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Glucuronidation, a crucial process in phase II metabolism, plays a vital role in the detoxification and elimination of endogenous substances and xenobiotics. A comprehensive and confident profiling of glucuronate-conjugated metabolites is imperative to understanding their roles in physiological and pathological processes. In this study, a chemical isotope labeling and dual-filtering strategy was developed for global profiling of glucuronide metabolites in biological samples. N , N -Dimethyl ethylenediamine (DMED- d 0 ) and its deuterated counterpart DMED- d 6 were used to label carboxylic acids through an amidation reaction. First, carboxyl-containing compounds were extracted based on a characteristic mass difference (Δ m / z , 6.037 Da) observed in MS between light- and heavy-labeled metabolites (filter I). Subsequently, within the pool of carboxyl-containing compounds, glucuronides were identified using two pairs of diagnostic ions ( m / z 247.1294/253.1665 and 229.1188/235.1559 for DMED- d 0 /DMED- d 6 -labeled glucuronides) originating from the fragmentation of the derivatized glucuronic acid group in MS/MS (filter II). Compared with non-derivatization, DEMD labeling significantly enhanced the detection sensitivity of glucuronides, as evidenced by a 3- to 55-fold decrease in limits of detection for representative standards. The strategy was applied to profiling glucuronide metabolites in urine samples from colorectal cancer (CRC) patients. A total of 685 features were screened as potential glucuronides, among which 181 were annotated, mainly including glucuronides derived from lipids, organic oxygen, and phenylpropanoids. Enzymatic biosynthesis was employed to accurately identify unknown glucuronides without standards, demonstrating the reliability of the dual-filtering strategy. Our strategy exhibits great potential for profiling the glucuronide metabolome with high coverage and confidence to reveal changes in CRC and other diseases.

Purpose: Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline)., Methods: The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation., Results: We found that the recombinant β-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r 2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%., Conclusions: We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations., (© 2024. The Author(s).)

Supercritical fluid chromatography-mass spectrometry (SFC-MS) has proved to be a beneficial tool for sample analysis for a wide variety of compounds and, as such, has recently gained the attention of the anti-doping community. We have tested the applicability of SFC-MS for routine doping control analysing approximately 3 × 1000 identical anti-doping samples utilising SFC-MS instruments from three different vendors: Agilent Technologies, Waters Corporation and Shimadzu Corporation. A 'dilute and inject' approach either without or after hydrolysis of glucuronide metabolites was applied. Most of the compounds included in our study demonstrated excellent chromatography, whereas some showed co-elution with endogenous interferences requiring MS discrimination. Retention times typically were very stable within batches (%CV ≤ 0.5%), although this appeared to be analyte and column dependent. Chromatographic peak shape was good (symmetrical) and stable over the period of the testing without any change of column. Our results suggest that SFC-MS is a sensitive, reproducible and robust analytical tool ready to be used in anti-doping laboratories alongside the currently applied techniques such as gas and liquid chromatography coupled to mass spectrometry. Even if instruments are designed slightly differently, all three setups demonstrated their fitness for the purpose in anti-doping testing., (© 2024 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

An untargeted metabolomic study identified four potential lung cancer diagnostic biomarkers in human urine. One of the potential biomarkers was an unidentified feature possessing a m / z value of 561+. "561+" was isolated from human urine and tentatively identified as 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol glucuronide with unknown C24,25 stereochemistry using 1 H NMR and mass spectrometry. In a prior report, the C24,25 stereochemistry of the aglycone, 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol, was found to be 24 S ,25 R through GC analysis of the acetonide-TMS derivative. An authentic sample was prepared and found not to have the same stereochemistry as "561+". To identify the C24,25 stereochemistry, four C24,C25 diastereoisomeric alcohols of 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol were prepared from chiral amino acids. Using an LCMS method, the C24,C25 stereochemistry of the "561+" aglycone was determined to be 24 R ,25 S . With the correct aglycone in hand, it was coupled with glucuronic acid to complete the first reported synthesis of 27-nor-5β-cholestane-3α,7α,12α,24 R ,25S pentol glucuronide. Deuterium labeled 27-nor-5β-cholestane-3α,7α,12α,24 R ,25 S pentol was also synthesized for use as an internal standard for MS quantitation.

Exposure to plants is known to improve physical and mental health and living in areas of high vegetation is associated with better health. The addition of quantitative measures of greenness exposure at individual-level to other objective and subjective study measures will help establish cause-and-effect relationships between greenspaces and human health. Because limonene is one of the most abundant biogenic volatile organic compounds emitted by plants, we hypothesized that urinary metabolites of inhaled limonene can serve as biomarkers of exposure to greenness. To test our hypothesis, we analyzed urine samples collected from eight human volunteers after limonene inhalation or after greenness exposure using liquid chromatography-high resolution mass spectrometry-based profiling. Eighteen isomers of nine metabolites were detected in urine after limonene inhalation, and their kinetic parameters were estimated using nonlinear mixed effect models. Urinary levels of most abundant limonene metabolites were elevated after brief exposure to a forested area, and the ratio of urinary limonene metabolites provided evidence of recent exposure. The identities and structures of these metabolites were validated using stable isotope tracing and tandem mass spectral comparison. Together, these data suggest that urinary metabolites of limonene, especially uroterpenol glucuronide and dihydroperillic acid glucuronide, could be used as individualized biomarkers of greenness exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

We present a quantitative clinical LC-MS/MS assay for the simultaneous analysis of buprenorphine, norbuprenorphine, and their glucuronide metabolites in human urine. The assay is based on the direct and hydrolysis-free sample preparation protocol, i.e., dilute and shoot, whereby clarified urine specimens are diluted in internal standard reagent and injected into the LC-MS/MS instrument. The analytical platform employs reversed-phase liquid chromatography for upfront separation and electrospray ionization multiple reaction monitoring MS detection via the triple-quadrupole (TSQ Quantiva) instrument. The assay has a quantitative analytical range of 5 ng/mL-1000 ng/mL represented at seven levels for each of the four analytes. A unique stable isotopically labeled analogue is used as internal standard for each analyte. For high-throughput performance, the assay can be multiplexed between two LC channels., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In the present study, the application and evaluation of Girard's Reagent T (GRT) derivatization for the simultaneous detection and significantly important identification of different phase II methenolone and mesterolone metabolites by LC-MS/(MS) are presented. For the LC-MS analysis of target analytes two complementary isolation methods were developed; a derivatization and shoot method in which native urine is diluted with derivatization reagent and is injected directly to LC-MS and a liquid-liquid extraction method, using ethyl acetate at pH 4.5, for the effective isolation of both sulfate and glucuronide metabolites of the named steroids as well as of their free counterparts. For the evaluation of the proposed protocols, urine samples from methenolone and mesterolone excretion studies were analyzed against at least one sample from a different excretion study. Retention times, along with product ion ratios, were evaluated according to the WADA TD2021IDCR requirements, in order to determine maximum detection and identification time windows for each metabolite. Established identification windows obtained after LC-MS/(MS) analysis were further compared with those obtained after GC-MS/(MS) analysis of the same samples from the same excretion studies, for the most common analytes monitored by GC-MS/(MS). Full validation was performed for the developed derivatization and shoot method for the identification of methenolone metabolite, 3α-hydroxy-1-methylen-5α-androstan-17-one-3-glucuronide (mth3). Overall, the GRT derivatization presented herein offers a tool for the simultaneous sensitive detection of free, intact glucuronide and sulfate metabolites by LC-MS/(MS) that enhance significantly the detection and identification time windows of specific methenolone and mesterolone metabolites for doping control analysis., (© 2023 John Wiley & Sons Ltd.)

