Metabolites In Human Urine Research Articles

Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine and Plasma by LC-MS/MS

Abstract The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl adulterated or associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine is a clonidine analog and is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect xylazine and xylazine metabolites in urine and plasma, and to investigate the presence and abundance of xylazine metabolites in remnant urine and plasma samples submitted for clinical testing. This is achieved by first collecting paired urine and serial plasma samples when urine tests positive for xylazine during routine clinical testing. Plasma samples were prepared for LC-MS/MS analysis by protein precipitation with methanol and xylazine-D6 internal standard. Urines were prepared by dilution with xylazine-D6, followed by a hydrolysis with beta-glucuronidase. All experiments were performed using an Aquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. LC separation utilized a C18 column with a 4-minute gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Multiple reaction monitoring for xylazine and 4-OH-xylazine was optimized with commercial standards. Calibration curves for both analytes were prepared from 0.5 – 2,000 ng/mL in plasma, and 5 – 5,000 ng/mL in urine. Product ion scanning was used to detect four additional xylazine metabolites by spectral matching with previously published full scan tandem mass spectra. The highest intensity fragment ions observed in the product ion spectra were used to develop multiple reaction monitoring experiments for four additional xylazine metabolites, previously described as: sulfone-xylazine, oxo-xylazine, OH-sulfone-xylazine, and OH-oxo-xylazine, in 50 remnant urines and 70 individual plasmas. In remnant urines, xylazine concentrations ranged from less than 5 to greater than 5,000 ng/mL. In remnant plasmas, xylazine ranged from less than 0.5 to 158 ng/mL. Previously reported xylazine metabolites were detected in both urine and plasma, and relative peak area ratios were calculated as metabolite-to-xylazine in both matrices. In urine, the highest average peak area ratio (3.1) was observed for sulfone xylazine, after hydrolysis. In plasma, the highest average peak area ratio (1.4) was observed for oxo-xylazine. We were unable to absolutely quantitate either metabolite due to lack of available standards. Future studies include investigation on the association of xylazine exposure and plasma concentrations with clinical presentation and patient outcomes.

B-271 High Sensitivity Homogeneous Enzyme Immunoassay for Benzodiazepines and Metabolites

Abstract Background Benzodiazepines are widely prescribed for treating various conditions such as anxiety, insomnia, and seizures due to their sedative and anxiolytic properties. Despite their therapeutic benefits, concerns persist regarding their potential for misuse, dependence, and addiction. Urine drug screening immunoassays serve as an important tool in monitoring benzodiazepine usage, ensuring adherence to prescribed regimens, identifying misuse or abuse, and facilitating necessary medical interventions. However, limitations of benzodiazepine immunoassays are well-documented in literature, with issues surrounding their lack of sensitivity to glucuronidated and 7-amino metabolites, inability to detect newer benzodiazepines, and poor cross-reactivity with different benzodiazepines. ARK Diagnostics has developed a highly sensitive homogeneous enzyme immunoassay capable of detecting both classic and designer benzodiazepines, in addition to their glucuronide and 7-amino metabolites in human urine at a cutoff concentration 200 ng/mL, without the need for glucuronidase or sample pretreatment. Methods The ARKTM Benzodiazepine Plus Assay is a liquid-stable homogenous enzyme immunoassay consisting of two reagents. The assay uses temazepam as the 200 ng/mL cutoff calibrator. The performance characteristics of this assay, including precision, spiked recovery, specificity, and method comparison to LC-MS/MS, were evaluated on the Beckman Coulter AU680 automated clinical analyzer. Results The ARKTM Benzodiazepine Plus demonstrated acceptable precision, with no overlap between cutoff (200 ng/mL) and ±25% control levels (150 ng/mL and 250 ng/mL) in a histogram overlap analysis, and ≤3.6% CV in semi-quantitative mode. Spiked temazepam samples spanning the semi-quantitative assay range of 1000 ng/mL were recovered between 95.0% and 103.7% of the spiked levels. Thirty-eight benzodiazepines produced cross-reactivities >80%, including the following benzodiazepines and metabolites: lorazepam, clonazepam, diazepam, alprazolam, temazepam, oxazepam glucuronide, lorazepam glucuronide, temazepam glucuronide, 7-aminoclonazepam, and 7-aminoflunitrazepam. In method comparison studies, a total of 103 urine samples containing benzodiazepines with LC-MS/MS values ranging from 1.3 ng/mL to 3257.6 ng/mL were tested with the ARKTM Benzodiazepine Plus Assay. Eighty samples tested positive with values ≥200 ng/mL and 23 samples tested negative with values <200 ng/mL by the ARKTM Benzodiazepine Plus assay. Of the 23 samples that tested negative, 21 had LC-MS/MS values <200 ng/mL and 2 samples were identified as midazolam samples with metabolite a-OH-midazolam levels >200 ng/mL. Conclusions The ARKTM Benzodiazepine Plus Assay enables the sensitive, rapid, and reliable measurement of benzodiazepines and their metabolites in human urine, without the need for hydrolysis or pretreatment. The assay addresses the present constraints of currently available benzodiazepine immunoassays, including their insufficient sensitivity, limited cross-reactivity to metabolites, and the requirement for glucuronidase pretreatment. The ARKTM Benzodiazepine Plus Assay is applicable to a wide range of clinical chemistry analyzers.

