems from Agilent, Sciex, or Waters. Superior to immunoassays for salivary melatonin due to lack of cross-reactivity [7].
Enzyme-Linked Immunosorbent Assay (ELISA) Kit	Immunoassay-based quantification of cortisol or melatonin.	Commercially available high-sensitivity Salivary Melatonin or Cortisol ELISA Kits. A more accessible alternative to LC-MS/MS but requires validation for circadian work [11].
Portable Lux Meter	Verifies adherence to dim light conditions during DLMO sampling protocols.	Essential for at-home studies to ensure ambient light is <10-30 lux, preventing melatonin suppression [9].
TimeTeller Kit	A specialized methodology for assessing circadian phase from saliva via core clock gene expression (e.g., ARNTL1, PER2) [6].	Provides an alternative molecular approach to hormonal phase assessment.
Stable Isotope-Labeled Internal Standards	Ensures quantification accuracy in mass spectrometry by correcting for matrix effects and recovery losses.	e.g., d4-melatonin, d4-cortisol. A critical component for reliable LC-MS/MS analysis [7].

The analytical validity of circadian hormone measurement is intrinsically linked to the choice of biological matrix. Blood remains the reference standard for definitive concentration measurements, while saliva offers an optimal balance of non-invasiveness, analytical reliability, and ecological validity for high-density sampling, making it the matrix of choice for determining phase markers like DLMO and CAR in real-world settings. Urine provides a robust measure of integrated 24-hour hormone production. The advancement of highly sensitive detection methods like LC-MS/MS has been pivotal in unlocking the full potential of saliva as a matrix for circadian endocrinology. By adhering to the detailed protocols and leveraging the appropriate tools outlined in this document, researchers can ensure the generation of valid, reproducible, and physiologically meaningful data in the study of circadian rhythms.

Within circadian biology research and the development of chronotherapeutic drugs, a critical step is the accurate assessment of an individual's circadian phase. This diagnostic precision is foundational for identifying circadian rhythm sleep-wake disorders (CRSWDs) and for timing drug administration to align with an individual's physiology to maximize efficacy and minimize adverse effects [71]. The gold standards for circadian phase assessment are the Dim Light Melatonin Onset (DLMO) and the Cortisol Awakening Response (CAR) [11] [71]. However, the measurement of these hormonal biomarkers can be performed using different biological matrices—blood, saliva, and urine—each with distinct methodological considerations, performance characteristics, and clinical validity. This application note synthesizes current evidence to provide a structured comparison of these matrices, detailing their diagnostic performance for specific disorders and offering standardized protocols for their use in research and drug development.

Comparative Analysis of Biological Matrices

The choice of biological matrix influences the cost, participant burden, analytical performance, and ultimately, the diagnostic validity of circadian phase assessment. The following section provides a quantitative and qualitative summary of these key considerations.

Table 1: Diagnostic Performance and Methodological Comparison of Circadian Hormone Matrices

Parameter	Blood (Plasma/Serum)	Saliva	Urine
Primary Analyte	Melatonin, Cortisol	Melatonin, Cortisol	6-Sulfatoxymelatonin (aMT6s)
Invasiveness & Burden	High (phlebotomy required) [6]	Low (non-invasive, home collection) [6]	Low (non-invasive)
Sampling Frequency	High-frequency for DLMO (e.g., hourly) [71]	High-frequency for DLMO (e.g., every 30-60 min) [71]	Spot or pooled samples over 8-24 hours
Key Diagnostic Marker	Plasma DLMO	Salivary DLMO [11]	aMT6s Acrophase or Total Output
Analytical Gold Standard	LC-MS/MS, Immunoassays [11]	LC-MS/MS, Immunoassays [11]	ELISA, LC-MS/MS
Sensitivity & Specificity	High sensitivity and specificity with LC-MS/MS [11]	High with LC-MS/MS; potential immunoassay interference [11]	Good correlation with melatonin production
Major Confounding Factors	Stress from venipuncture, posture [11]	Food debris, oral hygiene, blood contamination [6]	Renal function, hydration status, partial collection
Cost & Feasibility	High cost, requires clinical setting [71]	Lower cost, suitable for field studies [71]	Low cost, suitable for long-term monitoring

Table 2: Clinical Validity and Utility for Specific Disorders

Disorder / Application	Blood-Based Assessment	Saliva-Based Assessment	Urine-Based Assessment
Delayed Sleep-Wake Phase Disorder (DSWPD)	High validity for DLMO; confirms phase delay [71]	High validity and preferred method; high correlation with plasma, less invasive for evening sampling [11] [71]	Moderate validity; useful for estimating rhythm phase over 24h
Advanced Sleep-Wake Phase Disorder (ASWPD)	High validity for early DLMO identification	High validity and preferred method for identifying advanced phase	Moderate validity
Non-24-Hour Sleep-Wake Rhythm Disorder	Effective but burdensome for repeated assessment	Ideal for repeated phase assessments over time to track circadian period [71]	Useful for long-term rhythm tracking in free-living conditions
Shift Work Disorder	Effective but impractical for shift workers	Highly practical for assessment in the workplace/non-clinical settings [71]	Practical for assessing rhythm adaptation via aMT6s
Chronotherapy (Drug Timing)	Precise but high-burden for baseline measurement	Emerging as a practical tool for personalizing drug administration schedules [6]	Limited evidence for direct application

Detailed Experimental Protocols

Protocol for Salivary Dim Light Melatonin Onset (DLMO) Assessment

Salivary DLMO is a widely accepted and validated method for determining circadian phase in clinical and field research due to its non-invasive nature [11] [71].