In this work, the collision cross section (CCS) value of 103 steroids (including unconjugated metabolites and phase II metabolites conjugated with sulfate and glucuronide groups) was determined by liquid chromatography coupled to traveling wave ion mobility spectrometry (LC-TWIMS). A time of flight (QTOF) mass analyzer was used to perform the analytes determination at high-resolution mass spectrometry. An electrospray ionization source (ESI) was used to generate [M+H] + , [M + NH 4 ] + and/or [M - H] - ions. High reproducibility was observed for the CCS determination in both urine and standard solutions, obtaining RSD lower than 0.3% and 0.5% in all cases respectively. CCS determination in matrix was in accordance with the CCS measured in standards solution showing deviations below 2%. In general, CCS values were directly correlated with the ion mass and allowed differentiating between glucuronides, sulfates and free steroids although differences among steroids of the same group were less significant. However, more specific information was obtained for phase II metabolites observing differences in the CCS value of isomeric pairs concerning the conjugation position or the α/β configuration, which could be useful in the structural elucidation of new steroid metabolites in the anti-doping field. Finally, the potential of IMS reducing interferences from the sample matrix was also tested for the analysis of a glucuronide metabolite of bolasterone (5β-androstan-7α,17α-dimethyl-3α,17β-diol-3-glucuronide) in urine samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Numerous classes of endogenous and xenobiotic compounds are conjugated to uridine-5'-diphospho (UDP)-alpha-D-glucuronic acid which is catalyzed by human UDP-Glucuronosyltransferases (UGTs). The resulting beta-D-glucuronides can be hydrolyzed to β-D-glucuronic acid and the corresponding aglycone in a configuration retaining manner by beta-glucuronidases (GUSBs), which are widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants. This study investigates GUSBs' activity in the presence of ethanol (0-70% by volume) using different β-D-glucuronides (phenolphthalein-β-D-glucuronide, 4-nitrophenol-β-D-glucuronide, morphine-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide and 1-/2-propyl-β-D-glucuronide) as substrates. It was found that β-D-ethyl glucuronide (EtG), which is a minor UGT-derived metabolite of ethanol in man and one of the most frequently used biomarkers of alcohol consumption today, builds up from all investigated β-D-glucuronides by means of GUSBs in the presence of ethanol. The glucuronyl transfer reaction, which was neither detected in the absence of ethanol nor in absence of GUSBs, is minor at ethanol concentrations which are commonly observed in blood and tiβues after consumption of alcoholic beverages, but predominant at higher concentrations of ethanol. In spite of in vitro characteristics, our observations point to an additional biochemical path and another source of EtG, which should be further evaluated in the context of alcohol biomarker applications. The detection of EtG in several settings independent from of human UGT-metabolism (e.g. EtG post post-collection synthesis in E.coli coli-contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the described mechanism., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of "nitazenes." Etazene metabolites were identified through analysis of a human urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking a new psychoactive substances (NPS) cocktail purchased online and was analyzed by ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Etazene metabolites were predicted with BioTransformer 3.0, and the exact masses were added to the inclusion list. Eight possible metabolites were identified in the urine sample. N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene), and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites and etazene were also found. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. As N- and O-deethylated products seem to be the predominant urinary metabolites, the detection of these metabolites in urine can be useful to demonstrate etazene exposure., (© 2022 John Wiley & Sons Ltd.)

Silibinin is a mixture of two flavonoid lignan silibinins A and B from the seeds of milk thistle (Silybum marianum L.). Using ultra-performance liquid chromatography/quadrupole time-of-flight-MS (UPLC/Q-TOF-MS), a total of 18 metabolites were identified in rat and human urine samples after oral administration of silibinin capsule. Furthermore, nine glucuronides and/or sulfated metabolites and two prototype compounds were simultaneously quantified in rat urine after oral administration of silibinin capsule at 50 and 100 mg/kg. Over a 72-h period, 27.6% and 23.3% of silibinin were excreted in the form of metabolites (n = 11) in urine, among which five major metabolites, including silibinin A-7-O-β-glucuronide (SA-7G), silibinin B-7-O-β-glucuronide (SB-7G), silibinin A-5-O-β-glucuronide (SA-5G), silibinin B-5-O-β-glucuronide (SB-5G) and silibinin A-20-O-glucuronide (SA-20G), accounted for 20.5% and 15.5% of the dosages, respectively, when administered at doses of 50 and 100 mg/kg. These results suggested that glucuronidation at the C7-, C5- and C20-hydroxyls was the primary metabolic pathway of silibinin diastereoisomers in vivo. The present results provide helpful information about the in vivo metabolism and clinical usage of silibinin capsule., (© 2022 John Wiley & Sons Ltd.)

N '-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which always occur together and are present exclusively in tobacco products, are classified as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer. While 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) serves as an excellent biomarker for NNK exposure, the currently available biomarker for NNN exposure is urinary "total NNN" (free NNN plus its N -glucuronide). Quantitation of urinary NNN requires extensive precautions to prevent artifactual formation of NNN resulting from nitrosation of nornicotine during analysis. NNN itself can also be formed endogenously by the same nitrosation reaction, which may sometimes cause an overestimation of exposure to preformed NNN. It is thus important to develop an alternative biomarker to specifically reflect NNN metabolic fate and facilitate relevant cancer etiology studies. In this study, we report the first detection of N '-nitrosonornicotine-1 N -oxide (NNN- N -oxide) in human urine. Using a highly specific and sensitive MS 3 transition-based method, NNN- N -oxide was quantified with a mean level of 8.40 ± 6.04 fmol/mL in the urine of 10 out of 32 cigarette smokers. It occurred in a substantially higher level in the urine of 13 out of 14 smokeless tobacco users, amounting to a mean concentration of 85.2 ± 96.3 fmol/mL urine. No NNN- N -oxide was detected in any of the nonsmoker urine samples analyzed ( n = 20). The possible artifactual formation of NNN- N -oxide during sample preparation steps was excluded by experiments using added ammonium sulfamate. The low levels of NNN- N -oxide in the urine of tobacco users indicate that the pyridine N -oxidation pathway represents a minor detoxification pathway of NNN, which further supports the importance of the α-hydroxylation pathway of NNN metabolic activation in humans.

Urine is a common matrix for screening for cannabis use. Urine assays typically measure total 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THCCOOH) concentrations after hydrolysis cleaves the glucuronide. Urine THCCOOH concentration is adjusted by urine creatinine concentration or specific gravity, to account for variable hydration states. Therefore, we performed a population pharmacokinetic analysis of the urinary THCCOOH excretion, urinary flow rate, and creatinine excretion rate data. Urine was obtained over 168 h from six subjects who smoked low- (15.8 mg) and high-dose (33.8 mg) Δ 9 -tetrahydrocannabinol (THC) cigarettes on two occasions. Samples were analyzed for THCCOOH concentration by gas chromatography-mass spectrometry (GC-MS) and volume, time, and creatinine concentration measured. A population pharmacokinetic model of the urinary clearance of THCCOOH was created from these data, and potential covariates of urine creatinine concentration and urine creatinine excretion rate were assessed. Elimination clearance of THCCOOH was estimated as 0.104 ± 0.088 L/min, and its urinary clearance was 0.0022 ± 0.0015 L/min. Total urine excretion of THCCOOH was estimated at 2.3%. Urine flow rate and urine creatinine concentrations were significantly correlated, r 2  = 0.35. Creatinine excretion rate was 129.6 ± 71.0 ml/min, and the intrasubject variability was 31%-52% (SD%) during the week. Urinary creatinine excretion rate was a significant covariate for the urinary clearance of THCCOOH. Creatinine clearance is a significant covariate for urinary THCCOOH clearance. Only 2%-3% of bioavailable THC is excreted as THCCOOH and THCCOO-glucuronide via the urine. Correction of urine drug and/or metabolite concentration with urine creatinine concentration or specific gravity may be more problematic than previously appreciated., (© 2022 John Wiley & Sons, Ltd.)