B-298 Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine by LC-MS/MS

Abstract Background The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine, a clonidine analog is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing. Methods Remnant urine samples were identified when tested positive for xylazine during routine clinical LC-MS/MS testing. All experiments were performed using an Acquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. Urines were diluted with xylazine-D6 internal standard, hydrolyzed using β-glucuronidase and separated on a 100mm x 2.1mm, 2.6μm Kinetex C18 column (Phenomenex) with a 4 minute step gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Xylazine and 4-OH-xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards (Cayman Chemical and/or Sigma Aldrich), resulting in the following transitions: xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137. Xylazine and 4-OH-xylazine were quantified using calibration curves prepared from 1 - 5,000 ng/mL in negative urine. Product ion scanning (PIS) was used to detect four additional xylazine metabolites, based on spectral matching to previously published full scan high resolution product ion spectra. The highest abundance fragment ions and identified retention times from PIS experiments were used to perform targeted detection for sulfone-xylazine (253>181, 253>147), oxo-xylazine (235>122, 235>114), OH-sulfone-xylazine (269>197, 269>163), and OH-oxo-xylazine (251>197, 251>148). Results Xylazine was detected at >=1 ng/mL in 38% (54/138) of fentanyl positive urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested but xylazine was detected in only 1 urine that did not contain fentanyl.Xylazine concentrations quantified in 61 remnant urines ranged from <1 to >5,000 ng/mL. 4-OH-xylazine concentrations ranged from <1 to 509 ng/mL. We observed five previously reported xylazine metabolites: 4-OH-xylazine, oxo-xylazine, sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine. The mean concentration ratio of 4-OH-xylazine-to-xylazine was 0.11. The mean peak area ratios (metabolite-to-xylazine) for sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine, and oxo-xylazine were 3.5, 3.8, 2.1, and <0.1, respectively. The proposed sulfone-xylazine and OH-oxo-xylazine metabolites were observed to have the most abundant signal of all detected metabolites, although there is currently no commercially available standard to facilitate quantification. Conclusions Xylazine and 4-OH-xylazine were quantitated in 61 patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in plasma and association of xylazine detection with clinical presentation and patient outcomes.