1. Pre-Sampling Participant Preparation:

• Instruct participants to avoid alcohol, caffeine, and nicotine for at least 12 hours before and during sampling.
• Participants should not brush their teeth, floss, or eat a major meal 1 hour before each saliva sample to prevent blood contamination and interference with immunoassays [6].
• Adhere to a dim-light environment (< 10-30 lux) for at least 1-2 hours prior to and throughout the sampling period to prevent melatonin suppression [11].

2. Sample Collection Workflow:

• Timing: Begin collection 5-6 hours before habitual bedtime and continue until 1-2 hours after, typically sampling every 30-60 minutes [71].
• Procedure: Use sterile saliva collection aids (e.g., Salivettes). The participant passively drools or places the cotton swab in the mouth until saturated, without chewing.
• Storage: Immediately freeze samples at -20°C or lower until analysis.

3. Analytical Measurement:

• Preferred Method: Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) offers high sensitivity and specificity, minimizing cross-reactivity with analogous compounds [11].
• Alternative Method: Enzyme-Linked Immunosorbent Assay (ELISA) is cost-effective but may have lower specificity; validation against LC-MS/MS is recommended.

4. Data Analysis and DLMO Calculation:

• Plot melatonin concentration against clock time.
• The DLMO is typically defined as the time at which melatonin concentration crosses a fixed threshold (e.g., 3 pg/mL or 4 pg/mL) or a percentage (e.g., 2 SDs) above the baseline average [11] [71].

Protocol for Cortisol Awakening Response (CAR) Assessment

CAR is a dynamic marker of hypothalamic-pituitary-adrenal (HPA) axis activity and is sensitive to circadian phase and stress [11].

1. Pre-Sampling Participant Preparation:

• Provide participants with detailed written instructions and pre-programmed awakening alarms.
• Participants should avoid smoking, vigorous exercise, and eating breakfast until after the final sample is collected.

2. Sample Collection Workflow:

• Timing: Collect samples immediately upon awakening (S1), and then at 30 minutes (S2), 45 minutes (S3), and 60 minutes (S4) post-awakening.
• Procedure: Use salivettes for home collection. Participants must record exact awakening and sampling times for data validity.
• Storage: Freeze samples at -20°C immediately after collection.

3. Analytical Measurement:

• Analyze samples using commercially available and validated salivary cortisol ELISA or LC-MS/MS kits.

4. Data Analysis:

• Calculate the area under the curve with respect to ground (AUCg) to represent total cortisol secretion.
• Calculate the area under the curve with respect to increase (AUCi) to represent the dynamic change in cortisol.
• A blunted CAR is associated with poor health outcomes and may reflect circadian disruption.

Visualization of Circadian Rhythm Analysis Workflow

The following diagram illustrates the logical flow for selecting a biological matrix and conducting a circadian phase assessment, from research question to clinical interpretation.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Reagent Solutions for Circadian Hormone Assessment

Item	Function/Application	Examples & Notes
Salivette Collection Devices	Hygienic collection and recovery of saliva samples.	Sarstedt Salivette (cotton or polyester swabs). Polyester may be preferred for steroid hormone analysis.
LC-MS/MS Kit	High-sensitivity quantification of melatonin and cortisol.	Commercial kits from vendors like Chromsystems, Recipe. Offers superior specificity over immunoassays [11].
Salivary Melatonin/Cortisol ELISA Kit	Cost-effective immunoassay for hormone quantification.	Kits from Salimetrics, IBL International. Potential for cross-reactivity; validate for your specific research context [11].
aMT6s ELISA Kit	Quantifies the primary urinary melatonin metabolite.	Kits from Buhlmann, IBL International. Essential for assessing melatonin production via urine.
RNAprotect Reagent	Preserves RNA in saliva for gene expression studies.	Used in novel methods like TimeTeller to assess circadian phase from core clock gene expression (e.g., ARNTL1, PER2) [6].
Portable Lux Meter	Verifies dim light conditions during DLMO protocols.	Critical for protocol adherence; confirms ambient light levels are < 10-30 lux at the participant's eye level [11].

Comprehensive Comparison of Hormone Sampling Matrices

The selection of an appropriate biological matrix is fundamental to circadian rhythm research, impacting data reliability, participant burden, and clinical applicability. The table below provides a systematic comparison of blood, saliva, and urine for hormone circadian sampling.

Table 1: Comparative analysis of blood, saliva, and urine for circadian hormone sampling

Parameter	Blood (Serum/Plasma)	Saliva	Urine
Biological Matrix	Total hormone concentration (free + protein-bound) [72]	Free, bioavailable hormone fraction [72] [73]	Hormone metabolites and free cortisol [73] [8]
Key Strengths	- Considered gold standard for clinical diagnosis of major disorders [73]- High analyte concentration [12]- Broad clinical acceptance	- Non-invasive, stress-free collection [26] [72]- Ideal for free hormone measurement [72] [73]- Suitable for high-frequency, at-home sampling [12] [6]	- Non-invasive collection [73]- Provides integrated hormone metabolism data [74] [73]- Suitable for 24-hour cortisol rhythm assessment [8]
Major Limitations	- Invasive and stressful [73]- Single time-point snapshot [73]- Poorly reflects tissue uptake from topical therapies [73]	- Primarily limited to steroid hormones [73]- Requires rigorous quality control [73]- Low analyte concentration demands high-sensitivity assays [12]	- Affected by hydration status [73]- May not reflect real-time tissue levels [73]- Challenging for hormones from oral/topical supplements [73]
Ideal Circadian Use Cases	- Diagnosing endocrine disorders (e.g., Cushing's syndrome) [73]- Validation studies for novel biomarkers	- Dim Light Melatonin Onset (DLMO) assessment [12] [9]- Cortisol Awakening Response (CAR) and diurnal cortisol rhythm [12] [8]- Monitoring hormone replacement therapy [73]	- Long-term assessment of hormone metabolism pathways [74] [73]- 24-hour integrated cortisol output [8]
Key Circadian Analytes	Melatonin, Cortisol, Growth Hormone, Leptin [8]	Melatonin, Cortisol [12] [6] [8]	Cortisol, Melatonin metabolites [73] [8]
Sampling Feasibility	Low; requires clinical visits and trained phlebotomists	High; suitable for ambulatory and home-based collection over 24h [12] [6]	High; convenient for home collection, especially 24h pools [73]

Detailed Experimental Protocols for Circadian Assessment

Protocol for Salivary Dim Light Melatonin Onset (DLMO)

Dim Light Melatonin Onset is the gold standard marker for assessing the phase of the endogenous circadian clock [12].