Dehydrochloromethyltestosterone (DHCMT) is one of the most detected illicit used anabolic-androgenic steroids in professional sports. Therefore, a fast and accurate analysis of this substance is of great importance for a constructive fight against doping abuse. The conventional method for the analysis of this drug, GC-MSMS, is very sensitive and selective but also very time- and resource-consuming. With the presented work, a new approach for simple detection with LC-HRMSMS without any sample preparation is introduced. The method is based on the direct analysis of two newly described phase-II metabolites of the DHCMT long-term metabolite 4-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5β-androst-13-en-3α-ol (M3). LC-HRMSMS, GC-MSMS, fractionation and derivatization experiments are combined to identify and characterize for the first time two different glucuronide-acid conjugates of this metabolite in positive human urine samples. In addition, a third glucuronide metabolite was identified, however without isomeric structure determination. The detection of these metabolites is particularly interesting for confirmation analyses, as the method is rapid and requires little sample material., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In forensic toxicology, a marker of street heroin use is urgent especially in the absence of urinary 6-monoacetylmorphine. ATM4G, the Glucuronide of Acetylated product of Thebaine compound 4 Metabolite (ATM4), arising from byproducts of street heroin synthesis has been considered as a useful marker in some European studies. However, whether ATM4G is a universal marker particularly in Southeast Asia due to 'street' heroin with high purity, it's still unclear. To investigate putative markers for different regions, ATM4G and other metabolites including the Acetylated product of Thebaine compound 3 Metabolite (ATM3) and thebaol, also originated from thebaine were detected in 552 urine samples from heroin users in Taiwan. Results were compared with that from samples collected in the UK and Germany. Only a sulfo-conjugate of ATM4, ATM4S, was detected in 28 Taiwanese users using a sensitive MS 3 method whilst out of 351 samples from the UK and Germany, ATM4G was present in 91. Thebaol-glucuronide was first time detected in 118. No markers were detected in urine following herbal medicine use or poppy seed ingestion. The presence of ATM4S/ATM4G might be affected by ethnicities and heroin supplied in regions. Thebaol-glucuronide is another putative marker with ATM4G and ATM4S for street heroin use., (Copyright © 2021. Published by Elsevier Inc.)

The dietary exposure to the mycotoxin deoxynivalenol (DON) can be assessed by human biomonitoring (HBM). Here, we assessed the relation between dietary DON intake and the excretion of its major metabolite DON-15-glucuronide (DON15GlcA) through time, in an everyday situation. For 49 volunteers from the EuroMix biomonitoring study, the intake of DON from each meal was calculated and the excretion of DON and its metabolites was analyzed for each urine void collected separately throughout a 24-h period. The relation between DON and DON15GlcA was analyzed with a statistical model to assess the residence time and the excreted fraction of ingested DON as DON15GlcA (f abs_excr ). F abs_excr was treated as a random effect variable to address its heterogeneity in the population. The estimated time in which 97.5% of the ingested DON was excreted as DON15GlcA was 12.1 h, the elimination half-life was 4.0 h. Based on the estimated f abs_excr , the mean reversed dosimetry factor (RDF) of DON15GlcA was 2.28. This RDF can be used to calculate the amount of total DON intake in an everyday situation, based on the excreted amount of DON15GlcA. We show that urine samples collected over 24 h are the optimal design to study DON exposure by HBM.

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1'N-glucuronide and 17-epistanozolol-2'N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards. Examination of excretion study samples shows large detection windows for the phase-II metabolites stanozolol-1'N-glucuronide and 17-epistanozolol-1'N-glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid-phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), naloxone (NAL), and their glucuronide conjugates BUP-G, NBUP-G, and NAL-G in urine samples was developed. The method, omitting a hydrolysis step, involved non-polar solid-phase extraction, liquid chromatography on a C18 column, electrospray positive ionization, and mass analysis by multiple reaction monitoring. Quantification was based on the corresponding deuterium-labelled internal standards for each of the six analytes. The limit of quantification was 0.5 μg/L for BUP and NAL, 1 μg/L for NAL-G, and 3 μg/L for NBUP, BUP-G, and NBUP-G. Using the developed method, 72 urine samples from buprenorphine-dependent patients were analysed to cover the concentration ranges encountered in a clinical setting. The median (maximum) concentration was 4.2 μg/L (102 μg/L) for BUP, 74.7 μg/L (580 μg/L) for NBUP, 0.9 μg/L (85.5 μg/L) for NAL, 159.5 μg/L (1370 μg/L) for BUP-G, 307.5 μg/L (1970 μg/L) for NBUP-G, and 79.6 μg/L (2310 μg/L) for NAL-G., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Scope: The aim of the present work is to determine new biomarkers of the biological effects of hesperidin in orange juice (OJ) applying a non-targeted metabolomics approach validated by targeted metabolomics analyses of compliance biomarkers., Methods and Results: Plasma/serum and urine targeted (HPLC-MS/MS) and untargeted ( 1 H-NMR) metabolomics signatures are explored in a subsample with pre- and stage-1 hypertension subjects of the CITRUS study (N = 159). Volunteers received 500 mL day -1 of control drink, OJ, or hesperidin-enriched OJ (EOJ) for 12-weeks. A 6-h postprandrial study is performed at baseline. Targeted analyses reveals plasma and urine hesperetin 7-O-β-d-glucuronide as the only metabolite differing between OJ and EOJ groups after 12-weeks consumption, and in urine is correlated with a decreased systolic blood pressure level. The non-targeted approach shows that after single dose and 12-weeks consumption of OJ and EOJ change several metabolites related with an anti-inflammatory and antioxidant actions, lower blood pressure levels and uremic toxins., Conclusions: Hesperetin 7-O-β-d-glucuronide can be a candidate marker for distinguishing between the consumption of different hesperidin doses at 12-weeks consumption as well as a potential agent mediating blood pressure reduction. Moreover, changes in different endogenous metabolites can explain the mechanisms of action and the biological effects of hesperidin consumption., (© 2021 Wiley-VCH GmbH.)

Ochratoxin A (OTA) is a widespread food contaminant, with exposure estimated to range from 0.64 to 17.79 ng/kg body weight (bw) for average consumers and from 2.40 to 51.69 ng/kg bw per day for high consumers. Current exposure estimates are, however, associated with considerable uncertainty. While biomarker-based approaches may contribute to improved exposure assessment, there is yet insufficient data on urinary metabolites of OTA and their relation to external dose to allow reliable estimates of daily intake. This study was designed to assess potential species differences in phase II biotransformation in vitro and to establish a correlation between urinary OTA-derived glucuronides and mercapturic acids and external exposure in rats in vivo. In vitro analyses of OTA metabolism using the liver S9 of rats, humans, rabbits and minipigs confirmed formation of an OTA glucuronide but provided no evidence for the formation of OTA-derived mercapturic acids to support their use as biomarkers. Similarly, OTA-derived mercapturic acids were not detected in urine of rats repeatedly dosed with OTA, while indirect analysis using enzymatic hydrolysis of the urine samples prior to LC-MS/MS established a linear relationship between urinary glucuronide excretion and OTA exposure. These results support OTA-derived glucuronides but not mercapturic acids as metabolites suitable for biomonitoring.

Background: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,β-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged., Methods: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome., Results: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls., Conclusions: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques., (© 2021. The Author(s).)