Suspect screening candidate exposure biomarkers of acetyl tributyl citrate and acetyl triethyl citrate after human oral administration

Acetyl tributyl citrate (ATBC) and acetyl triethyl citrate (ATEC) are widely used as plasticizers, but their metabolites as exposure biomarkers for biomonitoring, as well as approximate human metabolic pathways, are not well understood. This study addresses this knowledge gap by conducting suspect screening to propose specific metabolites in human urine as potential biomarkers of exposure and explore their kinetic profiles. Ten volunteers were administered deuterium labeled ATBC (ATBC-d3) and seven received ATEC or deuterium labeled ATEC (ATEC-d3), with urine samples collected over 48 h post-administration. Employing ultra-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC-qTOF/MS), six metabolites of ATBC were consistently detected, including (OH)3-ATBC-d3, ADBC-d3, OH-ADBC-d3, DBC, OH-DBC, and OH-DBA. For ATEC, four metabolites were identified: ADEC-d3, AMEC-d3, OH-ADEC-d3, and DEC. Based on their high detection frequency, relative response, and specificity to their parent compounds, ADBC-d3 and OH-ADBC-d3 were identified as promising candidate biomarkers for ATBC exposure, while ADEC-d3 emerged as a suitable biomarker for ATEC. Estimated urinary elimination half-lives ranged from 1.0 to 9.9 h for ATBC metabolites and 1.6 to 3.0 h for ATEC metabolites. One-compartment kinetic modeling provided preliminary insights into metabolite kinetics. The study suggests that ATBC may undergo more hydroxylation compared to ATEC, but further quantitative analysis is needed to confirm this observation. This research advances the understanding of ATBC and ATEC metabolism in humans, providing a foundation for future exposure assessments and toxicological studies. The identified biomarkers and preliminary metabolic profiles offer valuable starting points for biomonitoring and risk assessment of these alternative plasticizers.

Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic Metabolites.

Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring >100-fold higher doses for tumour induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette (ABC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the GSH conjugate of the highly toxic monomethylarsonous acid (MMAIII), MMA(GS)2, and the major human urinary arsenic metabolite dimethylarsinic acid (DMAV). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M4I-10 epitope was first mapped to an octapeptide (411HVQDFTA418F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in HEK293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMAIII, monomethylarsonic acid, dimethylarsinous acid or DMAV], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)2 or DMAV, despite a >3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17β-estradiol-17-(β-D-glucuronide). Abcc4(+/+) mouse embryonic fibroblasts (MEFs) were ~3-fold more resistant to arsenate than Abcc4(-/-) MEFs; however, further characterization indicated this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to differences in human and mouse arsenic toxicokinetics. Significance Statement Toxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. The human ATP-binding cassette transporter hABCC4 is a high affinity transporter of toxicologically important arsenic metabolites. Here we used multiple cell models to demonstrate that mouse Abcc4 does not protect cells against, or transport, any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.

3-epi-18β-Glycyrrhetinic Acid or Its Glucuronide, the Metabolites of Glycyrrhizinic Acid with Individual Differences, Correlated with Diagnostic Marker for Licorice-Induced Pseudoaldosteronism in Humans.

Licorice is a crude drug that is used in traditional Japanese Kampo medicine and is also used as a sweetener. Occasionally, it causes pseudoaldosteronism (PsA) as a side effect. The major symptoms include hypokalemia, hypertension, edema, and low plasma aldosterone levels. PsA might be caused by the metabolites of glycyrrhizinic acid (GL), a component of licorice. The development of PsA markedly varies among individuals; however, the factors that cause these individual differences remain unknown. In this study, 78 patients who consumed Kampo medicines containing licorice were enrolled, and their laboratory data, including serum potassium levels, plasma aldosterone concentrations (PAC), and the concentrations of GL metabolites in the residual blood and/or urine samples were evaluated. Of the 78 participants, 18β-glycyrrhetinic acid (GA), 3-epi-GA, 3-oxo-GA, 18β-glycyrrhetinyl-30-O-glucuronide (GA30G), and 3-epi-GA30G were detected in the serum samples of 65, 47, 63, 62, and three participants, respectively. Of the 29 urine samples collected, GA30G and 3-epi-GA30G were detected in 27 and 19 samples. 3-epi-GA30G is a newly found GL metabolite. Moreover, 3-epi-GA, 3-oxo-GA, and 3-epi-GA30G were identified in human samples for the first time. High individual differences were found in the appearances of 3-epi-GA in serum and 3-epi-GA30G in urine, and the concentrations of these metabolites were correlated with serum PsA markers. The inhibitory titers of 3-epi-GA, 3-oxo-GA, GA30G, and 3-epi-GA30G on human 11β-hydroxysteroid dehydrogenase type 2 were almost similar. These findings suggest that 3-epi-GA and/or 3-epi-GA30G are associated with individual differences in the development of PsA. SIGNIFICANCE STATEMENT: In this study, we detected 3-epi-18β-glycyrrhetinic acid (3-epi-GA) in human serum for the first time. We also identified 3-epi-18β-glycyrrhetinyl-30-O-glucuronide (3-epi-GA30G) as a novel glycyrrhizinic acid (GL) metabolite in human urine. These GL metabolite levels showed correlations with markers of PsA. Additionally, there are individual differences in whether they appear in the serum/urine. In conclusion, 3-epi-GA/3-epi-GA30G correlates with individual differences in the development of PsA.

