Reliability of A Dried Urine Test for Comprehensive Assessment of Urine Hormones and Metabolites

Reliability of A Dried Urine Test for Comprehensive Assessment of Urine Hormones and Metabolites | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reliability of A Dried Urine Test for Comprehensive Assessment of Urine Hormones and Metabolites Mark Newman, Desmond Curran This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-122134/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Mar, 2021 Read the published version in BMC Chemistry → Version 1 posted 11 You are reading this latest preprint version Abstract Background Mass spectrometry allows for analysis of multiple hormone and organic acid metabolites from small urine volumes; however, to assess the full extent of daily hormone production, 24-hour urine collections are usually required. The aims of this study were, first, to confirm that mass spectrometric analysis of an array of hormones and organic acids would yield similar results in both liquid and dried urine, and, second, to determine if collection of four dried spot urine samples could be substituted for a 24-hour collection when measuring reproductive hormones. Methods Two study populations were included in this prospective observational study. Twenty individuals collected both a spot liquid urine and dried urine on filter paper to analyze eight organic acids. A second group of 26 individuals collected both a 24-hour urine and four dried spot urines during waking hours throughout the same day for evaluation of 17 reproductive hormones and metabolites; data from 18 of these individuals were available to compare liquid versus dried urine results. Dried urine was extracted, hydrolyzed, and derivatized before analysis by mass spectrometry; all analytes from dried urine were normalized to urine creatinine. Results Reproductive hormone results from dried and liquid urine were in excellent agreement with intraclass correlation coefficients (ICCs) greater than 0.90; comparison of dried to liquid urine for organic acids showed good to excellent agreement (ICC range: 0.75 to 0.99). Comparison between the 4-spot urine collection and 24-hour urine collection methods showed excellent agreement (ICC>0.9) for 14 of the 17 urine metabolites and good agreement for the others (ICC 0.78 to 0.85) with no systematic differences between the two methods of collection. Conclusions The burden of urine collection can be reduced using collection of four spot dried urines on filter paper without compromising comparability with hormone results from a 24-hour urine collection. A large number of urine analytes can be assessed from the dried urine with similar results to those from liquid urine. Given the ease of sample handling, this 4-spot dried urine assay would be useful for both clinical assessment of patients and for large epidemiologic studies. Biological Chemistry dried urine testing GC-MS/MS LC-MS/MS reproductive hormones estrogen testosterone androgens organic acids Figures Figure 1 Figure 2 Figure 3 Introduction Analysis of an array of hormones or metabolites may be useful for the clinician faced with a patient with a multitude of nonspecific symptoms that lead to a large differential of diagnoses, for screening of multiple diseases at once, for a patient on hormonal replacement therapy 1 , 2 , or for any patient where a complete picture of production and metabolism of a hormonal pathway is required 3 . Measurement of steroid hormones in urine can be an essential component to the diagnosis of hormone-related disorders 4 , 5 . Some patients may require sampling over multiple days to determine monthly variations or effects of change in treatment. For hormones with known circadian or pulsatile fluctuations, a representation of the entire day is essential 6 ; however, collection and storage of a 24-h urine can be cumbersome for patients 7 , 8 . Mass spectrometry technology, both liquid chromatography tandem mass spectrometry (LC-MS/MS) for water-soluble compounds and gas chromatography tandem mass spectrometry (GC-MS/MS) for non-polar compounds, is now routinely used to measure arrays of steroid hormones and organic acids because of its high assay sensitivity, accuracy with small volumes, and ability to evaluate multiple analytes at the same time 9 , 10 . This methodology allows for a complete profile of urine reproductive hormonal metabolites and multiple organic acids with high resolution of closely related structures 11 . Collection of urine on filter paper, which can then be dried and stored at room temperature until received by the laboratory 12 – 14 , offers a significant advance in patient convenience and may improve patient adherence. If multiple samples are collected throughout the day, there is potential to capture both the diurnal variation of hormones along with the full range of daily hormonal production 12 . Dried urine has already been shown to lead to equivalent measures as liquid urine in our hands for cortisol, cortisone, and the cortisol metabolites, a-tetrahydrocortisol, b-tetrahydrocortisol, and tetrahydrocortisone 15 as well as for estrone, estradiol, a-pregnanediol, and b-pregnanediol 12 . Others have found similar results between liquid and dried urine with organic acids 13 , 16 , 17 . The urinary analytes in this study included female and male reproductive hormones, 6-hydroxymelatoninsulfate, and a number of organic acids, which have a multitude of uses (Table 1 ). Inclusion of multiple metabolites from the estrogen and androgen pathways allows for a complete picture of estrogen and testosterone production and metabolism 18 , 19 . The primary goal of this study was to confirm that measurement of the urinary profile of reproductive hormones, 6-hydroxymelatoninsulfate, and an array of organic acids extracted from dried urine collected on filter paper as analyzed by tandem mass spectrometry would provide results in agreement with measurements from liquid urine. The secondary aim was to demonstrate that measurement of reproductive hormones in a collection of four dried urine samples over a 15-hour span throughout the day would accurately reflect the measurements of these hormones in a 24-h urine collection. Table 1 Partial list of urine hormones and analytes that can be measured from dried urine Variable Pathway Examples of Clinical Relevance Estrone Estrogen; estradiol metabolite Monitoring of hormonal replacement therapy; assessment of ovarian function or precocious puberty; evaluation of dietary effects 19 – 23 Estradiol Primary active estrogen Investigation of amenorrhea; assessment of precocious puberty; monitoring of hormonal replacement therapy; evaluation of dietary effects 19,23−26 Estriol Estrogen; estrogen metabolite via 16α-hydroxyestrone Monitoring of hormonal replacement therapy; evaluation of polycystic ovarian syndrome (PCOS); evaluation of dietary effects 19 , 23 , 27 2-hydroxyestrone Estrogen metabolite via estrone Estrogen receptor antagonist; antiproliferative; relative to other metabolites, may indicate health risks 24 , 25 , 28 , 29 2-hydroxyestradiol Estrogen metabolite via 2-hydroxyestrone Antiproliferative; relative to other metabolites, may indicate health risks; potential placental antioxidant 30 4-hydroxyestrone Estrogen metabolite via estrone Estrogen receptor agonist; relative to other metabolites, may indicate health risks; may be proangiogenic 29 16-hydroxyestrone Estrogen metabolite via estrone Estrogen receptor agonist; proliferative; relative to other metabolites, may indicate health risks; 2/16OHE1 ratio may be a marker of estrogen related cancer risk; together with estriol indicates activity of 16-hydroxylation pathway 24 , 25 , 28 , 29 2-methoxyestrone Estrogen metabolite via 2-hydroxyestrone May be anti-angiogenic; together with 2-hydroxyestrone and 2-hydroxyestradiol indicates activity of 2-hydroxylation pathway 31 , 32 Testosterone Primary active androgen Evaluation of male hypogonadism and infertility; monitoring of hormone therapy; anabolic to muscle and bone; evaluation of PCOS 18,19,33−35 Epitestosterone Inactive epimer of testosterone Evaluation of exogenous testosterone use; weak androgen antagonist 18 , 35 5α-Dihydrotestosterone Active form of testosterone in tissues Potent androgen; evaluation of hirsutism; evaluation of male infertility, hypogonadism, and low libido 18 , 36 Androsterone Metabolite of testosterone via 5αDHT Weak androgenic activity; neurosteroid; primary androgen metabolite in urine; evaluation of male infertility, hypogonadism, low libido, and hormone therapy; evaluation of PCOS 18 , 37 Etiocholanolone 5β epimer of androsterone Primary androgen metabolite in urine; inhibitory neurosteroid; evaluation of male infertility, hypogonadism, low libido, and hormone therapy; evaluation of PCOS 18 , 37 5a-Androstanediol Metabolite of testosterone and 5αDHT Weak androgenic and estrogenic properties; inhibitory neurosteroid; involved in prostate growth; evaluation of male infertility, hypogonadism, and low libido; evaluation of PCOS 18 , 27 5b-Androstanediol Metabolite of testosterone and 5αDHT; β epimer of 5α-androstanediol Has estrogen agonist properties; evaluation of male infertility, hypogonadism, and low libido; evaluation of PCOS 18 , 27 DHEA Major metabolite in urine is DHEAS Weak androgen produced by adrenal glands and brain; neurosteroid; implicated in neurocognitive declines and low libido; assessment of hirsutism and age-related changes in women; assessment of subclinical hypercortisolism 36 , 38 6-Hydroxymelatoninsulfate Metabolite of melatonin Representative of melatonin which is involved in the regulation of the sleep-wake cycle and promotes sleep; neuroprotective; evaluation of insomnia, fatigue, and infertility 39 , 40 Methylmalonic Acid Intermediate in the propionate pathway Increased in thiamine (B12) deficiency 41 Homovanillic Acid Metabolite of dopamine Elevated in cases of neuroblastoma; marker of metabolic stress in patients with psychiatric and neurologic diseases 42 Vanillylmandelic Acid Metabolite of norepinephrine Elevated in cases of neuroblastoma or pheochromocytoma 42 , 43 Kynurenic Acid Metabolite of tryptophan Part of the kynureninase pathway; increased in pyridoxine (B6) deficiency 44 – 47 Xanthurenic Acid Metabolite of tryptophan Part of the kynureninase pathway; increased in pyridoxine (B6) deficiency; possible measure of oxidative stress 46 – 48 5-HIAA Metabolite of tryptophan End product of serotonin metabolism; elevated in carcinoid syndrome; reduced in patients with major depressive disorder 42 , 49 , 50 Pyroglutamic Acid Intermediate in glutathione metabolism Increased in glutathione (an antioxidant) deficiency, as can occur with excess acetaminophen use 51 , 52 b-Hydroxyisovaleric Acid Metabolite of leucine Increased in biotin (B7) deficiency and in smokers 53 , 54 5-HIAA = 5-hydroxyindoleacetic acid; DHEA = dehydroepiandrosterone Methods Study populations A prospective observational study of urine collected from a population of healthy adult volunteers who agreed to participate in validation of urine analyses was conducted. This first study population included 26 individuals who provided data on hormonal measures (cortisol and cortisol metabolites, reproductive hormones, and 6-hydroxymelatoninsulfate) to compare samples from the 4-spot urine collection method to a 24-hour urine collection. A subset of these individuals (n = 18) had data available to compare measures from dried versus liquid urine. As cortisol and cortisol metabolites 15 and a and b-pregnanediol 12 were validated in previous analyses, only the following hormones were included in this analysis: estrone (E1), estradiol (E2), estriol (E3), 2-hydroxyestrone (2OHE1), 2-hydroxyestradiol (2OHE2), 4-hydroxyestrone (4OHE1), 16-hydroxyestrone (16OHE1), 2-methoxyestrone (2-methoxyE1), testosterone (T), epitestosterone (EpiT), 5a- dihydrotestosterone (DHT), androsterone, etiocholanolone, 5a-androstanediol, 5b-androstanediol, dehydroepiandrosterone (DHEA), and 6-hydroxymelatoninsulfate. The data were collected between February and November of 2015 and informed consent was obtained from all participants. The second analysis included 20 individuals whose deidentified data was pulled from the larger databank of 144,561 laboratory visits. Each of these samples included a single first-morning urine collection to compare results of dried versus liquid urine for the following organic acids: homovanillic acid (HVA), vanillylmandelic acid (VMA), kynurenic acid, xanthurenic acid, methymalonic acid (MMA), pyroglutamic acid, 5-hydroxyindoleacetic acid (5-HIAA), and b-hydroxyisovaleric (Hiv) acid. All data reports were deidentified during extraction so the IRB determined that written informed consent could be waived for this study population. Both studies (Clinical Trials ID: NCT04305093) were approved by the National University of Natural Medicine Institutional Review Board. All volunteers in both study populations reported no medical problems and were not pregnant. Individuals were not excluded based on current or recent use of any hormonal medications, as the goal was only to compare measurement values for differing methodologies. Eighty percent of women in the first study population and all women in the second study population were premenopausal. Sample Collection The 4-spot method involves urine samples collected at home at four times during the day: 1) the first urine of the day, 2) two hours after awakening, 3) in the afternoon (approximately 4 PM), and 4) before bed (10PM). Participants collected samples by completely saturating 2 × 3 inches of filter paper (Whatman Body Fluid Collection Paper) with urine. The paper was left exposed at room temperature for 24 hours to dry. The stability of analytes in dried urine at room temperature for as long as 84 days has previously been demonstrated by this laboratory 12 . Dried samples were stored at -80°C until analyzed. Reproductive hormones were assessed in all four samples collected, while only the first morning sample was used for 6-hydroxymelatoninsulfate and the organic acid tests. During the same day, all liquid urine samples for the 24-hour collection were added to a low-density polyethylene plastic container (ES Robbins, USA) container with approximately 1 g of boric acid and kept refrigerated for the duration of the collection. The four dried urine samples removed a total of about 8 mL of urine from the 24-hour collection. This was considered negligible and was not accounted for. The total volume of urine from 24-hour collections was measured, and an aliquot was frozen and stored at -80°C until tested. Urine Reproductive Hormone Analysis The urinary steroid hormones were analyzed using proprietary in-house CLIA (Clinical Laboratory Improvement Amendments) approved assays on the Agilent 7890/7000B GC-MS/MS (Agilent Technologies, Santa Clara, CA, USA). A 600 ul aliquot of liquid urine was taken from the sample collection and the equivalent of approximately 600 ul of urine was extracted from the filter paper using 2 mL of 100 mM ammonium acetate adjusted to a pH of 5.9. These aliquots of the conjugated hormones were transferred to a C18 solid phase extraction (SPE) column (UCT LLC, Briston, PA, USA), eluted using methanol, and the eluate was dried under nitrogen at 40 °C. The conjugated hormones were then hydrolyzed from their glucuronide and sulfate forms to free forms using enzymes from Helix pomatia (Sigma-Aldrich, St. Louis, MO, USA) in acetate buffer (55 °C, 90 minutes). The enzymatic reaction was quenched with sodium hydroxide and the hormones extracted with ethyl acetate. The ethyl acetate extracts were dried under nitrogen at 40 °C. The analytes were derivatized using a mixture of 100 ul acetonitrile (ACN) and 50 ul bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 70 °C. Internal standards (Steraloids, Newport, RI, USA) were added prior to ethyl acetate extraction, and the percentage recovery from all assays was greater than 90%. Derivatized extract (1.6 ul) was injected into the GC-MS/MS. Samples and controls were analyzed along with a standard curve spanning the expected range of concentrations. Instrument conditions for the oven were an initial temperature of 130 °C increasing to 200 °C at 25 °C /min, then to 230 °C at 4.3 °C/min, and finally to 