The biotransformation and excretion of darolutamide were investigated in a phase I study. Six healthy male volunteers received a single dose of 300 mg 14 C-darolutamide as an oral solution in the fasted state. Plasma, urine, and feces samples were analyzed for mass balance evaluation by liquid scintillation counting (LSC). Metabolite profiling and identification were determined using liquid chromatography mass-spectrometry with off-line radioactivity detection using LSC. Complete mass balance was achieved, with mean radioactivity recovery of 95.9% within 168 hours (63.4% in urine, 32.4% in feces). The administered 1:1 ratio of ( S , R )- and ( S , S )-darolutamide changed to approximately 1:5, respectively, in plasma. Darolutamide and the oxidation product, keto-darolutamide, were the only components quantifiable by LSC in plasma, accounting for 87.4% of total radioactivity, with a 2.1-fold higher plasma exposure for keto-darolutamide. Aside from darolutamide, the most prominent metabolites in urine were O -glucoronide (M-7a/b) and N -glucuronide (M-15a/b), as well as pyrazole sulfates (M-29, M-24) and glucuronides (M-21, M-22) resulting from oxidative cleavage of the parent. The darolutamide diastereomers were mainly detected in feces. In vitro assays showed that darolutamide metabolism involves a complex interplay between oxidation and reduction, as well as glucuronidation. Interconversion of the diastereomers involves oxidation to keto-darolutamide, primarily mediated by CYP3A4, followed by reduction predominantly catalyzed by cytosolic reductase(s), with aldo-keto reductase 1C3 playing the major role. The latter reaction showed stereoselectivity with preferential formation of ( S , S )-darolutamide. SIGNIFICANCE STATEMENT: The metabolism and excretion of darolutamide in humans revealed that oxidation (CYP3A4) and glucuronidation (UGT1A9, UGT1A1) were the main metabolic routes of elimination. Direct excretion also contributed to overall clearance. The two pharmacologically equipotent diastereomers of darolutamide interconvert primarily via oxidation to the active metabolite keto-darolutamide, followed by reduction predominantly by cytosolic reductase(s). The latter reaction showed stereoselectivity with preferential formation of ( S,S )-darolutamide. Data indicate a low drug-drug interaction potential of darolutamide with inducers or inhibitors of metabolizing enzymes., Competing Interests: Clemens-Jeremias von Bühler and Natalia Jungmann report employment by Bayer. Hille Gieschen, Karsten Denner, and Christian Zurth report employment by and stock ownership in Bayer. Michael Niehues and Olaf Prien are employed by NUVISAN, Inc., but were employees of Bayer, and Dr. Prien held stock in Bayer at the time that the work was performed. Marja Kähkönen, Pirjo Nykänen, Päivi Taavitsainen, and Annamari Vuorela report employment by Orion. Timo Korjamo and Mikko Koskinen report employment by and stock ownership in Orion., (Copyright © 2021 by The Author(s).)

Background: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders., Objective: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy., Study Design: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus., Results: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection., Conclusion: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1-3 G), 17β-estradiol 17-glucuronide (E2-17 G), 17β-estradiol 3-glucuronide (E2-3 G), estriol 16-glucuronide (E3-16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F-G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R 2 ≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F-G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC-MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC-MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC-MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15-20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0-1 month of age: 184.5 nmol/L; 15-20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Total urinary 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) concentrations are generally reported following cannabis administration. Few data are available for glucuronide and minor cannabinoid metabolite concentrations. All urine specimens from 11 frequent and 9 occasional cannabis users were analyzed for 11 cannabinoids for ~85 h by liquid chromatography with tandem mass spectrometry following controlled smoked, vaporized or oral 50.6 mg Δ9-tetrahydrocannabinol (THC) in a randomized, placebo-controlled, within-subject dosing design. No cannabidiol, cannabinol, cannabigerol, tetrahydrocannabivarin (THCV), THC, 11-OH-THC and Δ9-tetrahydrocannabinolic acid were detected in urine. Median THCCOOH-glucuronide maximum concentrations (Cmax) following smoked, vaporized and oral routes were 68.0, 26.7 and 360 μg/L for occasional and 378, 248 and 485 μg/L for frequent users, respectively. Median time to specific gravity-normalized Cmax (Tmax) was 5.1-7.9 h for all routes and all users. Median Cmax for THCCOOH, THC-glucuronide and 11-nor-9-carboxy-Δ9-THCV (THCVCOOH) were <7.5% of THCCOOH-glucuronide Cmax concentrations. Only THC-glucuronide mean Tmax differed between routes and groups, and was often present only in occasional users' first urine void. Multiple THCCOOH-glucuronide and THCCOOH peaks were observed. We also evaluated these urinary data with published models for determining recency of cannabis use. These urinary cannabinoid marker concentrations from occasional and frequent cannabis users following three routes of administration provide a scientific database to assess single urine concentrations in cannabis monitoring programs. New target analytes (CBD, CBN, CBG, THCV and phase II metabolites) were not found in urine. The results are important to officials in drug treatment, workplace and criminal justice drug monitoring programs, as well as policy makers with responsibility for cannabis regulations., (Published by Oxford University Press 2020.)

Detection of endogenous anabolic androgenic steroids (EAAS) misuse is a major challenge in doping control analysis. Currently, a number of endogenous steroids, which constitute the steroid profile, are quantified using gas chromatography (GC). With this methodology, only the sum of the free and glucuronidated steroids is measured together. A dilute-and-shoot LC-MS method, which is compliant with the quality requirements for measuring EAAS established by the World Anti-Doping Agency (WADA), was developed and validated containing glucuronidated and sulfated steroids in order to gain some extra information and to expand the existing steroid profile. The developed method is, to the best of our knowledge, the first method to combine both steroid glucuronides and sulfates, which is compliant with the quality standards of the technical document on EAAS, established by WADA. The first advantage of this new steroid profile is the reduced sample preparation time, as it is a direct injection method of diluted urine. A second advantage is the ability of the used gradient to separate 5α-androstane-3α,17β-diol-3-glucuronide (5ααβdiol3G), 5α-androstane-3α,17β-diol-17-glucuronide (5ααβdiol17G), 5β-androstane-3α,17β-diol-3-glucuronide (5βαβdiol3G) and 5β-androstane-3α,17β-diol-17-glucuronide (5βαβdiol17G) allowing to gain specific information on these isomers, which cannot be accomplished in GC-MS screening due to hydrolysis. This steroid profile also contains free testosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione as markers of degradation. In total, 17 compounds and 10 isotopically labelled internal standards are included in this method., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Stanozolol is still the most commonly used illicit anabolic-androgenic steroid (AAS) in professional sports. Therefore, accurate and fast analysis and long detection windows are of great interest in the field of antidoping analysis. In this work, a very simple, fast, and highly sensitive online solid-phase extraction method coupled with liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMSMS) for the analysis of stanozolol-N-glucuronides was developed. This fully validated procedure is characterized by only a few manual steps (dilution and addition of internal standard) in the sample preparation. A limit of identification (LOI) of 75 pg/mL, high accuracy (87.1%-102.1%), precision (3.1%-7.8%), and sensitivity was achieved. Furthermore, good linearity (> 0.99) and robustness, as well as no carry-over effects, could be observed. In addition to excellent confirmation analysis performance, this method shows sufficient potential for the identification and characterization of unknown metabolites. Using this method, it was possible to unambiguously confirm the presence of 1'N- and 2'N-stanozolol-glucuronide in human urine for the first time due to the access to reference material., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Animal sport doping control laboratories are constantly reviewing ways in which they can improve their service offering whilst ensuring that they remain economically viable. This paper describes the development and assessment of a rapid and economical method for the detection of intact glucuronide conjugates of three anabolic steroids and their metabolites along with three corticosteroids in canine urine. The analysis of intact drug conjugates for animal sport doping control is generally not performed routinely as it presents a number of analytical challenges, not least of which is the lack of availability of appropriate reference standards. Here, we report the development of a UHPLC-MS/MS method using APCI in the negative ion mode for the detection of intact phase II conjugates, including the importance of in vitro incubations in order to provide appropriate reference materials. Cross-validation of the developed method demonstrated that the detection capability of the intact phase II conjugates of stanozolol, boldenone, nandrolone, and their metabolites along with the corticosteroids dexamethasone and methylprednisolone was equivalent to that achieved in routine race-day screens. The new process has been in operation for approximately 2 years and has been used to analyze in excess of 13500 canine urine samples, resulting in a number of positive screening findings. To the best of our knowledge, this is the first reported use of a routine screen for intact drug conjugates within animal sport doping control., (© 2020 John Wiley & Sons, Ltd.)