In Vitro Metabolism of Quaternary Ammonium Compounds and Confirmation in Human Urine by Liquid Chromatography Ion-Mobility High-Resolution Mass Spectrometry.

Quaternary ammonium compounds (QACs) are high-production chemicals used as cleaning and disinfecting agents. Due to their ubiquitous presence in the environment and several toxic effects described, human exposure to these chemicals gained increasing attention in recent years. However, very limited data on the biotransformation of QACs is available, hampering exposure assessment. In this study, three QACs (dimethyl dodecyl ammonium, C10-DDAC; benzyldimethyl dodecylammonium, C12-BAC; cetyltrimethylammonium, C16-ATMAC) commonly detected in indoor microenvironments were incubated with human liver microsomes and cytosol (HLM/HLC) simulating Phase I and II metabolism. Thirty-one Phase I metabolites were annotated originating from 19 biotransformation reactions. Four metabolites of C10-DDAC were described for the first time. A detailed assessment of experimental fragmentation spectra allowed to characterize potential oxidation sites. For each annotated metabolite, drift-tube ion-mobility derived collision cross section (DTCCSN2) values were reported, serving as an additional identification parameter and allowing the characterization of changes in DTCCSN2 values following metabolism. Lastly, eight metabolites, including four metabolites of both C12-BAC and C10-DDAC, were confirmed in human urine samples showing high oxidation states through introduction of up to four oxygen atoms. This is the first report of higher oxidized C10-DDAC metabolites in human urine facilitating future biomonitoring studies on QACs.

Method optimization for a simultaneous determination of neonicotinoid, carbamate/thiocarbamate, triazole, organophosphate and pyrethroid pesticides and their metabolites in urine using UPLC-MS/MS

An accurate and sensitive method for the determination of a total of 23 pesticides and their metabolites in human urine has been optimised. The methodology is based on a previously published method based on solid-phase extraction with methanol and acetone followed by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in the selected reaction mode (SRM) with both positive and negative electrospray ionization (ESI+/-). The detection settings of the previous method, which allowed to determine the metabolites from 6 organophosphate and 2 pyrethroid pesticides, were optimised in order to include further pesticide groups, such as 11 neonicotinoids, 3 carbamates/thiocarbamates and 2 triazoles. The 5-windows method enduring 22 min was optimized with acceptable results in relation to accuracy (recoveries >75 %), precision (coefficients of variation <26 %) and linearity (R2> 0.9915). The limits of detection ranged between 0.012 ng/mL and 0.058 ng/mL. Samples from the German External Quality Assessment Scheme (G-EQUAS) encompassing 2 pyrethroids, 2 organophosphate and one neonicotinoid (6-chloronicotinic acid, a common metabolite of imidacloprid and acetamiprid) were analysed, and the latter, included in this newest optimization, provided good reference results. The method is optimal as a human biomonitoring tool for health risk assessment in large population surveys.