Primary Application: Determining the timing of the biological night in conditions like Delayed Sleep-Wake Phase Disorder, jet lag, and shift work studies [12].

Materials and Reagents:

• Saliva collection kits (e.g., Salivettes)
• Low-indoor-light environment (<10-30 lux) [9]
• Freezer (-20°C or lower) for sample storage
• LC-MS/MS kit or highly sensitive immunoassay for melatonin analysis

Step-by-Step Procedure:

• Participant Preparation: Instruct participants to avoid bright light for at least 1 hour prior to and during sampling. They must refrain from brushing teeth, eating, or drinking caffeinated beverages 1 hour before each sample. Consumption of alcohol, nicotine, and certain medications (e.g., NSAIDs, beta-blockers) should be restricted for 24 hours as they can suppress melatonin [12] [9].
• Sampling Schedule: Collect saliva samples every 30-60 minutes over a 4-6 hour window, typically starting 5 hours before and ending 1 hour after habitual bedtime [12].
• Sample Collection: Under dim light conditions, participants passively drool or use the provided collection device. They should note the exact collection time.
• Sample Handling: Centrifuge collection devices if required. Store samples immediately at -20°C or lower until analysis.
• Data Analysis: Determine DLMO using a fixed threshold method (e.g., the time when interpolated melatonin concentration exceeds 3-4 pg/mL in saliva) or a dynamic threshold (2 standard deviations above the mean of baseline samples) [12].

Protocol for Salivary Cortisol Awakening Response (CAR) and Diurnal Rhythm

The Cortisol Awakening Response is a distinct rise in cortisol levels following morning awakening and serves as an index of hypothalamic-pituitary-adrenal (HPA) axis reactivity [12].

Primary Application: Assessing HPA axis dynamics, circadian regulation, and the impact of psychological and metabolic stress [12] [8].

Materials and Reagents:

• Saliva collection kits
• Participant diary to log exact wake time and sample collection times
• Freezer for sample storage
• Sensitive LC-MS/MS or immunoassay for cortisol detection

Step-by-Step Procedure:

• Participant Preparation: Instruct participants on the critical importance of sampling immediately upon waking. They should avoid eating, drinking, smoking, or brushing teeth before completing the sampling series.
• Sampling Schedule: Collect samples at immediate awakening (S1), and then at 30 (S2), 45 (S3), and 60 (S4) minutes post-awakening. To assess the full diurnal profile, additional samples can be collected at 12:00 PM, 4:00 PM, and 8:00 PM [8].
• Sample Collection and Handling: Participants record the exact time of each sample. Samples are stored frozen after collection.
• Data Analysis: Calculate the area under the curve (AUC) with respect to ground (AUCg) and increase (AUCi) for the CAR. For the diurnal rhythm, calculate the slope from the peak to the nadir [8].

Diagram 1: Circadian hormone study workflow

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful execution of circadian hormone studies relies on a suite of reliable reagents and materials. The following table details key solutions for different stages of the research pipeline.

Table 2: Essential research reagents and materials for circadian hormone studies

Category	Product/Kit Examples	Function & Application Note
Sample Collection	Salivette (Sarstedt), Passive Drool Kits	Standardized saliva collection; Salivettes require centrifugation, while passive drool kits are often compatible with direct aspiration [6].
Sample Preservation	RNAprotect (Qiagen)	Preserves RNA integrity for transcriptomic studies of circadian genes (e.g., ARNTL1, PER2) from saliva [6].
Gold-Standard Assay	LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry)	Provides superior specificity and sensitivity for low-concentration hormones like salivary melatonin and cortisol, minimizing cross-reactivity issues of immunoassays [12].
High-Throughput Screening	ELISA (Enzyme-Linked Immunosorbent Assay)	A practical choice for high-throughput analysis of cortisol; requires validation against LC-MS/MS for accuracy [8].
Automated Processing	Freedom EVO (Tecan)	Automated liquid handling system that improves reproducibility and throughput for saliva-based immunoassays [72].
Novel Biosensors	Electrochemical Cortisol Sensors, ATR-FTIR Spectroscopy	Emerging technology for point-of-care (POC) and continuous monitoring of circadian biomarkers, though not yet widely available for clinical research [26] [8].

Diagram 2: Hormone pathway from SCN to saliva

In circadian rhythm research, the pursuit of a comprehensive understanding of hormonal fluctuations has led to the integration of multiple biological matrices. While blood serum has traditionally been the gold standard for hormonal assessment, saliva and urine sampling now provide complementary data that enable a more nuanced analysis of the body's 24-hour biological clocks. Saliva measures the biologically active, free fraction of hormones; blood serum captures total hormone levels including protein-bound fractions; and urine provides integrated measures of hormone excretion over time [8] [45]. This multi-matrix approach is particularly valuable for capturing the complex dynamics of circadian rhythms, where both pulsatile secretion and diurnal patterns play critical regulatory roles [8].