A rapid procedure for the determination of amphenicol antibiotics in human urine by liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is proposed. The presence of thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in the human body can be attributed to their administration to treat certain diseases or by eating food of animal origin. The TAP, FF and CAP excreted in urine is mainly in the form of glucuronide conjugates, although their free forms may also be excreted to a lesser extent. In the procedure described, the enzymatic hydrolysis of amphenicol glucuronide forms in urine was carried out using β-glucuronidase and sulfatase at pH 5 (37 °C, overnight) in order to discriminate the free and conjugated forms. Then, amphenicol antibiotics were submitted to dispersive liquid-liquid microextraction (DLLME) for preconcentration. All the parameters affecting DLLME efficiency were optimized, and the following conditions were selected: 0.9 g NaCl in 10 mL of urine, to which 1.2 mL methanol (as dispersant solvent) and 1 mL of 4-methyl-2-pentanone (as extractant solvent) were added. The absence of a matrix effect allowed quantification of the samples against aqueous standards. Detection limits were 29, 6 and 3 pg mL -1 for TAP, FF and CAP, respectively. Relative standard deviations were calculated to evaluate the intra- and inter-day precision and values lower than 10% were obtained in all cases. The trueness of the method was tested through recovery studies, obtaining satisfactory values (83-104%). Ten urine samples obtained from volunteers were analysed and all of them were free of the studied antibiotics., (Copyright © 2020 Elsevier B.V. All rights reserved.)

For an effective detection of doping with pseudo-endogenous anabolic steroids, the urinary steroid profile is of high value. In this work, the aim was to investigate steroid metabolism disruption after exogenous intramuscular administration of different testosterone esters. The investigation focused on both sulfo - and glucoro conjugated androgens. A single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido®) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanone®), was given to six healthy volunteers. Urine was collected throughout a testing period of 60 days. A LC-MS method was developed and validated for the analysis of eight conjugated steroids in their intact form. The results show that urinary changes in both sulfo - and glucuro conjugated steroid levels are prominent after the injection of testosterone esters. A promising potential marker for the intake of exogenous testosterone is the combined ratio of epitestosterone sulfate/epitestosterone glucuronide to testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) as a complementary biomarker for testosterone abuse. This represents a new piece of evidence to detect testosterone doping, representing a new approach and being independent from the metabolic connections of the markers in the steroid passport., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Oroxylin A (OA), as a natural flavonoid extracted from the root of Scutellaria baicalensis Georgi, is a candidate drug with multiple pharmacological activities. However, pharmacokinetic studies of OA have rarely been reported up to now. The present study aim to conduct a systemic evaluation on the pharmacokinetics, tissue distribution and excretion of OA in rats, with quantification of both OA and its two metabolites, Oroxylin A 7-O-glucuronide (OG) and Oroxylin A sodium sulfonate (OS) by the sensitive and rapid UPLC-MS/MS methods. The results show that OA was rapidly eliminated in vivo after a single-dose (2 mg/kg) i.v. administration of OA. The relative bioavailability of OA in all three i.g. administration groups (40, 120, and 360 mg/kg) were <2%. The AUC 0-t values of OA, OG, and OS in rats show an apparent dose-proportionality. OA, OG, and OS all underwent a rapid and widespread tissue distribution after i.g. administration (120 mg/kg) of OA. Except for stomach and intestine, the major distribution tissues of OA and its two metabolites in rats were liver, kidney, respectively. And OA was more widely distributed in tissue than its metabolites. After i.g. administration (120 mg/kg) of OA, it was mainly excreted from the feces, and OG mainly excreted from bile and urine, while OS almost free of excretion. This work present a comprehensive pharmacokinetics information for further investigation of OA and its two metabolites., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Dotinurad is a novel, selective urate reabsorption inhibitor, which reduces serum uric acid levels by inhibiting the urate transporter 1. The results of nonclinical studies indicated the possibility that the concomitant use of the non-steroidal anti-inflammatory drug oxaprozin affects the pharmacokinetics of dotinurad. We evaluated drug-drug interactions with respect to the pharmacokinetics and safety of dotinurad when co-administered with oxaprozin., Methods: This was an open-label, two-period, add-on study in healthy adult males. For a single dose of 4 mg of dotinurad with and without oxaprozin, we compared its pharmacokinetic parameters and evaluated safety., Results: This study enrolled 12 subjects, 11 of whom completed the study. The geometric mean ratio (90% confidence interval [CI]) of the urinary excretion rate of glucuronate conjugates of dotinurad after co-administration with oxaprozin compared to administration of dotinurad alone was 0.657 (0.624-0.692), while the geometric mean ratios (90% CIs) of the maximum plasma concentration and area under the plasma concentration-time curve from time zero to infinity (AUC 0-inf ) were 0.982 (0.945-1.021) and 1.165 (1.114-1.219), respectively. During the study, two adverse events occurred after administration of dotinurad alone and one occurred after administration of oxaprozin alone., Conclusions: In comparison with administration of dotinurad alone, co-administration with oxaprozin was associated with a 34.3% decrease in the urinary excretion rate of the glucuronate conjugates of dotinurad, and a 16.5% increase in AUC 0-inf of dotinurad. However, no clinically meaningful drug-drug interactions were observed. Administration of dotinurad alone was similar safety to co-administration with oxaprozin., Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT03350386.

Background: Dotinurad is a novel selective urate reabsorption inhibitor (SURI) that selectively inhibits the reabsorption of uric acid in renal tubules and promotes the excretion of uric acid into urine. In this study, the effects of age and gender on the pharmacokinetics (PK), pharmacodynamics (PD), and safety of dotinurad were evaluated in healthy subjects., Methods: An open-label study of a single oral administration of dotinurad 1 mg was conducted in elderly (≥ 65 years) Japanese males and females, and young (20-35 years) males and females (six patients each)., Results: Following a single-dose administration of dotinurad, the change in dotinurad plasma concentration showed a similar profile across groups. Regarding the PK parameters, there was no significant difference between elderly and young subjects. On comparing males and females, significant differences were observed in some parameters in elderly subjects. However, these differences in some parameters could not be detected by adjust for body weight. When PD parameters in elderly and young subjects were compared, significant differences were observed in some parameters in male subjects. On comparing males and females, significant differences were observed in some parameters in young subjects; however, the percent change in serum uric acid concentration decreased over time was relatively close for both groups. There were no clinically relevant safety problems., Conclusion: Age and gender had no clinically meaningful effect on the PK, PD, and safety of dotinurad., Clinical Trials: ClinicalTrials.gov identifier: NCT02344875.

Epidemiological studies have reported the relationship between bisphenol A (BPA) exposure and increased prevalence of asthma, but the mechanisms remain unclear. Here, we investigated whether BPA exposure and DNA methylation related to asthma in children. We collected urinary and blood samples from 228 children (Childhood Environment and Allergic Diseases Study cohort) aged 3 years. Thirty-three candidate genes potentially interacting with BPA exposure were selected from a toxicogenomics database. DNA methylation was measured in 22 blood samples with top-high and bottom-low exposures of BPA. Candidate genes with differential methylation levels were validated by qPCR and promoter associated CpG islands have been investigated. Correlations between the methylation percentage and BPA exposure and asthma were analyzed. According to our findings, MAPK1 showed differential methylation and was further investigated in 228 children. Adjusting for confounders, urinary BPA glucuronide (BPAG) level inversely correlated with MAPK1 promoter methylation (β = -0.539, p = 0.010). For the logistic regression analysis, MAPK1 methylation status was dichotomized into higher methylated and lower methylated groups with cut off continuous variable of median of promoter methylation percentage (50%) while performing the analysis. MAPK1 methylation was lower in children with asthma than in children without asthma (mean ± SD; 69.82 ± 5.88% vs. 79.82 ± 5.56%) ( p = 0.001). Mediation analysis suggested that MAPK1 methylation acts as a mediation variable between BPA exposure and asthma. The mechanism of BPA exposure on childhood asthma might, therefore, be through the alteration of MAPK1 methylation. The mechanism of BPA exposure on childhood asthma might, therefore, be through the alteration of MAPK1 methylation.

