Metabolites In Human Urine Samples Research Articles

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.

In Vitro and In Vivo Biotransformation Profiling of 6PPD-Quinone toward Their Detection in Human Urine.

The antioxidant N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its oxidized quinone product 6PPD-quinone (6PPD-Q) in rubber have attracted attention due to the ecological risk that they pose. Both 6PPD and 6PPD-Q have been detected in various environments that humans cohabit. However, to date, a clear understanding of the biotransformation of 6PPD-Q and a potential biomarker for exposure in humans are lacking. To address this issue, this study presents a comprehensive analysis of the extensive biotransformation of 6PPD-Q across species, encompassing both in vitro and in vivo models. We have tentatively identified 17 biotransformation metabolites in vitro, 15 in mice in vivo, and confirmed the presence of two metabolites in human urine samples. Interestingly, different biotransformation patterns were observed across species. Through semiquantitative analysis based on peak areas, we found that almost all 6PPD-Q underwent biotransformation within 24 h of exposure in mice, primarily via hydroxylation and subsequent glucuronidation. This suggests a rapid metabolic processing of 6PPD-Q in mammals, underscoring the importance of identifying effective biomarkers for exposure. Notably, monohydroxy 6PPD-Q and 6PPD-Q-O-glucuronide were consistently the most predominant metabolites across our studies, highlighting monohydroxy 6PPD-Q as a potential key biomarker for epidemiological research. These findings represent the first comprehensive data set on 6PPD-Q biotransformation in mammalian systems, offering insights into the metabolic pathways involved and possible exposure biomarkers.

An eco-friendly sample preparation procedure based on air-assisted liquid-liquid microextraction for the rapid determination of phthalate metabolites in urine samples by liquid chromatography-tandem mass spectrometry

Urinary phthalate metabolite (mPAEs) analysis is a reliable tool for assessing human exposure to phthalates. With growing interest in urinary biomonitoring of these metabolites, there is a need for fast and sensitive analytical methods. Therefore, a simple, rapid procedure for simultaneous determination of fifteen phthalate metabolites in human urine samples by liquid chromatography-tandem mass spectrometry was developed. The novelty of the present procedure is based on the use of diethyl carbonate as a green biobased extraction solvent and air-assisted liquid-liquid microextraction (AALLME) as a sample preparation step. A Plackett-Burman design was used for screening the factors that influence the AALLME extraction efficiency of mPAEs. The effective factors were then optimized by response surface methodology using a central composite rotatable design. Under the optimized conditions, good linearity can be achieved in a concentration range of 1.0–20.0 ng mL-1 with correlation coefficients higher than 0.99. The repeatability and reproducibility precision were in the range of 2–12% and 1–10% respectively. Recoveries ranging from 90% to 110%. This, and the low limits of detection, ranging from 0.01 to 0.05 ng mL-1, make the proposed procedure sensitive and suitable for human biomonitoring of phthalate exposures. For proof-of-principle, the new method was used to measure the urinary concentrations of mPAEs in 20 urine samples from Brazilian women. The high frequency of detections and in part high concentrations of mPAEs indicate to widespread exposure to several phthalates among Brazilian women.

Simultaneous biomonitoring of volatile organic compounds’ metabolites in human urine samples using a novel in-syringe based fast urinary metabolites extraction (FaUMEx) technique coupled with UHPLC-MS/MS analysis

Assessing the impact of human exposure to environmental toxicants is often crucial to biomonitoring the exposed dose. In this work, we report a novel fast urinary metabolites extraction (FaUMEx) technique coupled with UHPLC-MS/MS analysis for the highly sensitive and simultaneous biomonitoring of the five major urinary metabolites (thiodiglycolic acid, s-phenylmercapturic acid, t,t-muconic acid, mandelic acid, and phenyl glyoxylic acid) of common volatile organic compounds' (VOCs) exposure (vinyl chloride, benzene, styrene, and ethylbenzene) in human. FaUMEx technique comprises of two-steps, liquid-liquid microextraction was performed first in an extraction syringe using 1 mL of methanol (pH 3) as an extraction solvent and then, the extractant was passed through a clean-up syringe (pre-packed-with various sorbents including 500 mg anhydrous MgSO4, 50 mg C18, and 50 mg SiO2) to obtain the high order of matrice clean-up and preconcentration efficiency. The developed method displayed excellent linearity, and the correlation coefficients were >0.998 for all the target metabolites with detection and quantification limits of 0.02–0.24 ng mL−1 and 0.05–0.72 ng mL−1, respectively. Furthermore, the matrix effects were < ±5%, and inter and intra-day precision were <9%. Moreover, the presented method was applied and validated to real sample analysis for biomonitoring of VOC's exposure levels. The results showed that the developed FaUMEx-UHPLC-MS/MS method is fast, simple, low-cost, low-solvent consumption, high sensitivity with good accuracy and precision for five targeted urinary VOCs' metabolites. Therefore, the presented dual-syringe mode FaUMEx strategy with UHPLC-MS/MS technique can be applied to biomonitoring of various urinary metabolites to assess human exposure to environmental toxicants.