290 °C at 25 °C /min. Multiple reaction monitoring transitions for ion mass > ion product of fragmentation were 342.1 > 257.1 for E1, 416.2 > 285.1 for E2, 504.3 > 296.3 for E3, 430.2 > 345.0 for 2OHE1, 462.2.2 > 195.2 for 2OHE2, 430.2 > 354.0 for 4OHE1, 430.2 > 286.1 for 16OHE1, 372.2-342.1 for 2-methoxyE1, 360.2 > 174.1 for T, 360.2 > 174.1 for EpiT, 347.2 > 271.2 for DHT, 347.2 > 271.2 for androsterone, 347.2 > 253 for etiocholanolone, 421.2 > 331.2 for 5α-androstanediol, 241.2 > 185.2 for 5β-androstanediol, and 303.7 > 199.1 for DHEA. Creatinine was measured using a conventional colorimetric (Jaffe) method, after initial extraction from the filter paper. The average inter-assay coefficient of variation was 6.7% for creatinine. In addition to expressing the measures per mg of creatinine to correct for variations in filter paper saturation and hydration status, a secondary equation was applied to reduce bias related to the effects of age, sex, weight, and height on creatinine excretion 55 . Urine 6-hydroxymelatoninsulfate And Organic Acid Analysis The hydrophilic analytes were assessed by LC-MS/MS using proprietary in-house CLIA approved assays. For the 6-hydroxymelatoninsulfate assay, a 30 ul aliquot was taken from the methanol elution of both the liquid urine collection and the waking sample dried urine collected on filter paper. This extract was then reconstituted in 130 ul of deionized water. For the organic acids, a 100 ul aliquot of liquid urine was taken and an equivalent amount was extracted from the waking sample dried urine filter paper using 250 ul of water with the addition of 50 ul 100 mM ammonium acetate (Sigma-Aldrich, St. Louis, MO, USA) adjusted to a pH of 5.9 and 2% formic acid. For 6-hydroxymelatoninsulfate, 20 ul was injected into an ultra-performance liquid chromatography (UPLC) (Waters Corporation, Milford, MA, USA) column with a Waters™ tandem quadrupole mass spectrometer detector (TQD). The sample was eluted from a 1.8u 2.1 × 50 mm pentafluorophenyl (PFP) column (Agilent Technologies, Santa Clara, CA, USA) using a gradient of 95% 0.001% formic acid in 5% ACN to 45% 0.001% formic acid in 55% ACN. For the organic acids, 5 ul was injected into the Waters™ UPLC column with TQD. These analytes were eluted from a 1.6 um 2.1 × 50 mm Luna Omega PS C18 column (Phenomenex, Torrance, CA, USA) using a gradient 99.9% 0.2% formic acid in 0.1% ACN to 73% 0.2% formic acid in 27% ACN. Multiple reaction monitoring transitions for ion mass > ion product of fragmentation were 372.5 > 176.2 for 6-hydroxymelatoninsulfate, 331.5 > 179.2 for d4-6-hydroxymelatoninsulfate, 183.0 > 137.0 for HVA, 185.0 > 139.0 for d2-HVA, 197.0 > 138.0 for VMA, 199.8 > 140.9 for d3-VMA, 190.0 > 144.0 for kynurenic acid, 192.0 > 149.0 for d5-kynurenic acid, 206.0 > 160.0 for xanthurenic acid, 210.0 > 164.0 for d3-xanthurenic acid, 117.0 > 73.0 for MMA, 120.0 > 76.0 for d2-MMA, 130.0 > 84.0 for pyroglutamic acid, 135.0 > 89.0 for d5-pyroglutamic acid, and 192.0 > 146.0 for 5-HIAA. The same creatinine corrections of the measures used for the reproductive hormones were also used for 6-hydroxymelatoninsulfate and the organic acids. Statistical Methods A sample size of 18 individuals provides a power of greater than 80% to detect an intraclass correlation coefficient (ICC) of at least 0.6 with an alpha of 0.05 56 . The statistical analyses were performed using SAS/STAT® software, Version 9.3 (SAS Institute Inc., Cary, NC, USA) and generated 2-sided p-values. Variables are described as means ± standard deviation if normally distributed and median (interquartile range (IQR)) if the distribution was skewed. Student’s t-test (for normally distributed variables) or the Wilcoxon rank-sum test (for skewed variables) were used to determine differences between men and women. Spearman correlation coefficients (ρ) were used to determine inter class associations between variables. Consistency between liquid versus dried urine measures and 4-spot versus 24-h collection methodology was assessed using intra class correlation coefficients (ICC). ICCs, which range from 0 to 1 with proximity to 1 indicating better agreement, assess for agreement of a measure between two differing methodologies within individuals 57 . Skewed variables were log transformed to approximate a normal distribution prior to assessing ICCs. As 4-spot (ng/mg-Cr) and 24-h (ug/d) measures were expressed in differing units, sex-specific Z-scores ([individual measurement-mean]/standard deviation) were created to standardize the measures and allow for direct comparison. Comparisons of differences between measures within an individual were assessed using signed-rank tests (for skewed variables) or paired t-tests (for normally distributed variables). Because the hypotheses of this paper were intrinsically correlated, no adjustments were made for multiple comparisons. Results Study populations Characteristics of the first study population (n = 26) are shown in Table 2 . All of these individuals (58% female; 100% Caucasian) had data available for comparison of 4-spot versus 24-h urine samples for male and female reproductive hormones. A subset (10 female, 8 male) also had measurements to compare liquid versus dried urine samples for both the reproductive hormones and 6-hydroxymelatoninsulfate. Characteristics of the second study population (n = 20; 75% female; 80% Caucasian / 10% Hispanic / 10% Asian-Pacific Islander), data from whom were used to compare a single first morning collection of liquid versus dried urine samples for the organic acid tests, are provided in Table 3 . Table 2 Age and 24-h measures of reproductive hormones and 6-hydroxymelatoninsulfate of the first study population. Variable All Female (n = 15) Male (n = 11) Age (years) 36.8 ± 14.5 33.7 ± 7.8 37.8 ± 18 Estrone (ug/d) a 13.69 (6.32, 19.16) 18.75 (6.83, 23.68) 7.67 (6.26, 13.68) Estradiol (ug/d) a 1.73 (0.80, 3.26) 3.17 (0.89, 4.19) 1.14 (0.70, 1.62) Estriol (ug/d) a 6.82 (3.57, 12.80) 11.64 (6.14, 16.55) 5.56 (3.45, 6.91) 2-hydroxyestrone (ug/d) a 3.33 (1.27, 6.24) 5.82 (0.77, 8.46) 2.26 (1.27, 4.03) 2-hydroxyestradiol (ug/d) a 0.25 (0.11, 0.67) 0.58 (0.06, 0.81) 0.19 (0.11, 0.26) 4-hydroxyestrone (ug/d) a 0.37 (0.19, 0.75) 0.67 (0.19, 0.99) 0.30 (0.16, 0.36) 16-hydroxyestrone (ug/d) a 0.89 (0.42, 1.48) 1.40 (0.36, 1.58) 0.49 (0.42, 0.90) 2-methoxyestrone (ug/d) a 2.27 (1.30, 3.80) 3.56 (1.38, 5.50) 1.63 (1.05, 2.35) Testosterone (ug/d) b 9.15 (4.97, 51.92) 5.43 (4.51, 8.01) 61.40 (42.14, 97.70) Epitestosterone (ug/d) b 7.23 (4.01, 11.23) 4.44 (1.18, 7.49) 14.94 (7.99, 16.80) 5a-Dihydrotestosterone (ug/d) b 5.53 (2.72, 11.59) 2.72 (1.33, 5.46) 12.60 (9.88, 17.47) Androsterone (ug/d) a 1331.99 (854.41, 1940.29) 1032.77 (691.70, 1528.59) 2083.52 (1005.51, 2716.33) Etiocholanolone (ug/d) a 811.74 (463.17, 1045.23) 568.56 (430.73, 853.81) 1020.79 (716.22, 1423.92) 5a-Androstanediol (ug/d) b 102.50 (54.83, 197.14) 58.72 (46.49, 97.50) 229.01 (143.30, 292.53) 5b-Androstanediol (ug/d) a 35.58 (14.05, 60.75) 15.83 (9.38, 52.69) 60.75 (20.69, 122.18) DHEA (ug/d) 166.14 (80.45, 431.61) 136.42 (80.45, 317.83 351.32 (52.69, 732.63) 6-Hydroxymelatoninsulfate (ug/ml) 7.87 (5.93, 13.85) 7.48 (5.93, 8.43) 12.21 (5.11, 20.06) Age presented as mean± SD; hormone data presented as median (IQR) as these data were skewed. a p < 0.05; b p < 0.01 for differences between males and females as assessed by the Wilcoxon rank-sum test. DHEA = dehydroepiandrosterone Table 3 Age and measures of organic acids from an early morning spot urine collection of the second study population. Variable All Female (n = 15) Male (n = 5) Age (years) 34.4 ± 6.6 34.5 ± 6.6 34.0 ± 7.4 Methylmalonic Acid (ng/mg-Cr) 0.18 (0.16, 0.25) 0.20 (0.16, 0.29) 0.16 (0.16, 0.18) Homovanillic Acid (ng/mg-Cr) b 0.52 ± 0.16 0.57 ± 0.14 0.35 ± 0.05 Vanillylmandelic acid (ng/mg-Cr) 0.41 ± 0.12 0.43 ± 0.12 0.36 ± 0.13 Kynurenic Acid (ng/mg-Cr) 0.25 ± 0.08 0.26 ± 0.08 0.21 ± 0.05 Xanthurenic Acid (ng/mg-Cr) 0.06 (0.05, 0.07) 0.06 (0.04, 0.07) 0.06 (0.05, 0.07) 5-HIAA (ng/mg-Cr) a 0.44 (0.40, 0.55) 0.47 (0.41, 0.58) 0.37 (0.36, 0.41) Pyroglutamic Acid (ng/mg-Cr) 4.06 ± 0.69 4.11 ± 0.78 3.90 ± 0.33 b-Hydroxyisovaleric Acid (ng/mg-Cr) 0.27 ± 0.12 0.29 ± 0.13 0.21 ± 0.07 Data are presented as mean ± SD if normally distributed and median (IQR) if right-skewed. a p < 0.05; b p < 0.01 for differences between males and females as assessed by the Student t-test or Wilcoxon rank-sum test. 5-HIAA = 5-hydroxyindoleacetic acid Agreement Between Liquid And Dried Urine Measures For the majority of analytes, there was excellent agreement (ICC > = 0.90) between the liquid and dried measures (Table 4 ). The exceptions were VMA (ICC = 0.79) and pyroglutamic acid (ICC = 0.75), which still had good agreement. Similarly, for the majority of analytes, there was no systemic directionality to the difference in the dried urine compared to the liquid urine. However, estrone and estriol were consistently higher when measured in liquid urine, while some of the organic acids – VMA, kynurenic acid, pyroglutamic acid, and b-hydroxyisovaleric acid – were more concentrated in the dried urine sample (Table 4 ). Representative interclass correlations (Spearman) between the two methods are shown in Fig. 1 (the rest are available in Supplementary Fig. 1). Table 4 Comparison of liquid versus dried urine analytes (n = 18). Variable Dried Liquid Difference [95% CI] ICC [95% CI] Hormones (n = 18) Estrone (ug/d) 10.78 (6.26, 18.73) 12.78 (7.22, 20.65) 1.28 [0.09, 2.47] a 0.92 [0.80, 0.97] Estradiol (ug/d) 1.32 (0.70, 3.26) 1.29 (0.74, 3.36) 0.09 [-0.01, 0.19] 0.96 [0.89, 0.98] Estriol (ug/d) 6.82 (3.45, 11.64) 7.15 (4.76, 14.41) 0.86 [0.05, 1.67] a 0.90 [0.77, 0.96] 2-hydroxyestrone (ug/d) 2.22 (0.98, 6.24) 1.84 (1.17, 6.24) -0.01 [-0.18, 0.16] 0.98 [0.94, 0.99] 2-hydroxyestradiol (ug/d) 0.18 (0.09, 0.58) 0.18 (0.08, 0.47) 0.00 [-0.02, 0.03] 0.97 [0.92, 0.99] 4-hydroxyestrone (ug/d) 0.31 (0.16, 0.67) 0.40 (0.25, 0.62) 0.03 [-0.02, 0.09] 0.95 [0.87, 0.98] 16-hydroxyestrone (ug/d) 0.69 (0.42, 1.48) 0.61 (0.39, 1.24) -0.07 [-0.18, 0.05] 0.95 [0.87, 0.98] 2-methoxyestrone (ug/d) 1.64 (1.05, 3.56) 1.70 (1.50, 3.58) 0.12 [-0.07, 0.30] 0.97 [0.94, 0.99] Testosterone (ug/d) 7.64 (4.97, 49.46) 8.42 (5.80, 51.40) 2.07 [-0.26, 4.40] 0.99 [0.98, 1.00] Epitestosterone (ug/d) 7.23 (4.01, 11.23) 8.21 (4.23, 13.98) 0.51 [-0.06, 1.07] 0.99 [0.97, 1.00] 5a-Dihydrotestosterone (ug/d) 5.53 (2.72, 11.59) 4.94 (2.68, 10.35) -0.61 [-1.02, -0.19] a 0.99 [0.97, 1.00] Androsterone (ug/d) 1331.99 (929.43, 1933.30) 1237.63 (813.31, 1847.10) -33.02 [-105.70, 39.67] 0.99 [0.98, 1.00] Etiocholanolone (ug/d) 847.93 (430.73, 1088.18) 943.49 (414.13, 1161.61) 19.76 [-13.02, 52.54] 0.99 [0.98, 1.00] 5a-Androstanediol (ug/d) 110.09 (57.50, 197.14) 119.40 (65.63, 226.93) 15.67 [-12.67, 44.01] 0.90 [0.76, 0.96] 5b-Androstanediol (ug/d) 35.58 (14.05, 72.42) 46.39 (14.72, 65.39) -0.54 [-13.93, 12.85] 0.88 [0.73, 0.95] DHEA (ug/d) 131.42 (52.69, 444.35) 130.86 (53.27, 457.05) 8.65 [-36.66, 53.96] 1.00 [0.99, 1.00] 6-Hydroxymelatoninsulfate (ug/d) 7.87 (5.93, 14.33) 8.26 (5.93, 14.67) 0.04 [-1.14, 1.21] 0.99 [0.98, 1.00] Organic Acids (n = 20) Methylmalonic Acid (ng/mg-Cr) 0.18 (0.15, 0.23) 0.18 (0.16, 0.25) 0.00 [-0.01, 0.02] 0.96 [0.90, 0.98] Homovanillic Acid (ng/mg-Cr) 0.52 ± 0.17 0.52 ± 0.16 -0.01 [-0.05, 0.02] 0.88 [0.74, 0.95] Vanillylmandelic Acid (ng/mg-Cr) 0.45 ± 0.13 0.41 ± 0.12 -0.05 [-0.08, -0.02] a 0.79 [0.57, 0.91] Kynurenic Acid (ng/mg-Cr) 0.26 ± 0.08 0.25 ± 0.08 -0.01 [-0.02, -0.00] a 0.95 [0.88, 0.98] Xanthurenic Acid (ng/mg-Cr) 0.06 (0.05, 0.08) 0.06 (0.05, 0.07) -0.00 [-0.00, 0.00] 0.98 [0.95, 0.99] 5-HIAA (ng/mg-Cr) 0.46 (0.43, 0.57) 0.44 (0.40, 0.55) -0.01 [-0.05, 0.02] 0.98 [0.96, 0.99] Pyroglutamic Acid (ng/mg-Cr) 4.42 ± 0.72 4.06 ± 0.69 -0.41 [-0.58, -0.23] b 0.75 [0.51, 0.89] b-Hydroxyisovaleric Acid (ng/mg-Cr) 0.29 ± 0.12 0.27 ± 0.12 -0.02 [-0.03, -0.00] a 0.95 [0.88, 0.98] For liquid and dried measurements, data presented as mean±SD for normally distributed variables and median (IQR) for skewed variables; the differences were normally distributed and are presented as mean [95% CI]. a p<0.05; b p<0.0001 for the liquid minus dried difference by paired t-test 5-HIAA = 5-hydroxyindoleacetic acid, Cr = creatinine, DHEA = dehydroepiandrosterone, ICC = intraclass correlation coefficient Agreement between the DUTCH 4-spot and 24-hour urine collection for hormonal measures The measurement of reproductive hormones in urine samples collected four times throughout the day were comparable to the gold standard of a 24-h urine collection (Table 5 ). As the measures from the 4-spot urine collection are reported in ng/mg-Cr and the 24-h urine measures are reported in ug/d, sex-specific Z-scores were created for direct comparison of the two methodologies. There was excellent consistency (ICC > 0.9) for the majority of the analytes and good consistency for the remainder (estriol, 5a and 5b-androstanediol) (Table 5 ). There was no systematic directionality to the differences between any of the Z-scores (Table 5 ). Representative interclass (Spearman) correlations between the analytes are shown in Fig. 3 (the remainder are shown in Supplementary Fig. 3). A sensitivity analysis to verify the need for creatinine correction was done by calculating ICCs for the agreement between sex-specific Z-scores from the 24-h measures and the 4-spot assay without correction for creatinine. Without the creatinine correction, the ICCs were all lower than those observed with the creatinine correction (with the exception of estriol which had a slightly higher ICC by 0.04) with the degree of difference ranging from − 0.04 to 0.15 and averaging 0.07. An example of the interclass correlations (Spearman) between the 24-h measures and the 4-spot assay with and without the creatinine corrections is shown in Supplementary Fig. 4. Table 5 Comparison of the urinary hormone profile using the 4-spot (DUTCH) or 24-h urine collection method (n = 26). Variable 24-h urine collection (ug/d) 4-spot (ng/mg-Cr) Z-score difference [95% CI] a ICC [95% CI] Estrone 13.69 (6.32, 19.16) 14.37 (7.13, 18.75 0.00 [-0.13, 0.13] 0.95 [0.89, 0.97] Estradiol 1.73 (0.80, 3.26) 1.68 (0.92, 3.81) 0.00 [-0.16, 0.16] 0.92 [0.83, 0.96] Estriol 6.82 (3.57, 12.80) 7.39 (4.15, 12.96) 0.00 [-0.24, 0.24] 0.82 [0.64, 0.91] 2-hydroxyestrone 3.33 (1.27, 6.24) 3.72 (1.45, 6.75) -0.00 [-0.09, 0.09] 0.97 [0.94, 0.99] 2-hydroxyestradiol 0.25 (0.11, 0.67) 0.30 (0.13, 0.49) -0.00 [-0.10, 0.10] 0.97 [0.93, 0.99] 4-hydroxyestrone 0.37 (0.19, 0.75) 0.45 (0.27, 0.81) -0.00 [-0.15, 0.15] 0.93 [0.86, 0.97] 16-hydroxyestrone 0.89 (0.42, 1.48) 0.93 (0.57, 1.69) 0.00 [-0.19, 0.19] 0.90 [0.77, 0.95] 2-methoxyestrone 2.27 (1.30, 3.80) 2.11 (1.54, 4.15) 0.00 [-0.19, 0.19] 0.90 [0.78, 0.95] Testosterone 9.15 (4.97, 51.92) 9.33 (5.36, 54.73) -0.00 [-0.10, 0.10] 0.97 [0.93, 0.98] Epitestosterone 7.23 (4.01, 11.23) 7.53 (3.81, 13.90) -0.00 [-0.07, 0.07] 0.99 [0.97, 0.99] 5a-Dihydrotestosterone 5.53 (2.72, 11.59) 6.40 (2.16, 11.91) -0.00 [-0.09, 0.09] 0.97 [0.94, 0.99] Androsterone 1331.99 (854.41, 1940.29) 1346.46 (818.59, 2065.66) -0.00 [-0.09, 0.09] 0.97 [0.94, 0.99] Etiocholanolone 811.74 (463.17, 1045.23) 797.60 (475.43, 1061.66) -0.00 [-0.09, 0.09] 0.98 [0.95, 0.99] 5a-Androstanediol 102.50 (54.83, 197.14) 86.74 (51.31, 200.70) -0.00 [-0.27, 0.27] 0.78 [0.57, 0.89] 5b-Androstanediol 35.58 (14.05, 60.75) 34.49 (13.91, 62.77) 0.00 [-0.22, 0.22] 0.85 [0.70, 0.93] DHEA 166.14 (80.45, 431.61) 162.85 (78.17, 394.62) -0.00 [-0.05, 0.05] 0.99 [0.99, 1.00] The ICCs were calculated between measurements expressed as standardized sex-specific Z-scores (mean ± SD for all = 0.00 ± 0.98). Data are presented as median (IQR) for measurements and the difference (24-h minus 4-spot) in individual Z-scores is presented as mean [95% confidence interval]. a all p-values > 0.9 by paired t-test Cr = creatinine, DHEA = dehydroepiandrosterone, ICC = intraclass correlation coefficient Discussion This study demonstrated the feasibility of accurately measuring multiple (up to 32) analytes in dried urine samples collected on filter paper using assays that conform to CLIA criteria. All measurements from dried urine demonstrated at least good agreement with measures from liquid urine, and the majority (83%) demonstrated