The emerging field of circadian medicine recognizes that circadian disruption is linked to numerous pathological states, including sleep disorders, metabolic dysfunction, and immune-related conditions [6] [75]. Characterizing the individual circadian profile enables not only customized health routines but also chronotherapy—treatment regimens optimized to the patient's circadian clock to enhance efficacy and reduce adverse effects [6]. The integration of saliva, blood, and urine sampling provides researchers with a powerful toolkit to advance this promising area of personalized medicine.

Comparative Analysis of Biological Matrices

Technical Specifications and Applications

Table 1: Comparative analysis of biological matrices for circadian hormone assessment

Parameter	Saliva	Blood Serum	Urine
Hormones Measured	Free cortisol, melatonin, estradiol, progesterone [45]	Total cortisol (free + protein-bound), melatonin, FSH, LH [8] [45]	Cortisol metabolites, melatonin metabolites, steroid hormones [8]
Circadian Application	Diurnal rhythm with frequent sampling, especially for cortisol awakening response [8]	24-hour rhythm assessment with frequent sampling; gold standard for phase determination [8]	24-hour integrated measurement; suitable for long-term rhythm assessment [8]
Sample Collection	Non-invasive, self-administered, suitable for home collection [6] [45]	Invasive, requires trained phlebotomist, clinical setting [45]	Non-invasive, self-administered, collected over specific time intervals [8]
Collection Frequency	High frequency (multiple times daily) feasible [6]	Limited by invasiveness; continuous sampling possible in clinical settings [8]	Typically 24-hour collection or first-morning void [8]
Analyte Stability	Requires preservatives (e.g., RNAprotect for RNA) [6]	Stable with proper processing and storage [8]	Requires refrigeration during collection; preservatives often needed [8]
Key Advantages	Measures biologically active hormone fraction; ideal for chronotype assessment [8] [45]	Comprehensive hormone panel available; clinical gold standard [8] [45]	Integrated measure of hormone production; suitable for long-term monitoring [8]
Limitations	Limited biomarker portfolio compared to blood; potential contamination [6]	Inconvenient for frequent sampling; stress of venipuncture may affect hormone levels [45]	Does not capture pulsatile secretion; metabolite measures rather than native hormones [8]

Hormonal Dynamics Across Matrices

Table 2: Circadian characteristics of key hormones across different matrices

Hormone	Circadian Pattern	Detection in Saliva	Detection in Blood	Detection in Urine
Cortisol	Peaks early morning (30-45 min after awakening), declines throughout day, nadir during early sleep [8]	Free cortisol correlates with serum free cortisol; excellent for circadian rhythm assessment [8]	Total cortisol (free + protein-bound); remains elevated in early daytime [8]	24-hour urinary free cortisol; integrated measure of cortisol production [8]
Melatonin	Rises in evening, peaks during night (2-4 AM), decreases in early morning [8]	Nocturnal pattern correlates with serum melatonin; suitable for dim light melatonin onset (DLMO) [8]	Gold standard for DLMO assessment; clear nocturnal pattern [8]	Melatonin metabolites (6-sulfatoxymelatonin) reflect production [8]
Reproductive Hormones	Varies throughout menstrual cycle; perimenopausal fluctuations [45]	Estradiol, progesterone measurable; suitable for cycle mapping [45]	Comprehensive panel including FSH, LH, estradiol, progesterone [45]	Metabolites of steroid hormones; useful for longer-term assessment [45]

Experimental Protocols

Integrated Sampling Protocol for Circadian Assessment

Objective: To comprehensively characterize the circadian rhythm of cortisol and melatonin through simultaneous collection of saliva, blood, and urine samples over a 24-hour period.

Materials:

• Saliva: Salivettes or passive drool collection tubes with RNAprotect (1:1 ratio) [6]
• Blood: Serum separator tubes, venipuncture equipment, ice bath
• Urine: 24-hour collection containers with preservatives, graduated cylinder
• Laboratory equipment: Centrifuge, -80°C freezer, ELISA kits for hormone analysis [45]

Procedure:

• Participant Preparation:
  • Standardize light exposure, meal timing, and physical activity for 48 hours prior to sampling
  • Avoid medications that interfere with hormone measurements for appropriate washout period
  • For women, document menstrual cycle phase or menopausal status [45]

• Sampling Schedule:

  • Collect samples at 3-4 hour intervals throughout 24-hour period
  • Include additional samples during critical periods: cortisol awakening response (0, 30, 45, 60 minutes post-awakening) and melatonin rise (evening hours every 60 minutes) [8]

• Sample Collection:

  • Saliva: Collect 1.5 mL saliva using passive drool method, mix with RNAprotect in 1:1 ratio [6]
  • Blood: Draw 10 mL blood via venipuncture or indwelling catheter; process within 30 minutes
  • Urine: Collect all urine output in 24-hour container; record total volume [8]

• Sample Processing:

  • Saliva: Centrifuge at 1500 × g for 15 minutes, aliquot supernatant, store at -80°C
  • Blood: Allow to clot, centrifuge at 2000 × g for 10 minutes, aliquot serum, store at -80°C
  • Urine: Mix thoroughly, measure total volume, aliquot 5 mL samples, store at -80°C

• Hormonal Analysis:

  • Perform ELISA according to manufacturer protocols for each hormone [45]
  • Analyze cortisol and melatonin in all matrices
  • For gene expression analysis in saliva: Extract RNA, perform RT-qPCR for core clock genes (ARNTL1, PER2, NR1D1) [6]

Figure 1: Integrated multi-matrix sampling workflow for circadian assessment

Data Integration and Rhythm Analysis

Cosinar Analysis:

• Fit cosine curves to hormone data from each matrix using formula: y = mesor + amplitude * cos(2π*(time - acrophase)/24)
• Compare acrophase (peak time), amplitude, and mesor (mean) across matrices [6]