Automated online solid-phase extraction-tandem mass spectrometry detection for simultaneous analysis of acidic and alkaline catecholamines and their metabolites in human urine

Metabolic dysregulation of catecholamines (CAs) is implicated in various human diseases. Simultaneously analyzing these acidic and alkaline CAs and their metabolites poses a significant challenge for clinical detection. This study introduces an efficient method employing automated online solid-phase extraction coupled with tandem mass spectrometry (aoSPE-MS/MS). The method employs weak cation exchange (WCX) and mixed-mode anion exchange (MAX) adsorbents to fabricate an on-line solid-phase extraction (SPE) column, along with an automated injection and multi-valve switching capabilities. The setup allows for automated extraction and analysis of urine samples in 15 minutes while retaining a wide range of acidic and basic CAs and their metabolites. The applicability of this method was demonstrated by optimising the adsorbent dosage volume, extraction solvent, and extraction rate. The limits of detection (LODs) and limits of quantitation (LOQs) for the 8 CAs and their metabolites were determined using the aoSPE-MS/MS approach, with ranges of 0.0625 ∼ 62.5 ng/mL and 0.125 ∼ 125 ng/mL, respectively. Additionally, assessments were made on the linearity, accuracy, and precision within and between batches, as well as matrix and ionic effects, and spiked recoveries. The study discovered that the aoSPE-MS/MS technique simplifies operation, increases efficiency, saves time, and has low detection and quantification limits when detecting a wide range of acid and alkaline CAs and their metabolites in urine. The study successfully demonstrated the high-throughput and automated detection of the 8 CAs and their metabolites with varying acidity and alkalinity in human urine samples. This method is expected to be a potential powerful tool for clinical detection.

Method development with high-throughput enhanced matrix removal followed by UHPLC-QqQ-MS/MS for analysis of grape polyphenol metabolites in human urine

Grape and grape derived products contain many bioactive phenolics which have a variety of impacts on health. Following oral ingestion, the phenolic compounds and their metabolites may be detectable in human urine. However, developing a reliable method for the analysis of phenolic compounds in urine is challenging. In this work, we developed and validated a new high-throughput, sensitive and reproducible analytical method for the simultaneous analysis of 31 grape phenolic compounds and metabolites using Oasis PRiME HLB cleanup for sample preparation combined with ultra-performance liquid chromatography with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Using this new method, the accuracy achieved was 69.3 % ∼ 134.9 % (except for six compounds), and the recovery achieved was 52.4 % ∼ 134.7 % (except for two very polar compounds). For each of the 31 target analytes, the value of intra-day precision was less than 14.3 %. The value of inter-day precision was slightly higher than intra-day precision, with a range of 0.7 % ∼ 19.1 %. We report for the first time on the effect of gender and BMI on the accuracy and recovery of human urine samples, and results from analysis of variance (ANOVA), and principal component analysis (PCA) indicated there was no difference in the value of accuracy and recovery between different gender or BMI (>30) using our purposed cleanup and UHPLC-QqQ-MS/MS method. Overall, this newly developed method could serve as a powerful tool for analyzing grape phenolic compounds and metabolites in human urine samples.

2-Methyl-4'-(methylthio)-2-morpholinopropiophenone: A commercial photoinitiator being used as a new psychoactive substance