excellent agreement with intraclass correlations greater than 0.9. For most analytes, neither loss nor excess concentration occurred during the sample drying or laboratory extraction process. In addition, measurement of the reproductive steroid hormones, which are usually evaluated from a 24-hr collection due to their pulsatile release 58 , were well represented by the 4-spot dried urine collection with at least good agreement of the 4-spot measurement with the 24-h gold standard measure for all steroid metabolites and excellent agreement (> 0.9) for the majority (82%). There were no systematic differences between the relative amount of hormone collected by either methodology. The 4-spot dried urine (DUTCH) methodology allows for efficient, accurate assessment of numerous urine metabolites using a convenient collection method, while avoiding the need for a full 24-hr liquid urine collection. The 4-spot dried urine collection has previously been shown to be representative of select 24-h measures of steroidal hormones by our group 12 , as measurement of urinary a-pregnanediol, b-pregnanediol, estrone, and estradiol with this method are representative of both 24-h urine collections as well as serum hormone concentrations. In fact, repeated assessments over a month demonstrated that the dried urine collections could accurately recreate the changes observed in serum during the menstrual cycle 12 . In addition, not only does the 4-spot urine method accurately represent a 24-h urine collection for urinary cortisol, cortisone, and cortisol metabolites, but it can also be used to represent the expected diurnal pattern observed with salivary measures, if each of the four collections is considered individually 15 . Previous studies have validated the usefulness of high throughput GC-MS/MS for urine steroid profiling of more than 30 metabolites; however, this was done in liquid urine 5 . Conversely, measurement of organic acids in spot dried urine samples using filter paper has already been well-validated as a technique for screening neonates for metabolic disorders 16 , 59 , 60 and for screening for neuroblastoma 61 with similar recoveries obtained from liquid and dried urine. Dried urine has also been used to measure other urine analytes of interest, such as sodium and potassium, with similarly high levels of stability 62 . As with others who used either filter paper or cotton swabs 63 , we found that dried urine results are in agreement with liquid urine results, reduced the burden on patients, and had good stability over time 12 . Still, agreement between 24-h urine collections and early-morning, single spot urine collections for hormonal analysis are often poor 64 . This study now extends our prior findings 12 , 15 to show that the increase to four spot urines spaced throughout the waking hours provides better coverage of the hormonal output for all the male and female reproductive hormones and metabolites, resulting in strong agreement with 24-h urine measures. There are some caveats that must be considered when interpreting our results. During the urine collection, there may have been differences in saturation of the filter paper. Expressing the analyte concentration per mg of creatinine is designed to address this, while also correcting for hydration. The method used for creatinine adjustment also accounts for differences in creatinine excretion related to body size. This does rely on accurate self-reporting of age, height, and weight by the participants, so an estimate of expected creatinine excretion can be made. This may lead to the introduction of inaccuracies due to misreporting of individual characteristics; however, our statistical sensitivity analyses indicated that the DUTCH measurements were in better agreement with the 24-h urine results with the application of the creatinine corrections (see Supplementary Fig. 4), with the ICC for some metabolites increasing by as much as 0.15 and raising the level of agreement with 24-h measures from good to excellent. One of the limitations of this 4-spot dried urine method is that reference ranges are laboratory-specific and non-standardized. Still, interpretation of values above and below these reference ranges should be similar to that of other assays. In this study, there was some loss of hormone for estrone and estriol with the filter paper methodology, which may be related to differences in extraction efficiency between the steroid conjugates and creatinine, loss during the drying process, or incomplete saturation of the filter paper. Still, the difference was less than 12% of the total and would be compensated for by an adjustment of the reference range. In addition, we have previously shown that the dried urine measure of estrone has clinical utility because it is representative of serum estrogen measurements 12 . A number of organic acids plus DHT were more concentrated in the dried urine samples, on average. This may be due to differences in extraction efficiency, a matrix effect or analyte concentration during the drying process; however, this difference did represent less than 10% of the sample. Fortunately, due to the high level of agreement between the Z-scores from the DUTCH methodology and 24-h collections, laboratory reference ranges should account for these differences. Another issue is there are known genetic differences in glucuronidation of testosterone that may impact relative metabolism and urinary concentrations of testosterone and epitestosterone 65 , and this may mean urinary androgen measures are not fully representative of production rates in a small percentage of individuals. There are also genetic differences in the enzymes that metabolize estrogens, potentially shifting the ratio of 2-hyroxylation to 16-hydroxylation metabolites 66 , but these differences may be clinically relevant and indicative of cancer risk 67 . The methodology of a 4-spot urine collection on filter paper followed by GC-MS/MS or LC-MS/MS offers some advantages. The collection of dried urine on filter paper results in stable measurement of steroid hormones for extended periods of time both by us 12 and others 13 for up to one year 14 , even at ambient temperature. Concentrations of organic acids are also stable on dried filter paper for weeks 68 , 69 . Mass spectrometry assays, which are now the gold standard for measurement of steroid hormones in blood and urine 11 , allow for the use of small sample volumes with excellent sensitivity and accuracy along with simultaneous measurement of a relatively large number of analytes. In combination with chromatography, either gas or liquid, it provides precise separation of closely related molecules 70 by their chemical and physical properties. GC-MS/MS does not exclude any lipophilic steroids, and so a run will contain all excreted steroids 9 . The use of the H. pomatia enzymes adds to the accuracy of the quantification of the hormone conjugates, as these enzymes include both a sulfatase and a glucuronidase. The method of GC-MS/MS does require an additional extraction and derivatization step, but workflows can be optimized to maximize throughput. A 24-h urine collection may be difficult for some patients to fully collect, especially if they are not able to remain at home for an entire day, are disabled, or are incontinent. This methodology removes that barrier and provides the ability to measure multiple hormones at once with a noninvasive collection method, obtaining a complete picture of both production and clearance of the major steroidal hormones. A multitude of uses, both in research and in clinical scenarios, could be envisioned for assays that are able to measure multiple steroid hormones and organic acids in conveniently collected urine samples on filter paper. For example, the full range of hormonal changes in individuals related to disruption of the natural sleep cycle could be evaluated simultaneously. It is already known that the peak 6-sulfatoxymelatonin, as representative of melatonin, is lower in people working the night shift 71 ; a full appreciation of the urinary steroid profile in individuals who work at night could add to this prior research. Similarly, urine profiling may help to fully define the changes expected in genetic syndromes of steroidogenesis 5 , 72 and errors of metabolism 17 . Dried urine samples may be of particular benefit in screening neonates for organic acid disorders 16 , 17 , and there is recent interest in a possible association of organic acids with neuropsychiatric disorders 73 – 75 . The ability to look at a full urine profile can provide a more integrated view of the patient; for example, patients using oral contraceptives often have higher xanthurenic acid with concurrent pyroxidine deficiency 76 , both of which would be observable using dried urine analysis. A greater understanding of the full effect of changes in hormonal concentrations and metabolites or important clinical subgroups could be determined for both exogenous use of hormones and for exposure to endocrine disrupting compounds like bisphenol A 77 , 78 . Urine hormone profiling might also be used to fully describe age related changes, i.e. through puberty or menopause. Conclusions Mass spectrometry allows for the assessment of a full hormone profile in a small volume of urine such that an expanded view of both hormone production and clearance can be observed. In addition, results from dried urine are in strong agreement with those obtained from liquid urine. In combination with four spot urine collections on filter paper collected throughout the waking hours, we have shown that it is possible to accurately represent a 24-h urine collection. This technology may be useful to the clinician wishing to perform a large series of tests on patients to narrow the differential diagnosis, for those monitoring hormonal therapy or evaluating the menstrual cycle, or for those who need to reduce the burden of collection for their patients. This four-spot, dried urine method allows for assessment of both diurnal patterns 15 as well as total daily production, allowing for a comprehensive evaluation of adrenal and reproductive hormones and other urine metabolites. Abbreviations 2OHE1 2-hydroxyestrone 2-methoxyE1 2-methoxyestrone 4OHE1 4-hydroxyestrone 5-HIAA 5-hydroxyindoleacetic acid 16OHE1 16-hydroxyestrone CAN acetonitrile CLIA Clinical Laboratory Improvement Amendments Cr creatinine DHEA dehydroepiandrosterone DHT 5α-dihydrotestosterone DUTCH Dried Urine Testing for Comprehensive Hormones E1 estrone E2 estradiol E3 estriol EpiT epitestosterone GC-MS/MS gas chromatography with tandem mass spectrometry Hiv β-hydroxyisovaleric HVA homovanillic acid ICC intraclass correlation coefficient IQR interquartile range LC-MS/MS liquid chromatography with tandem mass spectrometry MMA methylmalonic acid T testosterone TQD tandem quadrupole mass spectrometer detector VMA vanillylmandelic acid Declarations Ethics approval and consent to participate The research was conducted on healthy adult volunteers at a commercial testing laboratory. Informed consent was obtained from all participants prior to the study or the IRB determined that written informed consent could be waived for the study population. All methods were performed in accordance with the relevant guidelines and regulations required by both the National University of Natural Medicine Institutional Review Board and CLIA. Availability of data and material The datasets acquired and/or analyzed during the current study are available from the corresponding author on reasonable request. Consent for publication Not applicable Competing interests Precision Analytical, Inc. is a commercial laboratory offering hormone testing to medical practitioners and individuals. All statistics were calculated and interpreted by an independent agent. Funding Precision Analytical Laboratory, Inc. provided funds to run urine and serum assays. Author’s contributions MN developed the methodology of the dried urine assay, designed the studies, interpreted the data, and made substantial contributions to the manuscript. DC acquired and interpreted the data and contributed to the preparation of the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors would like to acknowledge Danielle Martinot for writing the IRB proposal for this study and Marie Thearle, MD for creating tables and figures and assistance with statistical analysis and interpretation of the data. And thanks to the volunteers who provided samples for analysis. Author’s information MN, DC: Precision Analytical, Inc. 3138 NE Rivergate Street #301C, McMinnville, OR 97128 USA References Sansone A, Sansone M, Selleri R, et al. Monitoring testosterone replacement therapy with transdermal gel: when and how? Journal of Endocrinological Investigation . 2019;42(12):1491–1496. doi: 10.1007/s40618-019-01082-x Rodgers M. 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Bioanalysis . 2019;11(8):689–701. doi: 10.4155/bio-2018-0295 Monošík R, Dragsted LO. Dried urine swabs as a tool for monitoring metabolite excretion. Bioanalysis . 2018;10(17):1371–1381. doi: 10.4155/bio-2018-0042 Bileck A, Frei S, Vogt B, Groessl M. Urinary steroid profiles: comparison of spot and 24-hour collections. Journal of Steroid Biochemistry and Molecular Biology . 2020;200. doi: 10.1016/j.jsbmb.2020.105662 Sten T, Bichlmaier I, Kuuranne T, Leinonen A, Yli-Kauhaluoma J, Finel M. UDP-glucuronosyltransferases (UGTs) 2B7 and UGT2B17 display converse specificity in testosterone and epitestosterone glucuronidation, whereas UGT2A1 conjugates both androgens similarly. Drug Metabolism and Disposition . 2009;37(2):417–423. doi: 10.1124/dmd.108.024844 Yong M, Schwartz SM, Atkinson C, et al. Associations between polymorphisms in glucuronidation and sulfation enzymes and sex steroid concentrations in premenopausal women in the United States. Journal of Steroid Biochemistry and Molecular Biology . 2011;124(1–2):10–18. doi: 10.1016/j.jsbmb.2010.12.014 Sampson JN, Falk RT, Schairer C, et al. Association of estrogen metabolism with breast cancer risk in different cohorts of postmenopausal women. Cancer Research . 2017;77(4):918–925. doi: 10.1158/0008-5472.CAN-16-1717 Shinka T, Ohse M, Inoue Y, Kuhara T. Stability of 5-aminolevulinic acid on dried urine filter paper for a diagnostic marker of tyrosinemia type I. In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences . Vol 823. J Chromatogr B Analyt Technol Biomed Life Sci; 2005:44–46. doi: 10.1016/j.jchromb.2005.02.002 McCann M, Thompson M, Gueron IC, Lemieux B, Giguere R, Tuchman M. Methylmalonic acid quantification by stable isotope dilution gas chromatography-mass spectrometry from filter paper urine samples. Clin Chem . 1996;42(6 Pt 1):910–914. Homer N, Kothiya S, Rutter A, Walker BR, Andrew R. Gas chromatography tandem mass spectrometry offers advantages for urinary steroids analysis. Analytical Biochemistry . 2017;538:34–37. doi: 10.1016/j.ab.2017.09.002 Davis S, Mirick DK, Chen C, Stanczyk FZ. Night shift work and hormone levels in women. Cancer Epidemiology Biomarkers and Prevention . 2012;21(4):609–618. doi: 10.1158/1055-9965.EPI-11-1128 Pussard E, Travers S, Bouvattier C, et al. Urinary steroidomic profiles by LC-MS/MS to monitor classic 21-Hydroxylase deficiency. Journal of Steroid Biochemistry and Molecular Biology . 2020;198. doi: 10.1016/j.jsbmb.2019.105553 Marc DT, Ailts JW, Campeau DCA, Bull MJ, Olson KL. Neurotransmitters excreted in the urine as biomarkers of nervous system activity: Validity and clinical applicability. Neuroscience and Biobehavioral Reviews . 2011;35(3):635–644. doi: 10.1016/j.neubiorev.2010.07.007 Black CN, Bot M, Scheffer PG, Cuijpers P, Penninx BWJH. Is depression associated with increased oxidative stress? A systematic review and meta-analysis. Psychoneuroendocrinology . 2015;51:164–175. doi: 10.1016/j.psyneuen.2014.09.025 Abou-Saleh MT, Coppen A. Psychiatric progress. The biology of folate in depression: Implications for nutritional hypotheses of the psychoses. Journal of Psychiatric Research . 1986;20(2):91–101. doi: 10.1016/0022-3956(86)90009-9 Deac OM, Mills JL, Shane B, et al. Tryptophan catabolism and vitamin B-6 status are affected by gender and lifestyle factors in healthy young adults. Journal of Nutrition . 