Statistical Integration:

• Calculate cross-correlation functions between hormone profiles from different matrices
• Perform mixed-effects models to account for within-subject correlations across matrices

The Scientist's Toolkit: Essential Research Reagents

Table 3: Essential research reagents for multi-matrix circadian studies

Reagent/Category	Function	Application Notes
RNAprotect Solution	Preserves RNA integrity in saliva samples during storage and transport [6]	Use at 1:1 ratio with saliva; enables gene expression analysis of clock genes [6]
ELISA Kits	Quantifies hormone levels in saliva, serum, and urine [45]	Validate for each matrix; different sample dilutions may be required [45]
Salivette Collection Systems	Standardized saliva collection using cotton swabs or passive drool [6]	Choose material compatible with target analytes; some polymers interfere with steroid assays
Serum Separator Tubes	Facilitates clean serum separation after blood clotting [8]	Process within 30 minutes for optimal results; avoid hemolyzed samples [8]
24-Hour Urine Containers	Integrated collection of all urine output over 24-hour period [8]	Include preservatives appropriate for target hormones; record total volume [8]
Clock Gene Primers	RT-qPCR analysis of core clock gene expression (ARNTL1, PER2, NR1D1) [6]	Validate primer efficiency for saliva RNA; normalize to reference genes [6]

Circadian Signaling Pathways and Biomarkers

Figure 2: Circadian signaling pathways and biomarker distribution across matrices

The core molecular clock machinery consists of transcriptional-translational feedback loops involving core clock genes (CLOCK, BMAL1, PER, CRY) that generate approximately 24-hour oscillations [75]. This central pacemaker in the suprachiasmatic nucleus (SCN) regulates peripheral clocks throughout the body and coordinates the secretion of circadian hormones including cortisol and melatonin [8] [75].

Key Biomarkers by Matrix:

• Saliva: Free cortisol, melatonin, and clock gene expression (ARNTL1, PER2, NR1D1) provide insights into both hormonal rhythms and molecular clock function [6] [8]
• Blood: Total cortisol (free + protein-bound), melatonin, and reproductive hormones offer comprehensive hormonal profiling [8] [45]
• Urine: Cortisol and melatonin metabolites reflect integrated hormone production over time [8]

The correlation between acrophases (peak times) of salivary ARNTL1 gene expression and salivary cortisol demonstrates the potential for saliva to simultaneously capture both molecular and endocrine circadian aspects [6].

The integrative analysis of saliva, blood, and urine provides a powerful multidimensional approach to circadian rhythm assessment. Each matrix offers unique advantages: saliva captures the biologically active hormone fraction and enables non-invasive frequent sampling; blood provides the most comprehensive hormone panel; and urine offers integrated measures of hormone production. This multi-matrix approach enables researchers to capture the complex dynamics of circadian systems, from high-frequency pulsatile secretion to diurnal rhythms and long-term hormonal patterns. As circadian medicine advances, these integrated protocols will facilitate the development of personalized chronotherapeutic interventions optimized to an individual's circadian clock.

The accurate assessment of circadian rhythms is a cornerstone of chronobiology and is increasingly critical for precision medicine. Traditionally, circadian phase determination has relied on hormone measurements—particularly melatonin from blood, saliva, or urine—to track the output of the central pacemaker, the suprachiasmatic nucleus (SCN) [11] [7] [14]. While these hormonal markers remain gold standards, a novel frontier is emerging: the analysis of circadian gene expression directly from saliva. This approach leverages the fact that peripheral clocks, including those in oral tissues, are synchronized with the SCN and exhibit robust rhythmicity in core clock genes [6]. Salivary gene expression profiling offers a non-invasive, tissue-specific window into the molecular clockwork itself, providing a complementary biomarker to systemic hormonal signals. This Application Note details the protocols and analytical frameworks for implementing salivary gene expression analysis, positioning it within the broader context of biosampling for circadian research.

Comparative Analysis of Circadian Biosampling Matrices

The choice of biological matrix profoundly influences the feasibility, cost, and interpretability of circadian research. The table below summarizes key characteristics of blood, saliva, and urine for circadian hormone sampling, alongside the emerging approach of salivary gene expression.

Table 1: Comparison of Biosampling Matrices for Circadian Rhythm Assessment

Parameter	Blood (Plasma/Serum)	Saliva (Hormones)	Urine	Saliva (Gene Expression)
Primary Analytes	Melatonin, Cortisol, Blood Transcriptome	Melatonin, Cortisol	6-sulfatoxymelatonin (aMT6s), Cortisol metabolites	mRNA of core clock genes (e.g., BMAL1, PER1, PER2, NR1D1)
Invasiveness	High (venipuncture)	Low (non-invasive)	Low (non-invasive)	Low (non-invasive)
Feasibility for Frequent/Home Sampling	Low	High	High	High
Key Circadian Metrics	DLMO, CAR, hormonal amplitude	DLMO, CAR	aMT6s Acrophase, Total Output	Acrophase, Amplitude of gene expression rhythms
Key Analytical Platforms	LC-MS/MS, Immunoassays, RNA-Seq	LC-MS/MS, Immunoassays	Immunoassays	qRT-PCR, RNA-Seq
Major Advantages	High analyte concentration; "Gold standard" for melatonin; Access to transcriptome	Non-invasive; ideal for ambulatory and pediatric studies; reflects bioavailable hormone	Integrated hormone output over time; simple collection	Direct access to molecular clock machinery; tissue-specific information
Major Challenges	Stress of collection affects hormones; requires trained phlebotomist	Low hormone concentrations require sensitive assays; contaminated by food/blood	Phase estimates less precise than DLMO; hydration affects concentration	Low RNA yield and quality; requires RNA stabilizers; robust bioinformatics

Experimental Protocols for Salivary Circadian Biomarker Analysis

Protocol A: Salivary Collection for RNA and Hormone Analysis

This integrated protocol allows for the simultaneous analysis of transcriptional and endocrine rhythms from the same non-invasive sample [6].