Synthetic cathinones, which are novel psychoactive substances, have caused major social problems worldwide. A substance called 2-methyl-4′-(methylthio)-2-morpholinopropiophenone (MMMP), which is employed as a commercial industrial photoinitiator for triggering polymerization, has a basic cathinone backbone; however, few reports regarding MMMP have been published. In the current study, three potential metabolites of MMMP—namely hydroxy-MMMP (HO-MMMP), HO-MMMP-sulfoxide (HO-MMMP-SO), and HO-MMMP-sulfone (HO-MMMP-SO2)—were successfully synthesized, and MMMP and these three potential metabolites were used as standards to establish an analytic method based on liquid chromatography–tandem mass spectrometry for the quantitative analysis of urine. This analytic method and related parameters—including dynamic range, limit of quantification, selectivity, precision, accuracy, carryover effect, matrix effect, interference, and dilution integrity—were optimized and validated. Forty urine samples from 1,691 individuals who abused drugs were determined to contain MMMP, HO-MMMP, HO-MMMP-SO, or HO-MMMP-SO2; the results of this study indicate that approximately 2.37 % of drug abusers in Taiwan consumed MMMP in 2023. These 40 urine samples were analyzed to investigate the metabolism of MMMP in humans. The results indicate that HO-MMMP-SO is the main metabolite in human urine. This study recommends HO-MMMP-SO with a concentration of 2 ng/mL as a target and cutoff value, respectively, for identifying individuals who have consumed MMMP.

Simultaneous determination for 5 human urinary metabolites of UR-144 and XLR-11 by LC-MS/MS

This study aims to explore how the abused synthetic cannabinoids in human urine can be determined qualitatively and quantitatively by using the LC-MS/MS method. As a method validation based on the current FDA guideline, we tested 55 human urine samples (5 from drug-free volunteers and 50 from drug abusers) and evaluated the presence of parent drugs (UR144 and XLR11) and their five metabolites (XLR-11-N-4-Hydroxypentyl, XLR-11-6-Hydroxyindole, UR-144-N-4-Hydroxypentyl, UR-144-N-5-hydroxypentyl, and UR-144-N-pentanoic). The experimental data showed that the values of low limit of detection (LOD) for these analytes ranged from 0.05 to 0.15 ng/mL. Their low limit of quantitation (LLOQ) value was 1.0 ng/mL. The values of linear coefficients of determination (R2) for these analytes ranged from 0.9914 to 0.9988. Among these 50 urine testing samples of drug abusers, the parent XLR-11 and UR-144 analytes were detected only in 4 samples (with a concentration of 2.14 − 4.5 ng/mL) and 3 samples (with a concentration of 3.2 − 5.4 ng/mL), respectively. At the same time, the XLR-11-N-4-Hydroxypentyl (with a concentration of 4.5 − 57.2 ng/mL) and XLR-11-6-Hydroxyindole (with a concentration of 4.8 − 51.7 ng/mL) metabolites were detected in 24 and 11 samples, respectively. In addition, the UR-144-N-4-Hydroxypentyl (with a concentration of 3.5 − 87.24 ng/mL), UR-144-N-5-hydroxypentyl (with a concentration of 14.5-83.9 ng/mL), and UR-144-N-pentanoic (with a concentration of 2.5 − 104.4 ng/mL) metabolites were found in 26, 29, and 38 samples, respectively.

Simultaneous determination of twelve neonicotinoids and six metabolites in human urine with isotope-dilution UPLC-Q-Orbitrap HRMS

The extensive global use of neonicotinoid insecticides (NNIs) has led to widespread human exposure, necessitating the development of effective methods for large-scale biomonitoring. However, current methods are inadequate in simultaneously and accurately detecting various NNIs or their metabolites (m-NNIs). In this study, we aimed to establish a robust method using solid-phase extraction (SPE)-ultra high performance liquid chromatography tandem Q-Orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) for the simultaneous determination of 12 NNIs and 6 m-NNIs in human urine. Samples were prepared using Oasis HLB 96 well plate with Isopropanol: methanol (7:3, v/v) as the elution solvent. The target compounds were separated using the Accucore RP-MS column and subsequently analyzed under parallel reaction monitoring mode. NTN32692 (m/z = 255.06433) was confirmed to be the specific metabolite of cycloxaprid for the further detection. Satisfactory recoveries (81.6–122.4 %) of the NNIs and m-NNIs were observed, with intra- (n = 3) and inter-day (n = 9) relative standard deviation (RSD) ranging from 0.8 % to 13.7 % and from 1.1 % to 18.6 %, respectively. Good linearity (R2 > 0.99) was achieved for all analytes. The limits of detection (LODs) for all target analytes ranged from 0.01 ng/mL to 0.65 ng/mL. This method was applied to urine samples collected from 10 children recruited from an agricultural area in China. Our study provides an effective method to identify and assess human exposure to NNIs and their metabolites.