2015;145(4):701–707. doi: 10.3945/jn.114.203091 Kim EJ, Lee D, Chung BC, Pyo H, Lee J. Association between urinary levels of bisphenol-A and estrogen metabolism in Korean adults. Science of the Total Environment . 2014;470–471:1401–1407. doi: 10.1016/j.scitotenv.2013.07.040 Bloem LM, Storbeck KH, Swart P, du Toit T, Schloms L, Swart AC. Advances in the analytical methodologies: Profiling steroids in familiar pathways-challenging dogmas. Journal of Steroid Biochemistry and Molecular Biology . 2015;153:80–92. doi: 10.1016/j.jsbmb.2015.04.009 Supplementary Files ReliabilityofADriedUrineTestforComprehensiveAssessmentofUrineHormonesandMetabolitesSupplementaryFigures.docx Cite Share Download PDF Status: Published Journal Publication published 15 Mar, 2021 Read the published version in BMC Chemistry → Version 1 posted Editorial decision: Major revision 08 Feb, 2021 Reviews received at journal 05 Feb, 2021 Reviewers agreed at journal 25 Jan, 2021 Reviews received at journal 26 Dec, 2020 Reviewers agreed at journal 20 Dec, 2020 Reviewers agreed at journal 14 Dec, 2020 Reviewers invited by journal 09 Dec, 2020 Editor assigned by journal 08 Dec, 2020 Editor invited by journal 08 Dec, 2020 Submission checks completed at journal 08 Dec, 2020 First submitted to journal 04 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-122134","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":6060532,"identity":"0cd1de39-35da-4f86-97fb-27b7f205e330","order_by":0,"name":"Mark Newman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBADOYYDDAzMJGkxZjjADNSSwMDAQ6yWxAaitejObj4mdaPmTnrfjfyDnwt/2NjbMzA/fHQDjxazO8fSpHOOPcudeSOZWXpGQlpiDwObsXEOPi03csxu57Adzt1wI5mNmSfhcAIPAw+bNH4t+d9u5/w7nG4A0fLfnggtOWy3c9sOJ0C1HGDsIawlzfx3bt9hw5lnHhtL86QlJ/YcJuiX5MfGOd8Oy/MdT3z4mcfGzp69vfnhY3xasADS0sAoGAWjYBSMAmwAABijS/CN9X+LAAAAAElFTkSuQmCC","orcid":"","institution":"Precision Analytical, Inc.","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Newman","suffix":""},{"id":6060533,"identity":"9d4f9270-b8df-48b9-a304-673743c6287e","order_by":1,"name":"Desmond Curran","email":"","orcid":"","institution":"Precision Analytical, Inc.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Desmond","middleName":"","lastName":"Curran","suffix":""}],"badges":[],"createdAt":"2020-12-04 21:14:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-122134/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-122134/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13065-021-00744-3","type":"published","date":"2021-03-15T15:00:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4166302,"identity":"1f263095-6b6f-4e7e-beaa-1aea9a4311d5","added_by":"auto","created_at":"2020-12-10 17:21:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60367,"visible":true,"origin":"","legend":"Correlations between the liquid versus dried measurements for select urine steroid hormones. The remainder are available in Supplemental Figure 1. Reported correlation coefficients are Spearman correlations. Cr=creatinine, DHEA = dehydroepiandrosterone","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-122134/v1/ca9dd5d3a74e87ac856005f4.png"},{"id":4166303,"identity":"12b71c55-1be4-464e-ad01-ed62d06a60df","added_by":"auto","created_at":"2020-12-10 17:21:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38322,"visible":true,"origin":"","legend":"Correlations between the liquid versus dried measurements for select urine organic acids. The remainder are available in Supplemental Figure 2. Reported correlation coefficients are Spearman correlations. Cr=creatinine, Hiv = -Hydroxyisovaleric ","description":"","filename":"2.PNG","url":"https://assets-eu.researchsquare.com/files/rs-122134/v1/e0d2e52a169cb9e920648cef.PNG"},{"id":4166304,"identity":"6cefc97d-b6ad-484f-8992-3e12ab2e5be2","added_by":"auto","created_at":"2020-12-10 17:21:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58213,"visible":true,"origin":"","legend":"Correlations between the 24-h urine collection and 4-spot (DUTCH) urine collection measurements. The remainder are available in supplemental figure 3. Reported correlation coefficients are Spearman correlations. \nCr=Creatinine, DHEA = dehydroepiandrosterone, DUTCH = Dried Urine Testing for Comprehensive Hormones\n","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-122134/v1/1f8f6d887c2d7f2762954ed0.png"},{"id":13630731,"identity":"257318e5-9c71-47af-b7d6-a8131a681aed","added_by":"auto","created_at":"2021-09-17 08:13:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":971270,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-122134/v1/c74f5056-887f-4f92-bda7-5b0d5491c364.pdf"},{"id":4166305,"identity":"3fd54347-885e-480c-8437-9ab9de07ca02","added_by":"auto","created_at":"2020-12-10 17:21:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":162069,"visible":true,"origin":"","legend":"","description":"","filename":"ReliabilityofADriedUrineTestforComprehensiveAssessmentofUrineHormonesandMetabolitesSupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-122134/v1/b971faebd5a8c3408f08e48b.docx"}],"financialInterests":"","formattedTitle":"Reliability of A Dried Urine Test for Comprehensive Assessment of Urine Hormones and Metabolites","fulltext":[{"header":"Introduction","content":" \u003cp\u003eAnalysis of an array of hormones or metabolites may be useful for the clinician faced with a patient with a multitude of nonspecific symptoms that lead to a large differential of diagnoses, for screening of multiple diseases at once, for a patient on hormonal replacement therapy\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, or for any patient where a complete picture of production and metabolism of a hormonal pathway is required\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Measurement of steroid hormones in urine can be an essential component to the diagnosis of hormone-related disorders\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Some patients may require sampling over multiple days to determine monthly variations or effects of change in treatment. For hormones with known circadian or pulsatile fluctuations, a representation of the entire day is essential\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e; however, collection and storage of a 24-h urine can be cumbersome for patients\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMass spectrometry technology, both liquid chromatography tandem mass spectrometry (LC-MS/MS) for water-soluble compounds and gas chromatography tandem mass spectrometry (GC-MS/MS) for non-polar compounds, is now routinely used to measure arrays of steroid hormones and organic acids because of its high assay sensitivity, accuracy with small volumes, and ability to evaluate multiple analytes at the same time\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This methodology allows for a complete profile of urine reproductive hormonal metabolites and multiple organic acids with high resolution of closely related structures\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCollection of urine on filter paper, which can then be dried and stored at room temperature until received by the laboratory\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, offers a significant advance in patient convenience and may improve patient adherence. If multiple samples are collected throughout the day, there is potential to capture both the diurnal variation of hormones along with the full range of daily hormonal production\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Dried urine has already been shown to lead to equivalent measures as liquid urine in our hands for cortisol, cortisone, and the cortisol metabolites, a-tetrahydrocortisol, b-tetrahydrocortisol, and tetrahydrocortisone\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e as well as for estrone, estradiol, a-pregnanediol, and b-pregnanediol\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Others have found similar results between liquid and dried urine with organic acids\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The urinary analytes in this study included female and male reproductive hormones, 6-hydroxymelatoninsulfate, and a number of organic acids, which have a multitude of uses (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Inclusion of multiple metabolites from the estrogen and androgen pathways allows for a complete picture of estrogen and testosterone production and metabolism\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe primary goal of this study was to confirm that measurement of the urinary profile of reproductive hormones, 6-hydroxymelatoninsulfate, and an array of organic acids extracted from dried urine collected on filter paper as analyzed by tandem mass spectrometry would provide results in agreement with measurements from liquid urine. The secondary aim was to demonstrate that measurement of reproductive hormones in a collection of four dried urine samples over a 15-hour span throughout the day would accurately reflect the measurements of these hormones in a 24-h urine collection.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePartial list of urine hormones and analytes that can be measured from dried urine\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePathway\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExamples of Clinical Relevance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEstrone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen; estradiol metabolite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonitoring of hormonal replacement therapy; assessment of ovarian function or precocious puberty; evaluation of dietary effects\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEstradiol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimary active estrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInvestigation of amenorrhea; assessment of precocious puberty; monitoring of hormonal replacement therapy; evaluation of dietary effects\u003csup\u003e19,23\u0026minus;26\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEstriol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen; estrogen metabolite via 16α-hydroxyestrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMonitoring of hormonal replacement therapy; evaluation of polycystic ovarian syndrome (PCOS); evaluation of dietary effects\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2-hydroxyestrone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen metabolite via estrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstrogen receptor antagonist; antiproliferative; relative to other metabolites, may indicate health risks\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2-hydroxyestradiol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen metabolite via 2-hydroxyestrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAntiproliferative; relative to other metabolites, may indicate health risks; potential placental antioxidant\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4-hydroxyestrone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen metabolite via estrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstrogen receptor agonist; relative to other metabolites, may indicate health risks; may be proangiogenic\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16-hydroxyestrone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen metabolite via estrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstrogen receptor agonist; proliferative; relative to other metabolites, may indicate health risks; 2/16OHE1 ratio may be a marker of estrogen related cancer risk; together with estriol indicates activity of 16-hydroxylation pathway\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2-methoxyestrone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen metabolite via 2-hydroxyestrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMay be anti-angiogenic; together with 2-hydroxyestrone and 2-hydroxyestradiol indicates activity of 2-hydroxylation pathway\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTestosterone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimary active androgen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEvaluation of male hypogonadism and infertility; monitoring of hormone therapy; anabolic to muscle and bone; evaluation of PCOS\u003csup\u003e18,19,33\u0026minus;35\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEpitestosterone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInactive epimer of testosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEvaluation of exogenous testosterone use; weak androgen antagonist\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5α-Dihydrotestosterone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive form of testosterone in tissues\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePotent androgen; evaluation of hirsutism; evaluation of male infertility, hypogonadism, and low libido\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAndrosterone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of testosterone via 5αDHT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeak androgenic activity; neurosteroid; primary androgen metabolite in urine; evaluation of male infertility, hypogonadism, low libido, and hormone therapy; evaluation of PCOS\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEtiocholanolone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5β epimer of androsterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimary androgen metabolite in urine; inhibitory neurosteroid; evaluation of male infertility, hypogonadism, low libido, and hormone therapy; evaluation of PCOS\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5a-Androstanediol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of testosterone and 5αDHT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeak androgenic and estrogenic properties; inhibitory neurosteroid; involved in prostate growth; evaluation of male infertility, hypogonadism, and low libido; evaluation of PCOS\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5b-Androstanediol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of testosterone and 5αDHT; β epimer of 5α-androstanediol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHas estrogen agonist properties; evaluation of male infertility, hypogonadism, and low libido; evaluation of PCOS\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDHEA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMajor metabolite in urine is DHEAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeak androgen produced by adrenal glands and brain; neurosteroid; implicated in neurocognitive declines and low libido; assessment of hirsutism and age-related changes in women; assessment of subclinical hypercortisolism\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6-Hydroxymelatoninsulfate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of melatonin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRepresentative of melatonin which is involved in the regulation of the sleep-wake cycle and promotes sleep; neuroprotective; evaluation of insomnia, fatigue, and infertility\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethylmalonic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntermediate in the propionate pathway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIncreased in thiamine (B12) deficiency\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHomovanillic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of dopamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElevated in cases of neuroblastoma; marker of metabolic stress in patients with psychiatric and neurologic diseases\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVanillylmandelic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of norepinephrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElevated in cases of neuroblastoma or pheochromocytoma\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKynurenic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of tryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePart of the kynureninase pathway; increased in pyridoxine (B6) deficiency\u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eXanthurenic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of tryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePart of the