I. Materials and Reagents

• Saliva Collection Aid: Salivette cortisol tubes (Sarstedt) or similar. For RNA, use sterile polypropylene tubes.
• RNA Stabilization Reagent: RNAprotect Saliva Reagent (Qiagen) or PAXgene Saliva Container (PreAnalytiX).
• Portable Cooler: For temporary sample storage at 4°C during collection.
• Pre-labeled Cryovials: For long-term storage at -80°C.

II. Step-by-Step Procedure

• Participant Preparation: Instruct participants to avoid eating, drinking (except water), brushing teeth, or using mouthwash for at least 30 minutes prior to sampling.
• Sample Collection:
  • For hormone analysis, have the participant passively drool into a Salivette tube (~1-2 mL is sufficient).
  • For RNA analysis, have the participant drool into a tube containing an equal volume of RNA stabilization reagent (e.g., 1.5 mL saliva to 1.5 mL reagent) [6].
• Sample Processing:
  • Centrifuge Salivette tubes for 2-5 minutes at 1000 x g to separate clear saliva from the cotton roll.
  • For RNA-stabilized samples, mix thoroughly by inverting the tube 10 times before aliquoting.
• Storage: Immediately freeze all samples at -20°C or, ideally, -80°C. Avoid multiple freeze-thaw cycles.

III. Sampling Design for Circadian Profiling

• A minimum of 3-4 timepoints per day over 2 consecutive days is recommended to capture circadian waveform [6].
• Key timepoints should cover the expected peak and trough of expression. For example: 07:00 (awakening), 13:00 (afternoon), 19:00 (evening), and 23:00 (before bed).

Protocol B: RNA Extraction and Gene Expression Analysis via qRT-PCR

I. Materials and Reagents

• RNA Extraction Kit: Specifically validated for saliva, e.g., Norgen's Saliva RNA Purification Kit or Qiagen's RNeasy Protect Saliva Mini Kit.
• DNase I Treatment: To remove genomic DNA contamination.
• Nucleic Acid Quantification: Spectrophotometer (NanoDrop) or fluorometer (Qubit) with RNA assays.
• cDNA Synthesis Kit: High-capacity reverse transcription kit (e.g., from Applied Biosystems).
• qPCR Master Mix: SYBR Green or TaqMan-based.
• Primer/Probe Sets: Pre-validated primers for human core clock genes (ARNTL1/BMAL1, PER1, PER2, NR1D1, CLOCK) and housekeeping genes (e.g., GAPDH, B2M).

II. Step-by-Step Procedure

• RNA Extraction: Perform according to the manufacturer's protocol. The typical yield from 1-2 mL of saliva is 1-10 ng/μL.
• RNA Quality Control: Assess RNA concentration and purity (A260/280 ratio ~2.0). Due to low yield, quality can also be checked via the RNA Integrity Number (RIN) if sufficient material is available.
• cDNA Synthesis: Use 100-500 ng of total RNA as input for the reverse transcription reaction.
• Quantitative PCR (qPCR):
  • Set up reactions in duplicate or triplicate using 1-2 μL of cDNA per reaction.
  • Use the following standard cycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.
• Data Analysis: Calculate relative gene expression using the 2^(-ΔΔCt) method, normalizing to housekeeping genes and a reference sample.

Protocol C: Computational Analysis of Circadian Phase

TimeSignature and Similar ML Algorithms [76]: This machine learning method can infer circadian time from a minimal set of gene expression samples.

• Input: Gene expression data (e.g., from qPCR of a pre-defined gene set) from one or two timepoints.
• Process: The pre-trained algorithm compares the expression pattern of the input genes to a reference model of their circadian oscillation.
• Output: A precise estimate of the internal circadian time (e.g., in hours), which is robust to differences in sampling protocols and measurement platforms [76].

Cosine Fitting Analysis: For data from multiple timepoints, a cosine wave (e.g., cosinor analysis) can be fitted to the expression data for each gene to determine its acrophase (time of peak expression), amplitude (peak-to-trough difference), and mesor (rhythm-adjusted mean) [6] [77].

Visualization of Molecular Mechanisms and Workflows

The Core Circadian Clock Feedback Loop

This diagram illustrates the transcriptional-translational feedback loops (TTFLs) of the molecular clock, whose components can be measured in saliva [6] [78].

Figure 1: The core molecular clock is driven by interlocked feedback loops. The CLOCK:BMAL1 complex activates transcription of PER and CRY genes, which then form a complex that represses CLOCK:BMAL1 activity. A stabilizing loop involves CLOCK:BMAL1 activating REV-ERB, which represses BMAL1 transcription [78].

Experimental Workflow for Salivary Circadian Profiling

This flowchart outlines the end-to-end process from participant recruitment to data analysis.