An analytical procedure has been introduced to enable a study of the excretion of free bisphenol A (BPA), total BPA and its main metabolite bisphenol A glucuronide (BPA-GLUC). In the experiment, in which 100 μg/kg b. w. BPA was administered daily to one Istrian Pramenka sheep for 5 days with consecutive urine and feces samples being taken, BPA and total BPA were determined in samples using high-performance liquid chromatography (HPLC) with fluorescence detection. Because of their good recovery, precision, and sensitivity, the methods have also proved applicable to further ecotoxicological studies of free BPA, BPA-GLUC and total BPA. The results were subsequently compared with reported field studies of BPA in livestock excreta.

Menthol, which creates mint flavor and scent, is often added to tobacco in both menthol and nonmenthol cigarettes. A potent tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is extensively metabolized to its equally carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) as ( R )- or ( S )-NNAL enantiomers. NNAL is detoxified by UDP-glucuronosyltransferase (UGT) enzymes, with glucuronidation occurring on either NNAL's pyridine ring nitrogen (NNAL- N -Gluc) or the chiral alcohol [( R )- or ( S )-NNAL- O -Gluc]. To characterize a potential effect by menthol on NNAL glucuronidation, in vitro menthol glucuronidation assays and menthol inhibition of NNAL-Gluc formation assays were performed. Additionally, NNAL and menthol glucuronides (MG) were measured in the urine of smokers ( n = 100) from the Southern Community Cohort Study. UGTs 1A9, 1A10, 2A1, 2A2, 2A3, 2B4, 2B7, and 2B17 all exhibited glucuronidating activity against both l- and d-menthol. In human liver microsomes, both l- and d-menthol inhibited the formation of each NNAL-Gluc, with a stereospecific difference observed between the formation of ( R )-NNAL- O -Gluc and ( S )-NNAL- O -Gluc in the presence of d-menthol but not l-menthol. With the exception of three nonmenthol cigarette smokers, urinary MG was detected in all menthol and nonmenthol smokers, with l-MG comprising >98% of total urinary MG. Levels of urinary NNAL- N -Gluc were significantly ( P < 0.05) lower among subjects with high levels of total urinary MG; no significant changes in free NNAL were observed. These data suggest that the presence of menthol could lead to increases in alternative, activating metabolic pathways of NNAL in tobacco target tissues, increasing the opportunity for NNAL to damage DNA and lead to the development of tobacco-related cancers. SIGNIFICANCE STATEMENT: High levels of the major menthol metabolite, menthol-glucuronide, was observed in the urine of smokers of either menthol or nonmenthol cigarettes. The fact that a significant inverse correlation was observed between the levels of urinary menthol-glucuronide and NNAL-N-glucuronide, a major detoxification metabolite of the tobacco carcinogen, NNK, suggests that menthol may inhibit clearance of this important tobacco carcinogen., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

While 3-hydroxybenzo[a]pyrene (3-OHBaP) is a preferable biomarker to assess human exposure to benzo[a]pyrene (BaP), a sensitive and simple method is lacking. In this study, a specific and sensitive method based on liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed for direct analysis of 3-OHBaP glucuronide and sulfate conjugates in human urine samples without enzymatic hydrolysis. The limits of detection (LODs) were 0.06 ng L -1 for BaP-3-sulfate (BaP-3-S) and 0.16 ng L -1 for BaP-3-glucuronide (BaP-3-G), which showed high sensitivity. Both compounds showed excellent linearity ( r 2 > 0.99) in the range of 0.01-10 μg L -1 in the instrumental calibration. The absolute recoveries of the target analytes spiked in human urine for the entire analytical procedure were 68.3 ± 4.96% (mean ± SD) and 63.7 ± 5.47% for BaP-3-S and BaP-3-G, respectively. This method was applied to quantify BaP-3-G and BaP-3-S in 150 urine samples collected from healthy volunteers. The mean concentration of BaP-3-S was 0.67 ng g -1 creatinine (-1 creatinine), about 10-fold lower than that of BaP-3-G (6.73 ng g -1 creatinine, < LOD to 52.64 ng g -1 creatinine). For comparison, we also detected the concentration of free 3-OHBaP in 15 randomly selected samples without enzymatic hydrolysis and found at least >98% of 3-OHBaP is excreted mainly in these two conjugated forms in human urine. A statistically significant positive association was observed between urinary 3-OHBaP conjugates and urinary 8-OHdG levels ( p < 0.001) in the general population. This study developed a sensitive and simple method to determine urinary glucuronide/sulfate conjugated BaP metabolites and for the first time found that BaP exposure associated with 8-OHdG levels in the general population.

Glucuronidation is the most common phase II modification and plays an important role in human clearance metabolism. Glucuronidated metabolites have also been linked to disease development and microbiota-host co-metabolism. Although many of these compounds have been identified, the total number of unknown glucuronides and their impact on the human host's physiology can only be estimated. Herein, we describe the combination of an untargeted metabolomics analysis and enzymatic metabolic conversion for the selective detection of glucuronide conjugates by using UPLC-MS/MS in human urine samples. Our study demonstrates that this powerful strategy can be used for the selective identification of glucuronidated molecules and to discover unknown natural metabolites. In total, we identified 191 metabolites in a single sample including microbiota-derived compounds as well as previously unidentified molecules., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A liquid chromatography/electrospray ionization-tandem mass spectrometry-based method was developed for the identification of the conjugation positions of the monoglucuronides of 25-hydroxyvitamin D 3 [25(OH)D 3 ] and 24,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] in human urine. The method employed derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D 3 -3- and -25-glucuronides, or 24,25(OH) 2 D 3 -3-, -24- and -25-glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D 3 and 24,25(OH) 2 D 3 , respectively. The 3-glucuronide was not present for 24,25(OH) 2 D 3 , unlike 25(OH)D 3 , thus we found that selective glucuronidation occurs at the C24-hydroxy group for 24,25(OH) 2 D 3 ., (© 2019 John Wiley & Sons, Ltd.)

Formoterol is a long-acting beta2-adrenoceptor agonist (LABA) used for the treatment of asthma and exercise-induced bronchoconstriction. Formoterol is usually administered as a racemic (rac-) mixture of (R,R)- and (S,S)-enantiomers. While formoterol is restricted by the World Anti-Doping Agency (WADA), inhalation of formoterol is permitted to a predetermined dose (54 μg/24 hours) and a urine threshold of 40 ng/mL. However, chiral switch enantiopure (R,R)-formoterol is available, effectively doubling the therapeutic advantage for the same threshold. The aim of this study was to investigate whether formoterol exhibits enantioselective urinary pharmacokinetics following inhalation. Six healthy volunteers were administered a 12 μg inhaled dose of rac-formoterol. Urine was collected over 24 hours and analyzed by enantioselective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay. Total (free drug plus conjugated metabolite) median (min-max) rac-formoterol urine levels following inhalation were 1.96 (1.05-13.4) ng/mL, 1.67 (0.16-9.67) ng/mL, 0.45 (0.16-1.51) ng/mL, 0.61 (0.33-0.78) ng/mL, and 0.17 (0.08-1.06) ng/mL at 2, 4, 8, 12, and 24 hours, respectively, well below the 2019 urine threshold. The proportion of conjugation differed between enantiomers with glucuronide conjugation much greater for (R,R)-formoterol (around 30%-60% of total) compared to (S,S)-formoterol (0%-30%). There was clear evidence of inter-individual enantioselectivity observed in the ratios of (R,R):(S,S)-formoterol, where (S,S)- was predominant in free formoterol, and (R,R)- predominant in the conjugated metabolite. In conclusion, rac-formoterol delivered by inhalation exhibits enantioselective elimination in urine following single-dose administration. Enantioselective assays should be employed in doping control to screen for banned beta2-agonist chiral switch products such as (R,R)-formoterol, and total hydrolyzed rac-formoterol is warranted to account for inter-individual differences in enantioselective glucuronidation., (© 2019 John Wiley & Sons, Ltd.)