kynureninase pathway; increased in pyridoxine (B6) deficiency; possible measure of oxidative stress\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5-HIAA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of tryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnd product of serotonin metabolism; elevated in carcinoid syndrome; reduced in patients with major depressive disorder\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePyroglutamic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntermediate in glutathione metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIncreased in glutathione (an antioxidant) deficiency, as can occur with excess acetaminophen use\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eb-Hydroxyisovaleric Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolite of leucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIncreased in biotin (B7) deficiency and in smokers\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e5-HIAA\u0026thinsp;=\u0026thinsp;5-hydroxyindoleacetic acid; DHEA\u0026thinsp;=\u0026thinsp;dehydroepiandrosterone\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStudy populations\u003c/h2\u003e \u003cp\u003eA prospective observational study of urine collected from a population of healthy adult volunteers who agreed to participate in validation of urine analyses was conducted. This first study population included 26 individuals who provided data on hormonal measures (cortisol and cortisol metabolites, reproductive hormones, and 6-hydroxymelatoninsulfate) to compare samples from the 4-spot urine collection method to a 24-hour urine collection. A subset of these individuals (n\u0026thinsp;=\u0026thinsp;18) had data available to compare measures from dried versus liquid urine. As cortisol and cortisol metabolites\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and a and b-pregnanediol\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e were validated in previous analyses, only the following hormones were included in this analysis: estrone (E1), estradiol (E2), estriol (E3), 2-hydroxyestrone (2OHE1), 2-hydroxyestradiol (2OHE2), 4-hydroxyestrone (4OHE1), 16-hydroxyestrone (16OHE1), 2-methoxyestrone (2-methoxyE1), testosterone (T), epitestosterone (EpiT), 5a- dihydrotestosterone (DHT), androsterone, etiocholanolone, 5a-androstanediol, 5b-androstanediol, dehydroepiandrosterone (DHEA), and 6-hydroxymelatoninsulfate. The data were collected between February and November of 2015 and informed consent was obtained from all participants.\u003c/p\u003e \u003cp\u003eThe second analysis included 20 individuals whose deidentified data was pulled from the larger databank of 144,561 laboratory visits. Each of these samples included a single first-morning urine collection to compare results of dried versus liquid urine for the following organic acids: homovanillic acid (HVA), vanillylmandelic acid (VMA), kynurenic acid, xanthurenic acid, methymalonic acid (MMA), pyroglutamic acid, 5-hydroxyindoleacetic acid (5-HIAA), and b-hydroxyisovaleric (Hiv) acid. All data reports were deidentified during extraction so the IRB determined that written informed consent could be waived for this study population. Both studies (Clinical Trials ID: NCT04305093) were approved by the National University of Natural Medicine Institutional Review Board.\u003c/p\u003e \u003cp\u003eAll volunteers in both study populations reported no medical problems and were not pregnant. Individuals were not excluded based on current or recent use of any hormonal medications, as the goal was only to compare measurement values for differing methodologies. Eighty percent of women in the first study population and all women in the second study population were premenopausal.\u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eSample Collection\u003c/h2\u003e\n \u003cp\u003eThe 4-spot method involves urine samples collected at home at four times during the day: 1) the first urine of the day, 2) two hours after awakening, 3) in the afternoon (approximately 4 PM), and 4) before bed (10PM). Participants collected samples by completely saturating 2\u0026thinsp;\u0026times;\u0026thinsp;3 inches of filter paper (Whatman Body Fluid Collection Paper) with urine. The paper was left exposed at room temperature for 24 hours to dry. The stability of analytes in dried urine at room temperature for as long as 84 days has previously been demonstrated by this laboratory\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Dried samples were stored at -80\u0026deg;C until analyzed. Reproductive hormones were assessed in all four samples collected, while only the first morning sample was used for 6-hydroxymelatoninsulfate and the organic acid tests.\u003c/p\u003e \u003cp\u003eDuring the same day, all liquid urine samples for the 24-hour collection were added to a low-density polyethylene plastic container (ES Robbins, USA) container with approximately 1\u0026nbsp;g of boric acid and kept refrigerated for the duration of the collection. The four dried urine samples removed a total of about 8\u0026nbsp;mL of urine from the 24-hour collection. This was considered negligible and was not accounted for. The total volume of urine from 24-hour collections was measured, and an aliquot was frozen and stored at -80\u0026deg;C until tested.\u003c/p\u003e \n\u003ch2\u003eUrine Reproductive Hormone Analysis\u003c/h2\u003e\n \u003cp\u003eThe urinary steroid hormones were analyzed using proprietary in-house CLIA (Clinical Laboratory Improvement Amendments) approved assays on the Agilent 7890/7000B GC-MS/MS (Agilent Technologies, Santa Clara, CA, USA). A 600\u0026nbsp;ul aliquot of liquid urine was taken from the sample collection and the equivalent of approximately 600\u0026nbsp;ul of urine was extracted from the filter paper using 2\u0026nbsp;mL of 100\u0026nbsp;mM ammonium acetate adjusted to a pH of 5.9. These aliquots of the conjugated hormones were transferred to a C18 solid phase extraction (SPE) column (UCT LLC, Briston, PA, USA), eluted using methanol, and the eluate was dried under nitrogen at 40\u0026nbsp;\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe conjugated hormones were then hydrolyzed from their glucuronide and sulfate forms to free forms using enzymes from \u003cem\u003eHelix pomatia\u003c/em\u003e (Sigma-Aldrich, St. Louis, MO, USA) in acetate buffer (55\u0026nbsp;\u0026deg;C, 90 minutes). The enzymatic reaction was quenched with sodium hydroxide and the hormones extracted with ethyl acetate. The ethyl acetate extracts were dried under nitrogen at 40\u0026nbsp;\u0026deg;C. The analytes were derivatized using a mixture of 100\u0026nbsp;ul acetonitrile (ACN) and 50\u0026nbsp;ul bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 70\u0026nbsp;\u0026deg;C. Internal standards (Steraloids, Newport, RI, USA) were added prior to ethyl acetate extraction, and the percentage recovery from all assays was greater than 90%. Derivatized extract (1.6\u0026nbsp;ul) was injected into the GC-MS/MS. Samples and controls were analyzed along with a standard curve spanning the expected range of concentrations. Instrument conditions for the oven were an initial temperature of 130\u0026nbsp;\u0026deg;C increasing to 200\u0026nbsp;\u0026deg;C at 25\u0026nbsp;\u0026deg;C /min, then to 230\u0026nbsp;\u0026deg;C at 4.3\u0026nbsp;\u0026deg;C/min, and finally to 290\u0026nbsp;\u0026deg;C at 25\u0026nbsp;\u0026deg;C /min. Multiple reaction monitoring transitions for ion mass\u0026thinsp;\u0026gt;\u0026thinsp;ion product of fragmentation were 342.1\u0026thinsp;\u0026gt;\u0026thinsp;257.1 for E1, 416.2\u0026thinsp;\u0026gt;\u0026thinsp;285.1 for E2, 504.3\u0026thinsp;\u0026gt;\u0026thinsp;296.3 for E3, 430.2\u0026thinsp;\u0026gt;\u0026thinsp;345.0 for 2OHE1, 462.2.2\u0026thinsp;\u0026gt;\u0026thinsp;195.2 for 2OHE2, 430.2\u0026thinsp;\u0026gt;\u0026thinsp;354.0 for 4OHE1, 430.2\u0026thinsp;\u0026gt;\u0026thinsp;286.1 for 16OHE1, 372.2-342.1 for 2-methoxyE1, 360.2\u0026thinsp;\u0026gt;\u0026thinsp;174.1 for T, 360.2\u0026thinsp;\u0026gt;\u0026thinsp;174.1 for EpiT, 347.2\u0026thinsp;\u0026gt;\u0026thinsp;271.2 for DHT, 347.2\u0026thinsp;\u0026gt;\u0026thinsp;271.2 for androsterone, 347.2\u0026thinsp;\u0026gt;\u0026thinsp;253 for etiocholanolone, 421.2\u0026thinsp;\u0026gt;\u0026thinsp;331.2 for 5α-androstanediol, 241.2\u0026thinsp;\u0026gt;\u0026thinsp;185.2 for 5β-androstanediol, and 303.7\u0026thinsp;\u0026gt;\u0026thinsp;199.1 for DHEA.\u003c/p\u003e \u003cp\u003eCreatinine was measured using a conventional colorimetric (Jaffe) method, after initial extraction from the filter paper. The average inter-assay coefficient of variation was 6.7% for creatinine. In addition to expressing the measures per mg of creatinine to correct for variations in filter paper saturation and hydration status, a secondary equation was applied to reduce bias related to the effects of age, sex, weight, and height on creatinine excretion\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \n\u003ch2\u003eUrine 6-hydroxymelatoninsulfate And Organic Acid Analysis\u003c/h2\u003e\n \u003cp\u003eThe hydrophilic analytes were assessed by LC-MS/MS using proprietary in-house CLIA approved assays. For the 6-hydroxymelatoninsulfate assay, a 30\u0026nbsp;ul aliquot was taken from the methanol elution of both the liquid urine collection and the waking sample dried urine collected on filter paper. This extract was then reconstituted in 130\u0026nbsp;ul of deionized water. For the organic acids, a 100\u0026nbsp;ul aliquot of liquid urine was taken and an equivalent amount was extracted from the waking sample dried urine filter paper using 250\u0026nbsp;ul of water with the addition of 50\u0026nbsp;ul 100\u0026nbsp;mM ammonium acetate (Sigma-Aldrich, St. Louis, MO, USA) adjusted to a pH of 5.9 and 2% formic acid.\u003c/p\u003e \u003cp\u003eFor 6-hydroxymelatoninsulfate, 20\u0026nbsp;ul was injected into an ultra-performance liquid chromatography (UPLC) (Waters Corporation, Milford, MA, USA) column with a Waters\u0026trade; tandem quadrupole mass spectrometer detector (TQD). The sample was eluted from a 1.8u 2.1\u0026thinsp;\u0026times;\u0026thinsp;50\u0026nbsp;mm pentafluorophenyl (PFP) column (Agilent Technologies, Santa Clara, CA, USA) using a gradient of 95% 0.001% formic acid in 5% ACN to 45% 0.001% formic acid in 55% ACN. For the organic acids, 5\u0026nbsp;ul was injected into the Waters\u0026trade; UPLC column with TQD. These analytes were eluted from a 1.6 um 2.1\u0026thinsp;\u0026times;\u0026thinsp;50\u0026nbsp;mm Luna Omega PS C18 column (Phenomenex, Torrance, CA, USA) using a gradient 99.9% 0.2% formic acid in 0.1% ACN to 73% 0.2% formic acid in 27% ACN. Multiple reaction monitoring transitions for ion mass\u0026thinsp;\u0026gt;\u0026thinsp;ion product of fragmentation were 372.5\u0026thinsp;\u0026gt;\u0026thinsp;176.2 for 6-hydroxymelatoninsulfate, 331.5\u0026thinsp;\u0026gt;\u0026thinsp;179.2 for d4-6-hydroxymelatoninsulfate, 183.0\u0026thinsp;\u0026gt;\u0026thinsp;137.0 for HVA, 185.0\u0026thinsp;\u0026gt;\u0026thinsp;139.0 for d2-HVA, 197.0\u0026thinsp;\u0026gt;\u0026thinsp;138.0 for VMA, 199.8\u0026thinsp;\u0026gt;\u0026thinsp;140.9 for d3-VMA, 190.0\u0026thinsp;\u0026gt;\u0026thinsp;144.0 for kynurenic acid, 192.0\u0026thinsp;\u0026gt;\u0026thinsp;149.0 for d5-kynurenic acid, 206.0\u0026thinsp;\u0026gt;\u0026thinsp;160.0 for xanthurenic acid, 210.0\u0026thinsp;\u0026gt;\u0026thinsp;164.0 for d3-xanthurenic acid, 117.0\u0026thinsp;\u0026gt;\u0026thinsp;73.0 for MMA, 120.0\u0026thinsp;\u0026gt;\u0026thinsp;76.0 for d2-MMA, 130.0\u0026thinsp;\u0026gt;\u0026thinsp;84.0 for pyroglutamic acid, 135.0\u0026thinsp;\u0026gt;\u0026thinsp;89.0 for d5-pyroglutamic acid, and 192.0\u0026thinsp;\u0026gt;\u0026thinsp;146.0 for 5-HIAA. The same creatinine corrections of the measures used for the reproductive hormones were also used for 6-hydroxymelatoninsulfate and the organic acids.\u003c/p\u003e \n\u003ch2\u003eStatistical Methods\u003c/h2\u003e\n \u003cp\u003eA sample size of 18 individuals provides a power of greater than 80% to detect an intraclass correlation coefficient (ICC) of at least 0.6 with an alpha of 0.05\u003csup\u003e56\u003c/sup\u003e. The statistical analyses were performed using SAS/STAT\u0026reg; software, Version 9.3 (SAS Institute Inc., Cary, NC, USA) and generated 2-sided p-values.\u003c/p\u003e \u003cp\u003eVariables are described as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation if normally distributed and median (interquartile range (IQR)) if the distribution was skewed. Student\u0026rsquo;s t-test (for normally distributed variables) or the Wilcoxon rank-sum test (for skewed variables) were used to determine differences between men and women. Spearman correlation coefficients (ρ) were used to determine \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003einter\u003c/span\u003eclass associations between variables.\u003c/p\u003e \u003cp\u003eConsistency between liquid versus dried urine measures and 4-spot versus 24-h collection methodology was assessed using \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eintra\u003c/span\u003eclass correlation coefficients (ICC). ICCs, which range from 0 to 1 with proximity to 1 indicating better agreement, assess for agreement of a measure between two differing methodologies within individuals\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Skewed variables were log transformed to approximate a normal distribution prior to assessing ICCs. As 4-spot (ng/mg-Cr) and 24-h (ug/d) measures were expressed in differing units, sex-specific Z-scores ([individual measurement-mean]/standard deviation) were created to standardize the measures and allow for direct comparison. Comparisons of differences between measures within an individual were assessed using signed-rank tests (for skewed variables) or paired t-tests (for normally distributed variables). Because the hypotheses of this paper were intrinsically correlated, no adjustments were made for multiple comparisons.\u003c/p\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eStudy populations\u003c/h2\u003e\n\u003cp\u003eCharacteristics of the first study population (n\u0026thinsp;=\u0026thinsp;26) are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. All of these individuals (58% female; 100% Caucasian) had data available for comparison of 4-spot versus 24-h urine samples for male and female reproductive hormones. A subset (10 female, 8 male) also had measurements to compare liquid versus dried urine samples for both the reproductive hormones and 6-hydroxymelatoninsulfate. Characteristics of the second study population (n\u0026thinsp;=\u0026thinsp;20; 75% female; 80% Caucasian / 10% Hispanic / 10% Asian-Pacific Islander), data from whom were used to compare a single first morning collection of liquid versus dried urine samples for the organic acid tests, are provided in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAge and 24-h measures of reproductive hormones and 6-hydroxymelatoninsulfate of the first study population.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVariable\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAll\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFemale (n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMale (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36.8\u0026thinsp;\u0026plusmn;\u0026thinsp;14.