Figure 2: The integrated workflow for salivary circadian profiling, from participant screening through molecular and computational analysis [79] [6] [76].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Research Reagent Solutions for Salivary Circadian Biomarker Studies

Item	Function/Application	Example Products/Brands
Saliva RNA Collection & Stabilization	Preserves RNA integrity immediately upon collection, preventing degradation.	RNAprotect Saliva Reagent (Qiagen), PAXgene Saliva Container (PreAnalytiX)
Saliva RNA Extraction Kit	Isolves high-quality, inhibitor-free total RNA from small saliva volumes.	Norgen Saliva RNA Purification Kit, Qiagen RNeasy Protect Saliva Mini Kit
cDNA Synthesis Kit	Converts extracted RNA into stable cDNA for subsequent PCR amplification.	High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
qPCR Assays	Quantifies expression of specific target genes. Available as SYBR Green or more specific TaqMan assays.	TaqMan Gene Expression Assays (Applied Biosystems), PrimePCR Assays (Bio-Rad)
Pre-designed Circadian Gene Panels	Focused panels of core clock and control genes for streamlined qPCR workflow.	TimeTeller Panel [6], custom PrimePCR Panels (Bio-Rad)
Circadian Computational Tools	Software/Packages for phase, amplitude, and period determination from gene expression data.	TimeSignature [76], cosinor2 (R package), BioDare2 (online platform)
Salivette Tubes	Simple and hygienic collection system for saliva, suitable for hormone analysis.	Salivette Cortisol (Sarstedt)

Conclusion

The choice between blood, saliva, and urine for circadian hormone sampling is not a matter of identifying a single superior method, but of strategically aligning the matrix with the specific research or clinical question. Saliva excels in quantifying free, bioavailable hormones for real-time circadian phase assessment, urine provides an unparalleled window into hormone metabolism and clearance pathways, while blood offers a robust measure of total hormone levels. The integration of multiple matrices can yield a systems-level understanding of circadian endocrinology. Future directions point toward the standardization of protocols, the validation of novel salivary biomarkers like core clock gene expression, and the application of these insights into chronotherapy to enhance drug efficacy and safety in development pipelines. For biomedical researchers, a nuanced mastery of these sampling methodologies is fundamental to advancing both circadian biology and precision medicine.