Buprenorphine is a commonly used opioid in pain therapy as well as in opiate maintenance therapy. Immunoassays are quick and cost-effective methods for the necessary toxicological urine analysis of maintenance therapy patients. In this study a novel enzymatic immunoassay, the Thermo Fisher Scientific CEDIA Buprenorphine II assay (Bup2) was evaluated for the detection of buprenorphine, norbuprenorphine and their conjugated metabolites in human urine samples. The Bup2 assay has a cut-off of 10 ng/mL with ±25% controls, whereas the existing CEDIA Buprenorphine assay (Bup1) has a cut-off of 5 ng/mL and ±40% controls. Both assays were analyzed on a Thermo Scientific Indiko Plus benchtop analyzer. Seven-day precision studies of Bup2 assay demonstrated excellent precision of 7.2-10.6%. No crossover between control samples and the cut-off level were observed. Urine samples of 120 patients undergoing opiate maintenance therapy were collected. Immunoassay results of Bup1 and Bup2 were confirmed by gas chromatography mass spectrometry (GC/MS) for buprenorphine and norbuprenorphine as well as for their glucuronides. Comparison showed a specificity of 0.99 between the Bup2 assay and GC/MS, whereas the Bup1 assay had a specificity 0.70 due to 21 false positive samples. The reason is a known cross-reactivity of the Bup1 assay to opiate compounds. The Bup2 assay revealed one false positive result close to the cut-off value; no specific candidate possibly causing a cross-reaction was detected by GC/MS and liquid chromatography tandem mass-spectrometry (LC/MS/MS) methods. The data presented demonstrate an excellent correlation of the Bup2 assay to GC/MS, showing improved specificity and sensitivity when compared to the Bup1 assay. Thus, the Bup2 assay is highly suitable for urine testing, even for opiate maintenance patients receiving high doses of morphine., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Glucuronidation is a major phase II conjugation pathway in mammals, playing an important role in the detoxification and biotransformation of xenobiotics including mycotoxins such as deoxynivalenol (DON). Culmorin (CUL), a potentially co-occurring Fusarium metabolite, was recently found to inhibit the corresponding detoxification reaction in plants, namely DON-glucoside formation, raising the question whether CUL might affect also the mammalian counterpart. Using cell-free conditions, CUL when present equimolar (67 µM) or in fivefold excess, suppressed DON glucuronidation by human liver microsomes, reducing the formation of DON-15-glucuronide by 15 and 50%, and DON-3-glucuronide by 30 and 50%, respectively. Substantial inhibitory effects on DON glucuronidation up to 100% were found using the human recombinant uridine 5'-diphospho-glucuronosyltransferases (UGT) 2B4 and 2B7, applying a tenfold excess of CUL (100 µM). In addition, we observed the formation of a novel metabolite of CUL, CUL-11-glucuronide, identified for the first time in vitro as well as in vivo in piglet and human urine samples. Despite the observed potency of CUL to inhibit glucuronidation, no significant synergistic toxicity on cell viability was observed in combinations of CUL (0.1-100 µM) and DON (0.01-10 µM) in HT-29 and HepG2 cells, presumably reflecting the limited capacity of the tested cell lines for DON glucuronidation. However, in humans, glucuronidation is known to represent the main detoxification pathway for DON. The present results, including the identification of CUL-11-glucuronide in urine samples of piglets and humans, underline the necessity of further studies on the relevance of CUL as a potentially co-occurring modulator of DON toxicokinetics in vivo.

Background: Monitoring of medication compliance and drug abuse is used by clinicians to increase patient prescription drug compliance and reduce illicit drug abuse and diversion. Despite available immunoassays, chromatography-mass spectrometry-based methods are considered the gold standard for urine drug monitoring owing to higher sensitivities and specificities. Herein, we report a fast, convenient ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect or quantify 37 clinically relevant prescription drugs, drugs of abuse, and related glucuronides and other metabolites in human urine by single diluted sample injection., Methods: Analytes consisted of prescription and illicit opioids, benzodiazepines, and drugs of abuse, including parent compounds and glucuronidated and nonglucuronidated metabolites. Urine samples were diluted with water and supplemented with deuterated internal standards without enzymatic hydrolysis, analyte extraction, or sample purification. Analytes were separated by reversed-phase UPLC and quantified by positive-mode electrospray ionization and collision-induced dissociation MS. Assay validation followed Food and Drug Administration bioanalytical guidelines., Results: Total analytical run time was 5.5 min. All analytes demonstrated acceptable inter- and intraassay accuracy, imprecision, and linearity throughout clinically relevant analytical ranges (1-2000 ng/mL, depending on analyte). All analytes demonstrated acceptable selectivity, stability, matrix effects, carryover, and performance compared to national reference laboratory or previously validated in-house methods. A total of 23 and 14 analytes were validated for quantitative and qualitative testing, respectively., Conclusions: A convenient UPLC-MS/MS assay for simultaneously monitoring 37 analytes in human urine was validated for use in pain management testing. Advantages of this multiplex assay include facile sample preparation and higher-throughput definitive detection including glucuronide metabolite quantification., (© 2018 American Association for Clinical Chemistry.)

Buprenorphine and its metabolites are routinely monitored to assess patient compliance with drug detoxification programs or as pain killers. A rapid method for the simultaneous analysis of buprenorphine, norbuprenorphine, and glucuronides in urine using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. Urine samples were diluted in water containing formic acid 0.1% and directly injected into the UHPLC-MS/MS system without any sample pretreatment. Quality control (QC) samples, prepared using 20 different urine matrices, fortified at 3 concentration levels, were quantified using four deuterated internal standards. The accuracy values obtained spanned from 90 to 114% with repeatability lower than 10% also in the inter-day between batch experiments. Matrix effects (ME), evaluated before correction with internal standards using Matuszewski procedure, mainly affected the analysis of buprenorphine glucuronide. The use of deuterated internal standards (IS) for each analyte was necessary to compensate for ME and was essential in the determination of glucuronides. The method was applied to 30 real urine samples from patients under a detoxification therapy. Duplicate analyses were performed with the presented method and compared with another method which involves a standard hydrolysis procedure. Real sample results were compared showing a good performance agreement, with differences between the two methods lower than ±20% in the quantification results., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There have been many reports regarding toxic chemicals in birds. Chemicals are mainly metabolized in the liver through phase I oxidation by cytochrome P450 (CYP) and phase II conjugation by conjugated enzymes, such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), glutathione-S-transferase (GST), etc. Xenobiotic metabolism differs among bird species, but little detailed information is available. In the present study, the four-ring polycyclic aromatic hydrocarbon (PAH), pyrene, was used as a model xenobiotic to clarify the characteristics of xenobiotic metabolism in birds compared with laboratory animals by in vivo and in vitro studies. Plasma, bile, and excreta (urine and feces) were collected after oral administration of pyrene and analyzed to clarify xenobiotic metabolism ability in chickens and quails. Interestingly, pyrenediol-glucuronide sulfate (PYDOGS) and pyrenediol-diglucuronide (PYDOGG) were present in chickens and quails but not in rats. In addition, the area under the curve (AUC), maximum plasma concentration (C max ), and time to maximum plasma concentration (T max ) of pyrene-1-sulfate (PYOS) were higher than those of the parent molecule, pyrene, while the elimination half-life (t 1/2 ) and mean residence time (MRT) were faster than those of the parent pyrene. With regard to sulfation of 1-hydroxypyrene (PYOH), the maximum velocity (V max ) and Michaelis constant (K m ) of rat liver cytosol were greater than those of chicken and quail liver cytosol. Furthermore, V max /K m of UGT activity in rat liver microsomes was also greater than those of chicken and quail liver microsomes. Characterization of xenobiotic metabolism revealed species differences between birds and mammals, raising concerns about exposure to various xenobiotics in the environment., (Copyright © 2018. Published by Elsevier Inc.)