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37.8\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstrone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.69 (6.32, 19.16)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.75 (6.83, 23.68)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.67 (6.26, 13.68)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstradiol (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.73 (0.80, 3.26)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.17 (0.89, 4.19)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.14 (0.70, 1.62)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstriol (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.82 (3.57, 12.80)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.64 (6.14, 16.55)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.56 (3.45, 6.91)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyestrone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.33 (1.27, 6.24)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.82 (0.77, 8.46)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.26 (1.27, 4.03)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyestradiol (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25 (0.11, 0.67)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.58 (0.06, 0.81)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.19 (0.11, 0.26)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e4-hydroxyestrone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.37 (0.19, 0.75)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.67 (0.19, 0.99)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.30 (0.16, 0.36)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e16-hydroxyestrone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.89 (0.42, 1.48)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.40 (0.36, 1.58)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.49 (0.42, 0.90)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-methoxyestrone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.27 (1.30, 3.80)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.56 (1.38, 5.50)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.63 (1.05, 2.35)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTestosterone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.15 (4.97, 51.92)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.43 (4.51, 8.01)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e61.40 (42.14, 97.70)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEpitestosterone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.23 (4.01, 11.23)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.44 (1.18, 7.49)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.94 (7.99, 16.80)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5a-Dihydrotestosterone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.53 (2.72, 11.59)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.72 (1.33, 5.46)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.60 (9.88, 17.47)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAndrosterone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1331.99 (854.41, 1940.29)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1032.77 (691.70, 1528.59)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2083.52 (1005.51, 2716.33)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEtiocholanolone (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e811.74 (463.17, 1045.23)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e568.56 (430.73, 853.81)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1020.79 (716.22, 1423.92)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5a-Androstanediol (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e102.50 (54.83, 197.14)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.72 (46.49, 97.50)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e229.01 (143.30, 292.53)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5b-Androstanediol (ug/d)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35.58 (14.05, 60.75)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.83 (9.38, 52.69)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60.75 (20.69, 122.18)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDHEA (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e166.14 (80.45, 431.61)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e136.42 (80.45, 317.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e351.32 (52.69, 732.63)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e6-Hydroxymelatoninsulfate (ug/ml)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.87 (5.93, 13.85)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.48 (5.93, 8.43)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.21 (5.11, 20.06)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003eAge presented as mean\u0026plusmn; SD; hormone data presented as median (IQR) as these data were skewed.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003csup\u003eb\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for differences between males and females as assessed by the Wilcoxon rank-sum test.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003eDHEA\u0026thinsp;=\u0026thinsp;dehydroepiandrosterone\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAge and measures of organic acids from an early morning spot urine collection of the second study population.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVariable\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAll\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFemale (n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMale (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMethylmalonic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.18 (0.16, 0.25)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.20 (0.16, 0.29)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.16 (0.16, 0.18)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHomovanillic Acid (ng/mg-Cr)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVanillylmandelic acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eKynurenic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eXanthurenic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.06 (0.05, 0.07)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.06 (0.04, 0.07)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.06 (0.05, 0.07)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5-HIAA (ng/mg-Cr)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.44 (0.40, 0.55)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.47 (0.41, 0.58)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.37 (0.36, 0.41)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePyroglutamic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eb-Hydroxyisovaleric Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD if normally distributed and median (IQR) if right-skewed.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003csup\u003eb\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for differences between males and females as assessed by the Student t-test or Wilcoxon rank-sum test.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e5-HIAA\u0026thinsp;=\u0026thinsp;5-hydroxyindoleacetic acid\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch2\u003eAgreement Between Liquid And Dried Urine Measures\u003c/h2\u003e\n\u003cp\u003eFor the majority of analytes, there was excellent agreement (ICC\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;0.90) between the liquid and dried measures (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The exceptions were VMA (ICC\u0026thinsp;=\u0026thinsp;0.79) and pyroglutamic acid (ICC\u0026thinsp;=\u0026thinsp;0.75), which still had good agreement. Similarly, for the majority of analytes, there was no systemic directionality to the difference in the dried urine compared to the liquid urine. However, estrone and estriol were consistently higher when measured in liquid urine, while some of the organic acids \u0026ndash; VMA, kynurenic acid, pyroglutamic acid, and b-hydroxyisovaleric acid \u0026ndash; were more concentrated in the dried urine sample (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Representative interclass correlations (Spearman) between the two methods are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (the rest are available in Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of liquid versus dried urine analytes (n\u0026thinsp;=\u0026thinsp;18).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr style=\"height: 59px;\"\u003e\n\u003cth style=\"height: 59px;\" align=\"left\"\u003e\n\u003cp\u003eVariable\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px;\" align=\"left\"\u003e\n\u003cp\u003eDried\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px;\" align=\"left\"\u003e\n\u003cp\u003eLiquid\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px;\" align=\"left\"\u003e\n\u003cp\u003eDifference\u003c/p\u003e\n\u003cp\u003e[95% CI]\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"height: 59px;\" align=\"left\"\u003e\n\u003cp\u003eICC [95% CI]\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHormones (n\u0026thinsp;=\u0026thinsp;18)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstrone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e10.78 (6.26, 18.73)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e12.78 (7.22, 20.65)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e1.28 [0.09, 2.47]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.92 [0.80, 0.97]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstradiol (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.32 (0.70, 3.26)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.29 (0.74, 3.36)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.09 [-0.01, 0.19]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.96 [0.89, 0.98]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstriol (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e6.82 (3.45, 11.64)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e7.15 (4.76, 14.41)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.86 [0.05, 1.67]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.90 [0.77, 0.96]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyestrone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e2.22 (0.98, 6.24)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.84 (1.17, 6.24)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e-0.01 [-0.18, 0.16]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.98 [0.94, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyestradiol (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.18 (0.09, 0.58)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.18 (0.08, 0.47)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.00 [-0.02, 0.03]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.97 [0.92, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e4-hydroxyestrone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.31 (0.16, 0.67)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.40 (0.25, 0.62)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.03 [-0.02, 0.09]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.95 [0.87, 0.98]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e16-hydroxyestrone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.69 (0.42, 1.48)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.61 (0.39, 1.24)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e-0.07 [-0.18, 0.05]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.95 [0.87, 0.98]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-methoxyestrone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.64 (1.05, 3.56)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.70 (1.50, 3.58)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.12 [-0.07, 0.30]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.97 [0.94, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTestosterone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e7.64 (4.97, 49.46)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e8.42 (5.80, 51.40)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e2.07 [-0.26, 4.40]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.99 [0.98, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEpitestosterone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e7.23 (4.01, 11.23)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e8.21 (4.23, 13.98)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.51 [-0.06, 1.07]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.99 [0.97, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5a-Dihydrotestosterone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e5.53 (2.72, 11.59)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e4.94 (2.68, 10.35)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e-0.61 [-1.02, -0.19]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.99 [0.97, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAndrosterone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1331.99 (929.43, 1933.30)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1237.63 (813.31, 1847.10)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e-33.02 [-105.70, 39.67]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.99 [0.98, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEtiocholanolone (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e847.93 (430.73, 1088.18)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e943.49 (414.13, 1161.61)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e19.76 [-13.02, 52.54]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.99 [0.98, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5a-Androstanediol (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e110.09 (57.50, 197.14)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e119.40 (65.63, 226.93)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e15.67 [-12.67, 44.01]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.90 [0.76, 0.96]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5b-Androstanediol (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e35.58 (14.05, 72.42)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e46.39 (14.72, 65.39)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e-0.54 [-13.93, 12.85]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.88 [0.73, 0.95]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDHEA (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e131.42 (52.69, 444.35)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e130.86 (53.27, 457.05)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e8.65 [-36.66, 53.96]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e1.00 [0.99, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e6-Hydroxymelatoninsulfate (ug/d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e7.87 (5.93, 14.33)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e8.26 (5.93, 14.67)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.04 [-1.14, 1.21]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.99 [0.98, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eOrganic Acids (n\u0026thinsp;=\u0026thinsp;20)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMethylmalonic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.18 (0.15, 0.23)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.18 (0.16, 0.25)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.00 [-0.01, 0.02]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.96 [0.90, 0.98]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHomovanillic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e-0.01 [-0.05, 0.02]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.88 [0.74, 0.95]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eVanillylmandelic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e-0.05 [-0.08, -0.02]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.79 [0.57, 0.91]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eKynurenic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e-0.01 [-0.02, -0.00]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.95 [0.88, 0.98]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eXanthurenic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.06 (0.05, 0.08)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.06 (0.05, 0.07)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e-0.00 [-0.00, 0.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.98 [0.95, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5-HIAA (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.46 (0.43, 0.57)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.44 (0.40, 0.55)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e-0.01 [-0.05, 0.02]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n\u003cp\u003e0.98 [0.96, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003ePyroglutamic Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e4.