Sample preparation for suspect screening of persistent, mobile and toxic substances and their phase II metabolites in human urine by mixed-mode liquid chromatography

Persistent, mobile and toxic substances have drawn attention nowadays due to their particular properties, but they are overlooked in human monitorization works, limiting the knowledge of the human exposome. In that sense, human urine is an interesting matrix since not only parent compounds are eliminated, but also their phase II metabolites that could act as biomarkers. In this work, 11 sample preparation procedures involving preconcentration were tested to ensure maximum analytical coverage in human urine using mixed-mode liquid chromatography coupled with high-resolution tandem mass spectrometry. The optimized procedure consisted of a combination of solid-phase extraction and salt-assisted liquid-liquid extraction and it was employed for suspect screening. Additionally, a non-discriminatory dilute-and-shoot approach was also evaluated. After evaluating the workflow in terms of limits of identification and type II errors (i.e., false negatives), a pooled urine sample was analysed. From a list of 1450 suspects and in-silico simulated 1568 phase II metabolites (i.e. sulphates, glucuronides, and glycines), 44 and 14 substances were annotated, respectively. Most of the screened suspects were diverse industrial chemicals, but biocides, natural products and pharmaceuticals were also detected. Lastly, the complementarity of the sample preparation procedures, columns, and analysis conditions was assessed. As a result, dilute-and-shoot and the Acclaim Trinity P1 column at pH = 3 (positive ionization) and pH = 7 (negative ionization) allowed the maximum coverage since almost 70 % of the total suspects could be screened using those conditions.

Capillary Zone Electrophoresis with Diode-Array Detection as a Tool to Monitor Variations in the Concentration of Organic Acids from the Tricarboxylic Acid Cycle in Human Urine

The tricarboxylic acid (TCA) cycle is responsible for the generation of energy in the human body and may inform about the energy status in different physiological and pathological conditions. It was developed a method to quantify the TCA cycle intermediates (α-ketoglutaric, citric, fumaric, lactic, malic, and succinic acids) by capillary zone electrophoresis with diode array detector (CZE-DAD) in indirect mode. The background electrolyte consisted of 24 mmol L–1 2,6-pyridine carboxylic acid, 76 mmol L–1 β-alanine, and 4 mmol L–1 hexadecyltrimethylammonium hydroxide (pH 4). The method was validated and presented a good linearity (R2 &gt; 0.9922) and adequate limits of detection (0.24-1.84 mg L–1) and quantification (0.74-5.57 mg L–1). The method was successfully applied to samples of individuals with obesity before being submitted to bariatric surgery and one year after the surgery. The citric, lactic, and malic acids were detected in the analyzed samples. To evaluate the changes in concentrations of the TCA intermediates, principal component analysis was carried out, and there was no significant difference between the two sets of samples. Altogether, the data obtained after applying this new method suggest that CZE-DAD could be used to detect and quantify the TCA cycle metabolites in human urine.

Human Urinary Metabolomics as Biomarkers in Tobacco Users: A Systematic Review.

Urine as a biofluid has been rarely used as a diagnostic fluid in oral diseases. The article aims to systematically review the utility of human urinary carcinogen metabolites as an approach for obtaining important information about tobacco and cancer. The following article reviews the use of urine and its metabolites as biomarkers in various lesions of the oral cavity including oral squamous cell carcinoma and as a screening method in evaluating tobacco and its components. A bibliographic comprehensive search was carried out in the main databases: PUBMED, SciELO, Google Scholar, VHL, and LILACS for articles that were published from 1985 to 2020. The inclusion criteria were "urinary metabolites," "oral cancer/HNSCC," "body fluids," "tobacco," and "metabolomics." A total of 55 articles were collected which included laboratory studies, systematic reviews, and literature of urinary metabolites in tobacco users. Most of the studies carried out show accurate results with high sensitivity of urinary metabolite biomarkers in individuals with tobacco-based habits and lesions caused by them. The review indicates that urinary metabolite analysis demonstrates its applicability for the diagnosis and prognosis of disease. Urine is a remarkable and useful biofluid for routine testing and provides an excellent resource for the discovery of novel biomarkers, with an advantage over tissue biopsy samples due to the ease and less invasive nature of collection.