References

• 1. Mechanism of the circadian clock in physiology - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC4073891/]
• 2. Circadian clock [https://en.wikipedia.org/wiki/Circadian_clock]
• 3. Molecular regulations of circadian rhythm and implications ... [https://www.nature.com/articles/s41392-022-00899-y]
• 4. Circadian rhythm in cardiovascular diseases: a bibliometric ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10290417/]
• 5. The Biology of Central and Peripheral Circadian Clocks [https://www.sciencedirect.com/science/article/pii/S0896627312003327]
• 6. Comprehensive integrative analysis of circadian rhythms in ... [https://www.nature.com/articles/s44323-025-00035-3]
• 7. Circadian Biomarkers in Humans: Methodological Insights ... [https://www.mdpi.com/2218-273X/15/7/1006]
• 8. Cortisol Detection Methods and the Hormone's Role in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12470794/]
• 9. Human circadian rhythm studies: Practical guidelines for ... [https://www.sciencedirect.com/science/article/pii/S2451994422000062]
• 10. Methods for Assessment of Sleep and Circadian Rhythms in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12540570/]
• 11. Methodological Insights into the Detection of Melatonin and ... [https://pubmed.ncbi.nlm.nih.gov/40723877/]
• 12. Circadian Biomarkers in Humans: Methodological Insights into ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12293921/]
• 13. Effect of night-shift work on cortisol circadian rhythm and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9210564/]
• 14. Keeping an eye on circadian time in clinical research and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9790849/]
• 15. Circadian inference of rhythmicity using comparative ... [https://www.sciencedirect.com/science/article/pii/S2590137025000834]
• 16. Human circadian rhythm studies: Practical guidelines for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9382328/]
• 17. Methods for Assessment of Sleep and Circadian Rhythms ... [https://link.springer.com/article/10.1007/s11906-025-01345-4]
• 18. Shift Right: Assessing Circadian Rhythm Sleep Disorders ... [https://salimetrics.com/salivary-melatonin-and-dim-light-melatonin-onset-dlmo/]
• 19. Home-based dim light melatonin onset assessment among ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12413864/]
• 20. Feasibility and acceptability of self-directed, remote dim ... [https://www.frontiersin.org/journals/sleep/articles/10.3389/frsle.2025.1593196/full]
• 21. Cortisol Detection Methods and the Hormone's Role in ... [https://www.mdpi.com/1422-0067/26/18/9141]
• 22. The cortisol awakening response: Fact or fiction? - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC12035071/]
• 23. Cortisol awakening response proactively modulates fronto- ... [https://www.sciencedirect.com/science/article/pii/S105381192500254X]
• 24. Biomarkers in development of psychotropic drugs - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC3182003/]
• 25. Multimodal Noninvasive Assessment of C-Reactive Protein for ... [https://formative.jmir.org/2025/1/e77108]
• 26. From Saliva to Diagnosis: A Scoping Review of Conventional ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12427769/]
• 27. Salivary Hormone Balance Testing Panel [https://vibrant-wellness.com/tests/hormones/salivary-hormone]
• 28. Dutch Plus [https://livingyoungcenter.com/wellness-health/specialty-testing/dutch-plus/]
• 29. Saliva Testing [https://www.zrtlab.com/sample-types/saliva/]
• 30. Five key points to ensure reliable and accurate saliva testing [https://www.news-medical.net/whitepaper/20240506/5-key-points-to-ensure-reliable-and-accurate-saliva-testing-for-female-hormones.aspx]
• 31. Validation of Saliva as the Clinical Specimen Type for a ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11437455/]
• 32. 5 steps to robust saliva testing for female hormones [https://www.tecan.com/blog/saliva-hormone-testing-for-female-hormonal-imbalance-treatment]
• 33. Circadian Rhythm: Biological Functions, Diseases, and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12547082/]
• 34. HUMAP Urine Hormone & Metabolite Test: Complete 2025 ... [https://revolutionhealth.org/blogs/news/humap-urine-hormone-metabolite-test]
• 35. Complexities in salivary and urinary methodology for ... [https://gremjournal.com/journal/01-2025/complexities-in-salivary-and-urinary-methodology-for-ovulation-and-menstrual-cycle-hormone-detection-a-scoping-review/]
• 36. Updates to the DUTCH Report [https://dutchtest.com/articles/updates-to-the-dutch-report]
• 37. Your 2025 Hormone Balance Reset Plan [https://www.healthsourceimc.com/blog/your-2025-hormone-balance-reset-plan/]
• 38. Reliable in-house LC-MS/MS method for steroid hormone ... [https://pubmed.ncbi.nlm.nih.gov/40946765/]
• 39. HuMap™ Hormone & Urinary Metabolites Assessment ... [https://iclabs.ca/test/1885/]
• 40. Using Urine Hormone Testing to Personalize Dietary ... [https://dutchtest.com/articles/urine-hormone-testing-to-personalize-dietary-supplementation]
• 41. Preparation for blood tests: what can go wrong before the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12377977/]
• 42. Sleep Quality & Circadian Rhythm: The Best Biomarkers to ... [https://superpower.com/best-biomarkers/sleep-quality-circadian-rhythm?srsltid=AfmBOoqvEcA4v1Z8W5-8-ng088DeEUHjns-n0lX9SLg-NQdTrGfddhNj]
• 43. Comparing 17β‐estradiol and progesterone concentrations in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12314634/]
• 44. Recommendations for sample selection, collection and ... [https://link.springer.com/article/10.1007/s11306-025-02259-7]
• 45. Saliva testing for hormonal diagnosis: The facts and basics [https://www.news-medical.net/whitepaper/20240506/Saliva-testing-for-hormonal-diagnosis-The-facts-and-basics.aspx]
• 46. Circadian fluctuations in saliva biochemical composition [https://pubmed.ncbi.nlm.nih.gov/40932231/]
• 47. Comparative evaluation of four new immunoassays and LC-MS/MS for the measurement of urinary free cortisol in Cushing's syndrome diagnosis - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC12205834/]
• 48. Comparing immunoassay and mass spectrometry ... [https://www.sciencedirect.com/science/article/pii/S0306453025001027]
• 49. Comparisons of Immunoassay and Mass Spectrometry Measurements of Serum Estradiol Levels and Their Influence on Clinical Association Studies in Men - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC3667264/]
• 50. Optimizing Hormone Therapy With Circadian Rhythms [https://www.medscape.com/viewarticle/optimizing-hormone-therapy-circadian-rhythms-2025a1000nnn]
• 51. Procedures for numerical analysis of circadian rhythms - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC3663600/]
• 52. Screening and Diagnostic Testing for Cushing's Syndrome [https://csrf.net/understanding-cushings/diagnostic-testing/]
• 53. Practice Parameters for the Clinical Evaluation and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC2082098/]
• 54. THE PREANALYTICAL ERRORS: A CONTINUOUS… [https://ascls.org/the-preanalytical-errors/]
• 55. Preanalytical Variables and Their Influence on the Quality ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6238383/]
• 56. An overview of ELISA: a review and update on best laboratory ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11808753/]
• 57. Optimization, Validation and Standardization of ELISA [https://www.intechopen.com/chapters/74297]
• 58. QuEChERS analytical approach for monitoring endocrine ... [https://link.springer.com/article/10.1007/s00216-025-06099-1]
• 59. NORMAN guidance on suspect and non-target screening in ... [https://enveurope.springeropen.com/articles/10.1186/s12302-023-00779-4]
• 60. Quality assurance and quality control for human biomonitoring ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12206187/]
• 61. Optimization of experimental designs for biological rhythm ... [https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1013662]
• 62. Circadian Variation of Salivary Oxytocin in Young Adult ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12402539/]
• 63. A method for simultaneous analysis of urinary melatonin ... [https://pubmed.ncbi.nlm.nih.gov/40720929/]
• 64. Performance of Blood-Based Biomarkers for Human Circadian ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12572356/]
• 65. Strategies to enhance internal validity in multi ... - PubMed [https://pubmed.ncbi.nlm.nih.gov/9919302/]
• 66. A word on standardization in longitudinal studies: don't - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC4569815/]
• 67. Advancing clinical liquid chromatography-mass ... [https://www.sciencedirect.com/science/article/pii/S2772391725000131]
• 68. Screens may be upping your risk of cancer, heart disease ... [https://nypost.com/2025/11/19/health/screens-may-be-upping-your-risk-of-cancer-heart-disease-and-obesity/]
• 69. Endocrine regulation of circadian rhythms [https://www.nature.com/articles/s44323-025-00024-6]
• 70. Circadian phase, circadian period and chronotype are ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6055478/]
• 71. Assessment of Circadian Rhythms - PMC - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC6857846/]
• 72. saliva vs. blood diagnostics for hormone testing - The Blog [https://www.tecan.com/blog/saliva-hormone-testing-versus-blood-diagnostics]
• 73. Understanding Hormone Testing: Serum, Saliva, or Urine [https://www.espritwellness.com/understanding-hormone-testing-serum-saliva-or-urine-whats-best-for-your-health/]
• 74. Hormone testing: Trends to watch in 2025 - Health Journal [https://healthjournal.uk/5-trends-that-are-shaping-the-future-of-hormone-testing/]
• 75. Circadian clock genes and insomnia: molecular mechanisms ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12548078/]
• 76. Universal method for robust detection of circadian state from ... [https://pubmed.ncbi.nlm.nih.gov/30201705/]
• 77. Circadian rhythms revealed: unraveling the genetic ... [https://www.frontiersin.org/journals/sleep/articles/10.3389/frsle.2025.1544945/full]
• 78. Circadian regulation of extracellular vesicle biogenesis, ... [https://www.nature.com/articles/s44323-025-00053-1]
• 79. Salivary Circadian Gene Expression as a Molecular ... [https://pubmed.ncbi.nlm.nih.gov/40986222/]