Aim, materials & methods: Urinary cortisol profile has the potential as a diagnostic biomarker. We therefore developed a stable-isotope dilution ultraperformance chromatography multistage MS-based method to quantify cortisol and 16 metabolites in human urines. Results & conclusion: The LOD for cortisol and its metabolites ranges from 0.02 to 5.81 pg/μl urine. The inter- and intraday variations were 3.7-12.9% and 3.5-15.6%, respectively. Among the metabolites analyzed, significant person-to-person heterogeneity was observed, demonstrating the need for comprehensive metabolite profiling in diagnosis. Nevertheless, the glucuronides of dihydrocortisol, dihydrocortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone are the major ones. The sum of the glucuronidated and free forms constitute >93% of the metabolites analyzed, which is termed as total cortisol equivalent. Total cortisol equivalent may serve as a surrogate of cortisol secretion. Clinical trial registration number: NCT02500472.

The present study compared and validated two analytical methods, HPLC-HRMS, and GC-MS using MSTFA as derivatization agent, for the analysis of microbiota-derived phenolic acids and aromatic compounds accumulating in urine, collected over a 24 h period after the consumption of 500 mL of orange juice. In addition, purification procedures using SDB-L and HLB solid phase cartridges were compared when HPLC-HRMS technique was used. Both HPLC-HRMS and GC-MS methodologies were successfully validated in terms of specificity, sensitivity, limit of detection and quantification, recovery and matrix effects. HPLC-HRMS, unlike GC-MS, does not require sample derivatization prior to analysis. GC-MS was not suitable for the analysis of phenolic sulfate and glucuronide metabolites because of their lack of volatility. These phase II metabolites could, however, be analysed by HPLC-HRMS which, as a consequence, provided more detailed and complete information on the phenolic compounds derived from microbiota-mediated degradation of orange juice (poly)phenols. Furthermore, the use of SDB-L and HLB cartridges for sample purification prior to HPLC-HRMS analysis is suitable for free phenolics and glucuronide metabolites but not sulfate derivatives. These findings highlight that the use of an inappropriate analytical protocol can adversely affect studies on the bioavailability of dietary (poly)phenols in which microbiota-derived phenolic catabolites play an important role., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ortho-phenylphenol (OPP) has been widely used as a fungicide and preservative. Although low-dose studies have demonstrated its low toxicity in animals and humans, high-dose exposure to this contaminant has toxic effects that range from skin irritation to bladder cancer. Thus far, monitoring of OPP exposure in the general population has been performed by measuring OPP after urine hydrolysis with the β-glucuronidase/arylsulfatase enzyme and sometimes by the use of a mineral acid. We developed a sensitive, accurate, and robust method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to specifically measure two-phase II OPP metabolites excreted in human urine, OPP sulfate (OPP-S), and OPP glucuronide (OPP-G). Comparative analysis of urine samples from 50 volunteers living in the Quebec City area using a direct method and phosphoric acid hydrolysis method previously developed in our laboratory showed no statistically significant difference (p value for paired t test = 0.701) in OPP concentrations. Moreover, a significant difference showed that underestimation (p value for paired t test = 0.025) occurs when β-glucuronidase/arylsulfatase enzyme deconjugation is used. The LOD achieved by the direct method permits the detection of OPP-S and OPP-G metabolites in urine at the submicrogram per liter level. Graphical abstract ᅟ.

Among the emerging new psychoactive substances (NPS), compounds carrying an N-ortho-methoxybenzyl substituent, the so-called NBOMes, represented a highly potent group of new hallucinogens. Recently, 3,4-dimethoxyamphetamine (3,4-DMA)-NBOMe and 4-methylmethamphetamine (4-MMA)-NBOMe occurred, but no data on their pharmacokinetics were available. According to other NBOMes, they are expected to be extensively metabolized. For detection and identification of their phase I and II metabolites, nano liquid chromatography coupled to high resolution tandem mass spectrometry (nanoLC-HRMS/MS) was used. Rat urine was prepared by simple dilution and incubation mixtures with pooled human liver S9 fraction by precipitation. Furthermore, the results concerning detectability using the new nanoLC approach were compared to those obtained by conventional ultra-high performance LC (UHPLC). In addition, the detectability of the compounds by standard urine screening approaches (SUSAs) routinely used by the authors with UHPLC-HRMS/MS, LC-MS n , and GC-MS was tested. Both NBOMes were extensively metabolized mainly by O-demethylation and conjugation with glucuronic acid (3,4-DMA-NBOMe) or oxidation of the tolyl group to the corresponding carboxylic acid (4-MMA-NBOMe). The developed nanoLC-HRMS/MS approach was successfully applied for identification of 38 3,4-DMA-NBOMe metabolites and 33 4-MMA-NBOMe metabolites confirming its detection power. Furthermore, the solvent saving nanoLC system showed comparable results to the UHPLC-HRMS/MS approach. In addition, an intake of an estimated low common user's dose of the compounds was detectable by all SUSAs only via their metabolites. Suggested targets for urine screening procedures were O-demethyl- and O,O-bis-demethyl-3,4-DMA-NBOMe and their glucuronides and carboxy-4-MMA-NBOMe and its glucuronide and N-demethyl-carboxy-4-MMA-NBOMe., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Di-(2-propylheptyl) phthalate (DPHP) is used as a plasticizer for polyvinyl chloride products. A tolerable daily intake of DPHP of 0.2 mg/kg body weight has been derived from rat data. Because toxicokinetic data of DPHP in humans were not available, it was the aim of the present work to monitor DPHP and selected metabolites in blood and urine of 6 male volunteers over time following ingestion of a single DPHP dose (0.7 mg/kg body weight). Concentration-time courses in blood were obtained up to 24 h for DPHP, mono-(2-propylheptyl) phthalate (MPHP), mono-(2-propyl-6-hydroxyheptyl) phthalate (OH-MPHP), and mono-(2-propyl-6-oxoheptyl) phthalate (oxo-MPHP); amounts excreted in urine were determined up to 46 h for MPHP, OH-MPHP, oxo-MPHP, and mono-(2-propyl-6-carboxyhexyl) phthalate (cx-MPHP). All curves were characterized by an invasion and an elimination phase the kinetic parameters of which were determined together with the areas under the concentration-time curves in blood (AUCs). AUCs were: DPHP > MPHP > oxo-MPHP > OH-MPHP. The amounts excreted in urine were: oxo-MPHP > OH-MPHP> > cx-MPHP > MPHP. The AUCs of MPHP, oxo-MPHP, or OH-MPHP could be estimated well from the cumulative amounts of urinary OH-MPHP or oxo-MPHP excreted within 22 h after DPHP intake. Not considering possible differences in species-sensitivity towards unconjugated DPHP metabolites, it was concluded from a comparison of their AUCs in DPHP-exposed humans with corresponding earlier data in rats that there is no increased risk of adverse effects associated with the internal exposure of unconjugated DPHP metabolites in humans as compared to rats when receiving the same dose of DPHP per kg body weight., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)