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e-0.41 [-0.58, -0.23]\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.75 [0.51, 0.89]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eb-Hydroxyisovaleric Acid (ng/mg-Cr)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e-0.02 [-0.03, -0.00]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" align=\"left\"\u003e\n\u003cp\u003e0.95 [0.88, 0.98]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr style=\"height: 26px;\"\u003e\n\u003ctd style=\"height: 26px;\" colspan=\"5\"\u003eFor liquid and dried measurements, data presented as mean\u0026plusmn;SD for normally distributed variables and median (IQR) for skewed variables; the differences were normally distributed and are presented as mean [95% CI].\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 15px;\"\u003e\n\u003ctd style=\"height: 15px;\" colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003ep\u0026lt;0.05; \u003csup\u003eb\u003c/sup\u003ep\u0026lt;0.0001 for the liquid minus dried difference by paired t-test\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 13px;\"\u003e\n\u003ctd style=\"height: 13px;\" colspan=\"5\"\u003e5-HIAA\u0026thinsp;=\u0026thinsp;5-hydroxyindoleacetic acid, Cr\u0026thinsp;=\u0026thinsp;creatinine, DHEA\u0026thinsp;=\u0026thinsp;dehydroepiandrosterone, ICC\u0026thinsp;=\u0026thinsp;intraclass correlation coefficient\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;Agreement between the DUTCH 4-spot and 24-hour urine collection for hormonal measures\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe measurement of reproductive hormones in urine samples collected four times throughout the day were comparable to the gold standard of a 24-h urine collection (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). As the measures from the 4-spot urine collection are reported in ng/mg-Cr and the 24-h urine measures are reported in ug/d, sex-specific Z-scores were created for direct comparison of the two methodologies. There was excellent consistency (ICC\u0026thinsp;\u0026gt;\u0026thinsp;0.9) for the majority of the analytes and good consistency for the remainder (estriol, 5a and 5b-androstanediol) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). There was no systematic directionality to the differences between any of the Z-scores (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Representative interclass (Spearman) correlations between the analytes are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026nbsp;(the remainder are shown in Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\n\u003cp\u003eA sensitivity analysis to verify the need for creatinine correction was done by calculating ICCs for the agreement between sex-specific Z-scores from the 24-h measures and the 4-spot assay without correction for creatinine. Without the creatinine correction, the ICCs were all lower than those observed with the creatinine correction (with the exception of estriol which had a slightly higher ICC by 0.04) with the degree of difference ranging from \u0026minus;\u0026thinsp;0.04 to 0.15 and averaging 0.07. An example of the interclass correlations (Spearman) between the 24-h measures and the 4-spot assay with and without the creatinine corrections is shown in Supplementary Fig.\u0026nbsp;4.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of the urinary hormone profile using the 4-spot (DUTCH) or 24-h urine collection method (n\u0026thinsp;=\u0026thinsp;26).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eVariable\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e24-h urine collection (ug/d)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e4-spot (ng/mg-Cr)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eZ-score difference [95% CI]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eICC [95% CI]\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstrone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13.69 (6.32, 19.16)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.37 (7.13, 18.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e0.00 [-0.13, 0.13]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.95 [0.89, 0.97]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstradiol\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.73 (0.80, 3.26)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.68 (0.92, 3.81)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e0.00 [-0.16, 0.16]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.92 [0.83, 0.96]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEstriol\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.82 (3.57, 12.80)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.39 (4.15, 12.96)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e0.00 [-0.24, 0.24]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.82 [0.64, 0.91]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyestrone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.33 (1.27, 6.24)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.72 (1.45, 6.75)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.09, 0.09]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.97 [0.94, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-hydroxyestradiol\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.25 (0.11, 0.67)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.30 (0.13, 0.49)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.10, 0.10]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.97 [0.93, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e4-hydroxyestrone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.37 (0.19, 0.75)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.45 (0.27, 0.81)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.15, 0.15]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.93 [0.86, 0.97]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e16-hydroxyestrone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.89 (0.42, 1.48)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.93 (0.57, 1.69)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e0.00 [-0.19, 0.19]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.90 [0.77, 0.95]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2-methoxyestrone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.27 (1.30, 3.80)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.11 (1.54, 4.15)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e0.00 [-0.19, 0.19]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.90 [0.78, 0.95]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTestosterone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.15 (4.97, 51.92)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.33 (5.36, 54.73)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.10, 0.10]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.97 [0.93, 0.98]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEpitestosterone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.23 (4.01, 11.23)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.53 (3.81, 13.90)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.07, 0.07]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.99 [0.97, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5a-Dihydrotestosterone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.53 (2.72, 11.59)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.40 (2.16, 11.91)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.09, 0.09]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.97 [0.94, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAndrosterone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1331.99 (854.41, 1940.29)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1346.46 (818.59, 2065.66)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.09, 0.09]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.97 [0.94, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eEtiocholanolone\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e811.74 (463.17, 1045.23)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e797.60 (475.43, 1061.66)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.09, 0.09]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.98 [0.95, 0.99]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5a-Androstanediol\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.50 (54.83, 197.14)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e86.74 (51.31, 200.70)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.27, 0.27]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.78 [0.57, 0.89]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5b-Androstanediol\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.58 (14.05, 60.75)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34.49 (13.91, 62.77)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e0.00 [-0.22, 0.22]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.85 [0.70, 0.93]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDHEA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e166.14 (80.45, 431.61)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e162.85 (78.17, 394.62)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026minus;\"\u003e\n\u003cp\u003e-0.00 [-0.05, 0.05]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.99 [0.99, 1.00]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe ICCs were calculated between measurements expressed as standardized sex-specific Z-scores (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for all =\u0026thinsp;0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98). Data are presented as median (IQR) for measurements and the difference (24-h minus 4-spot) in individual Z-scores is presented as mean [95% confidence interval].\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eall p-values\u0026thinsp;\u0026gt;\u0026thinsp;0.9 by paired t-test\u003c/p\u003e\n\u003cp\u003eCr\u0026thinsp;=\u0026thinsp;creatinine, DHEA\u0026thinsp;=\u0026thinsp;dehydroepiandrosterone, ICC\u0026thinsp;=\u0026thinsp;intraclass correlation coefficient\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eThis study demonstrated the feasibility of accurately measuring multiple (up to 32) analytes in dried urine samples collected on filter paper using assays that conform to CLIA criteria. All measurements from dried urine demonstrated at least good agreement with measures from liquid urine, and the majority (83%) demonstrated excellent agreement with intraclass correlations greater than 0.9. For most analytes, neither loss nor excess concentration occurred during the sample drying or laboratory extraction process. In addition, measurement of the reproductive steroid hormones, which are usually evaluated from a 24-hr collection due to their pulsatile release\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, were well represented by the 4-spot dried urine collection with at least good agreement of the 4-spot measurement with the 24-h gold standard measure for all steroid metabolites and excellent agreement (\u0026gt;\u0026thinsp;0.9) for the majority (82%). There were no systematic differences between the relative amount of hormone collected by either methodology. The 4-spot dried urine (DUTCH) methodology allows for efficient, accurate assessment of numerous urine metabolites using a convenient collection method, while avoiding the need for a full 24-hr liquid urine collection.\u003c/p\u003e \u003cp\u003eThe 4-spot dried urine collection has previously been shown to be representative of select 24-h measures of steroidal hormones by our group\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, as measurement of urinary a-pregnanediol, b-pregnanediol, estrone, and estradiol with this method are representative of both 24-h urine collections as well as serum hormone concentrations. In fact, repeated assessments over a month demonstrated that the dried urine collections could accurately recreate the changes observed in serum during the menstrual cycle\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In addition, not only does the 4-spot urine method accurately represent a 24-h urine collection for urinary cortisol, cortisone, and cortisol metabolites, but it can also be used to represent the expected diurnal pattern observed with salivary measures, if each of the four collections is considered individually\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have validated the usefulness of high throughput GC-MS/MS for urine steroid profiling of more than 30 metabolites; however, this was done in liquid urine\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Conversely, measurement of organic acids in spot dried urine samples using filter paper has already been well-validated as a technique for screening neonates for metabolic disorders\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e and for screening for neuroblastoma\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e with similar recoveries obtained from liquid and dried urine. Dried urine has also been used to measure other urine analytes of interest, such as sodium and potassium, with similarly high levels of stability\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. As with others who used either filter paper or cotton swabs\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, we found that dried urine results are in agreement with liquid urine results, reduced the burden on patients, and had good stability over time\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Still, agreement between 24-h urine collections and early-morning, single spot urine collections for hormonal analysis are often poor\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. This study now extends our prior findings\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e to show that the increase to four spot urines spaced throughout the waking hours provides better coverage of the hormonal output for all the male and female reproductive hormones and metabolites, resulting in strong agreement with 24-h urine measures.\u003c/p\u003e \u003cp\u003eThere are some caveats that must be considered when interpreting our results. During the urine collection, there may have been differences in saturation of the filter paper. Expressing the analyte concentration per mg of creatinine is designed to address this, while also correcting for hydration. The method used for creatinine adjustment also accounts for differences in creatinine excretion related to body size. This does rely on accurate self-reporting of age, height, and weight by the participants, so an estimate of expected creatinine excretion can be made. This may lead to the introduction of inaccuracies due to misreporting of individual characteristics; however, our statistical sensitivity analyses indicated that the DUTCH measurements were in better agreement with the 24-h urine results with the application of the creatinine corrections (see Supplementary Fig.\u0026nbsp;4), with the ICC for some metabolites increasing by as much as 0.15 and raising the level of agreement with 24-h measures from good to excellent.