Innovative analysis of diazepam, zolpidem and their main metabolites in human urine by micelle-to-solvent stacking in capillary electrophoresis

Diazepam and zolpidem are the most widely used medications for managing insomnia. However, significant concerns regarding the potential risks of misuse and abuse problems arose in many literatures. While urine analysis is a valuable diagnostic tool, a challenge arises from the fact that some parent drugs may remain undetectable in urine. This necessitates concurrent monitoring of their metabolites. Here, we described an innovative on-line sample preconcentration technique known as micelle to solvent stacking (MSS) for the analysis of diazepam, zolpidem, and their main metabolites in urine. Several key parameters warrant further discussion to optimize the MSS model, enhancing its performance in terms of sensitivity and resolution. After optimizing the conditions, we conducted a validation test, achieving high correlation coefficients (greater than 0.9977) for intra-day and inter-day regression lines. Additionally, both the relative standard deviation (RSD) and relative error (RE) remained below 6.10% and 12.55%, respectively. The limits of detection (LODs, S/N = 3) for all five analytes ranged from 2.0 to 56 ng/mL. Compared to the conventional capillary zone electrophoresis method, this new approach exhibited remarkable sensitivity enhancements, ranging from 123 to 235-fold. Upon applying this method to actual urine samples from patients, we successfully detected nordiazepam, zolpidem, and its metabolites. This simple and sensitive approach has promising applications in supporting patient medication safety and bolstering forensic investigations.

Quantitative Analysis of Buprenorphine, Norbuprenorphine, and Their Glucuronide Metabolites in Human Urine.

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 5ng/mL-1000ng/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.

Stable Isotope Labeling-Based Nontargeted Strategy for Characterization of the In Vitro Metabolic Profile of a Novel Doping BPC-157 in Doping Control by UHPLC-HRMS

Traditional strategies for the metabolic profiling of doping are limited by the unpredictable metabolic pathways and the numerous proportions of background and chemical noise that lead to inadequate metabolism knowledge, thereby affecting the selection of optimal detection targets. Thus, a stable isotope labeling-based nontargeted strategy combined with ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) was first proposed for the effective and rapid metabolism analysis of small-molecule doping agents and demonstrated via its application to a novel doping BPC-157. Using 13C/15N-labeled BPC-157, a complete workflow including automatic 13C0,15N0-13C6,15N2 m/z pair picking based on the characteristic behaviors of isotope pairs was constructed, and one metabolite produced by a novel metabolic pathway plus eight metabolites produced by the conventional amide-bond breaking metabolic pathway were successfully discovered from two incubation models. Furthermore, a specific method for the detection of BPC-157 and the five main metabolites in human urine was developed and validated with satisfactory detection limits (0.01~0.11 ng/mL) and excellent quantitative ability (linearity: 0.02~50 ng/mL with R2 &gt; 0.999; relative error (RE)% &lt; 10% and relative standard deviation (RSD)% &lt; 5%; recovery &gt; 90%). The novel metabolic pathway and the in vitro metabolic profile could provide new insights into the biotransformation of BPC-157 and improved targets for doping control.

Determination of Bisphenols and Phthalate Metabolites in Human Urine with Ultra-performance Liquid Chromatography/Tandem Mass Spectrometry

Determination of Bisphenols and Phthalate Metabolites in Human Urine with Ultra-performance Liquid Chromatography/Tandem Mass Spectrometry