\u003c/p\u003e \u003cp\u003eOne of the limitations of this 4-spot dried urine method is that reference ranges are laboratory-specific and non-standardized. Still, interpretation of values above and below these reference ranges should be similar to that of other assays. In this study, there was some loss of hormone for estrone and estriol with the filter paper methodology, which may be related to differences in extraction efficiency between the steroid conjugates and creatinine, loss during the drying process, or incomplete saturation of the filter paper. Still, the difference was less than 12% of the total and would be compensated for by an adjustment of the reference range. In addition, we have previously shown that the dried urine measure of estrone has clinical utility because it is representative of serum estrogen measurements\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. A number of organic acids plus DHT were more concentrated in the dried urine samples, on average. This may be due to differences in extraction efficiency, a matrix effect or analyte concentration during the drying process; however, this difference did represent less than 10% of the sample. Fortunately, due to the high level of agreement between the Z-scores from the DUTCH methodology and 24-h collections, laboratory reference ranges should account for these differences. Another issue is there are known genetic differences in glucuronidation of testosterone that may impact relative metabolism and urinary concentrations of testosterone and epitestosterone\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, and this may mean urinary androgen measures are not fully representative of production rates in a small percentage of individuals. There are also genetic differences in the enzymes that metabolize estrogens, potentially shifting the ratio of 2-hyroxylation to 16-hydroxylation metabolites\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, but these differences may be clinically relevant and indicative of cancer risk\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe methodology of a 4-spot urine collection on filter paper followed by GC-MS/MS or LC-MS/MS offers some advantages. The collection of dried urine on filter paper results in stable measurement of steroid hormones for extended periods of time both by us\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and others\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e for up to one year\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, even at ambient temperature. Concentrations of organic acids are also stable on dried filter paper for weeks\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Mass spectrometry assays, which are now the gold standard for measurement of steroid hormones in blood and urine\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, allow for the use of small sample volumes with excellent sensitivity and accuracy along with simultaneous measurement of a relatively large number of analytes. In combination with chromatography, either gas or liquid, it provides precise separation of closely related molecules\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e by their chemical and physical properties. GC-MS/MS does not exclude any lipophilic steroids, and so a run will contain all excreted steroids\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The use of the \u003cem\u003eH. pomatia\u003c/em\u003e enzymes adds to the accuracy of the quantification of the hormone conjugates, as these enzymes include both a sulfatase and a glucuronidase. The method of GC-MS/MS does require an additional extraction and derivatization step, but workflows can be optimized to maximize throughput. A 24-h urine collection may be difficult for some patients to fully collect, especially if they are not able to remain at home for an entire day, are disabled, or are incontinent. This methodology removes that barrier and provides the ability to measure multiple hormones at once with a noninvasive collection method, obtaining a complete picture of both production and clearance of the major steroidal hormones.\u003c/p\u003e \u003cp\u003eA multitude of uses, both in research and in clinical scenarios, could be envisioned for assays that are able to measure multiple steroid hormones and organic acids in conveniently collected urine samples on filter paper. For example, the full range of hormonal changes in individuals related to disruption of the natural sleep cycle could be evaluated simultaneously. It is already known that the peak 6-sulfatoxymelatonin, as representative of melatonin, is lower in people working the night shift\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e; a full appreciation of the urinary steroid profile in individuals who work at night could add to this prior research. Similarly, urine profiling may help to fully define the changes expected in genetic syndromes of steroidogenesis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e and errors of metabolism\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Dried urine samples may be of particular benefit in screening neonates for organic acid disorders\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and there is recent interest in a possible association of organic acids with neuropsychiatric disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. The ability to look at a full urine profile can provide a more integrated view of the patient; for example, patients using oral contraceptives often have higher xanthurenic acid with concurrent pyroxidine deficiency\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, both of which would be observable using dried urine analysis. A greater understanding of the full effect of changes in hormonal concentrations and metabolites or important clinical subgroups could be determined for both exogenous use of hormones and for exposure to endocrine disrupting compounds like bisphenol A\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Urine hormone profiling might also be used to fully describe age related changes, i.e. through puberty or menopause.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eMass spectrometry allows for the assessment of a full hormone profile in a small volume of urine such that an expanded view of both hormone production and clearance can be observed. In addition, results from dried urine are in strong agreement with those obtained from liquid urine. In combination with four spot urine collections on filter paper collected throughout the waking hours, we have shown that it is possible to accurately represent a 24-h urine collection. This technology may be useful to the clinician wishing to perform a large series of tests on patients to narrow the differential diagnosis, for those monitoring hormonal therapy or evaluating the menstrual cycle, or for those who need to reduce the burden of collection for their patients. This four-spot, dried urine method allows for assessment of both diurnal patterns\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e as well as total daily production, allowing for a comprehensive evaluation of adrenal and reproductive hormones and other urine metabolites.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e2OHE1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-hydroxyestrone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e2-methoxyE1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-methoxyestrone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e4OHE1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4-hydroxyestrone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e5-HIAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e5-hydroxyindoleacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e16OHE1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e16-hydroxyestrone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacetonitrile\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLIA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClinical Laboratory Improvement Amendments\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCr\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecreatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDHEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edehydroepiandrosterone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDHT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e5α-dihydrotestosterone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDUTCH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDried Urine Testing for Comprehensive Hormones\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eE1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eestrone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eE2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eestradiol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eE3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eestriol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEpiT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eepitestosterone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC-MS/MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egas chromatography with tandem mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHiv\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eβ-hydroxyisovaleric\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomovanillic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintraclass correlation coefficient\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIQR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterquartile range\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC-MS/MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eliquid chromatography with tandem mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethylmalonic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etestosterone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTQD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etandem quadrupole mass spectrometer detector\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evanillylmandelic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was conducted on healthy adult volunteers at a commercial testing laboratory. Informed consent was obtained from all participants prior to the study or the IRB determined that written informed consent could be waived for the study population. All methods were performed in accordance with the relevant guidelines and regulations required by both the National University of Natural Medicine Institutional Review Board and CLIA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets acquired and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrecision Analytical, Inc. is a commercial laboratory offering hormone testing to medical practitioners and individuals. All statistics were calculated and interpreted by an independent agent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrecision Analytical Laboratory, Inc. provided funds to run urine and serum assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMN developed the methodology of the dried urine assay, designed the studies, interpreted the data, and made substantial contributions to the manuscript. DC acquired and interpreted the data and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge Danielle Martinot for writing the IRB proposal for this study and Marie Thearle, MD for creating tables and figures and assistance with statistical analysis and interpretation of the data. And thanks to the volunteers who provided samples for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMN, DC: Precision Analytical, Inc. 3138 NE Rivergate Street #301C, McMinnville, OR 97128 USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSansone A, Sansone M, Selleri R, et al. Monitoring testosterone replacement therapy with transdermal gel: when and how? \u003cem\u003eJournal of Endocrinological Investigation\u003c/em\u003e. 2019;42(12):1491\u0026ndash;1496. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s40618-019-01082-x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodgers M. Adequacy of hormone replacement therapy for osteoporosis prevention assessed by serum oestradiol measurement, and the degree of association with menopausal symptoms. \u003cem\u003eBritish Journal of General Practice\u003c/em\u003e. 1997;47(416):161\u0026ndash;165. Accessed October 11, 2020. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/9167320/\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Medeiros, Sebasti\u0026atilde;o Freitas, Barbosa, Jacklyne Silva, Yamamoto MMW. Comparison of steroidogenic pathways among normoandrogenic and hyperandrogenic polycystic ovary syndrome patients and normal cycling women. \u003cem\u003eJ Obstet Gynaecol Res\u003c/em\u003e. 2015;41(2):254\u0026ndash;263. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jog.12524\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKetha H, Kaur S, Grebe SK, Singh RJ. Clinical applications of LC-MS sex steroid assays: Evolution of methodologies in the 21st century. \u003cem\u003eCurrent Opinion in Endocrinology, Diabetes and Obesity\u003c/em\u003e. 2014;21(3):217\u0026ndash;226. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/MED.0000000000000068\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJong WHAD, Buitenwerf E, Pranger AT, et al. 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The aims of this study were, first, to confirm that mass spectrometric analysis of an array of hormones and organic acids would yield similar results in both liquid and dried urine, and, second, to determine if collection of four dried spot urine samples could be substituted for a 24-hour collection when measuring reproductive hormones. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTwo study populations were included in this prospective observational study. Twenty individuals collected both a spot liquid urine and dried urine on filter paper to analyze eight organic acids. A second group of 26 individuals collected both a 24-hour urine and four dried spot urines during waking hours throughout the same day for evaluation of 17 reproductive hormones and metabolites; data from 18 of these individuals were available to compare liquid versus dried urine results. Dried urine was extracted, hydrolyzed, and derivatized before analysis by mass spectrometry; all analytes from dried urine were normalized to urine creatinine.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eReproductive hormone results from dried and liquid urine were in excellent agreement with intraclass correlation coefficients (ICCs) greater than 0.90; comparison of dried to liquid urine for organic acids showed good to excellent agreement (ICC range: 0.75 to 0.99). Comparison between the 4-spot urine collection and 24-hour urine collection methods showed excellent agreement (ICC\u0026gt;0.9) for 14 of the 17 urine metabolites and good agreement for the others (ICC 0.78 to 0.85) with no systematic differences between the two methods of collection. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe burden of urine collection can be reduced using collection of four spot dried urines on filter paper without compromising comparability with hormone results from a 24-hour urine collection. A large number of urine analytes can be assessed from the dried urine with similar results to those from liquid urine. Given the ease of sample handling, this 4-spot dried urine assay would be useful for both clinical assessment of patients and for large epidemiologic studies.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Reliability of A Dried Urine Test for Comprehensive Assessment of Urine Hormones and Metabolites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-10 17:21:01","doi":"10.21203/rs.3.rs-122134/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-02-09T03:52:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-05T07:38:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f9aab76f-d545-4518-9caa-35b6f3bb3943","date":"2021-01-26T01:57:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-26T11:20:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c8c377fb-e40b-4593-9f40-2b0ad07df08c","date":"2020-12-20T14:09:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9421d5d6-4b68-42c8-9e0e-5f7ef5213a67","date":"2020-12-14T20:24:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-12-09T16:10:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-08T16:45:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-08T15:44:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-08T12:41:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Chemistry","date":"2020-12-04T21:10:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"05f209a0-a35e-4526-ac42-81b4aea244dc","owner":[],"postedDate":"December 10th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1413838,"name":"Biological Chemistry"}],"tags":[],"updatedAt":"2021-03-27T15:01:00+00:00","versionOfRecord":{"articleIdentity":"rs-122134","link":"https://doi.org/10.1186/s13065-021-00744-3","journal":{"identity":"bmc-chemistry","isVorOnly":false,"title":"BMC Chemistry"},"publishedOn":"2021-03-15 15:00:33","publishedOnDateReadable":"March 15th, 2021"},"versionCreatedAt":"2020-12-10 17:21:01","video":"","vorDoi":"10.1186/s13065-021-00744-3","vorDoiUrl":"https://doi.org/10.1186/s13065-021-00744-3","workflowStages":[]},"version":"v1","identity":"rs-122134","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-122134","identity":"rs-122134","version":["v1"]},"buildId":"oE6Zbj460LM0Up2FdVbMZ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}