Development of a high-throughput differential mobility separation–tandem mass spectrometry (DMS-MS/MS) method for clinical urine drug testing

IntroductionDifferential mobility separation (DMS) is an analytical technique used for rapid separation of ions and isomers based on gas phase mobility prior to entering a mass spectrometer for analysis. The entire DMS process is accomplished in fewer than 20 ms and can be used as a rapid alternative to chromatographic separation. ObjectiveThe primary objective was to evaluate the utility of DMS-tandem mass spectrometry (DMS-MS/MS) as a replacement for immunoassay-based clinical toxicology testing. MethodsA sensitive DMS-MS/MS method was developed and validated for simultaneous identification of 33 drugs and metabolites in human urine samples. After DMS optimization, the method was validated and used to screen 56 clinical urine samples. These results were compared to results obtained by immunoassay. ResultsThe DMS-MS/MS method achieved limits of detection ranging from 5 to 100 ng/mL. Moreover, the total analysis time was 2 min per sample. For the method performance evaluation, DMS-MS/MS results were compared with previously obtained urine toxicology immunoassay results. DMS-MS/MS showed higher sensitivity and identified 20% more drugs in urine, which were confirmed by LC-MS/MS. ConclusionThe DMS-MS/MS as applied in our lab demonstrated the capability for rapid drug screening and provided better analytical performance than immunoassay.

Human Biomonitoring of T-2 Toxin, T-2 Toxin-3-Glucoside and Their Metabolites in Urine through High-Resolution Mass Spectrometry.

The metabolic profile of T-2 toxin (T-2) and its modified form T-2-3-glucoside (T-2-3-Glc) remain unexplored in human samples. Therefore, the present study aimed to investigate the presence of T-2, T-2-3-Glc and their respective major metabolites in human urine samples (n = 300) collected in South Italy through an ultra-high performance liquid chromatography (UHPLC) coupled to Q-Orbitrap-HRMS methodology. T-2 was quantified in 21% of samples at a mean concentration of 1.34 ng/mg Crea (range: 0.22–6.54 ng/mg Crea). Almost all the major T-2 metabolites previously characterized in vitro were tentatively found, remarking the occurrence of 3′-OH-T-2 (99.7%), T-2 triol (56%) and HT-2 (30%). Regarding T-2-3-Glc, a low prevalence of the parent mycotoxin (1%) and its metabolites were observed, with HT-2-3-Glc (17%) being the most prevalent compound, although hydroxylated products were also detected. Attending to the large number of testing positive for T-2 or its metabolites, this study found a frequent exposure in Italian population.

CE-DAD-MS/MS in the simultaneous determination and identification of selected antibiotic drugs and their metabolites in human urine samples.

In this study, a new analytical method was developed and validated for the simultaneous analysis of antibiotic drugs (amoxicillin, cefotaxime, ciprofloxacin, clindamycin, linezolid, metronidazole) and their metabolites (amoxycilloic acid, amoxicillin diketopiperazine, 3-desacetyl cefotaxime lactone, clindamycin sulfoxide, ciprofloxacin piperazinyl-N4-sulfate, linezolid N-oxide, metronidazole-OH) in human urine. Capillary electrophoresis (CE) along with the tandem mass spectrometry (MS/MS) was used to determine and identify all analytes. Appropriate conditions for MS/MS measurements along with the use of the central composite design were optimized. The effects of different analytical conditions (the composition, the concentration, and the pH value of the background electrolyte, the time and pressure of the injection, the capillary temperature and influence of the organic modifier) on the migration and separation of antibiotic drugs and metabolites were examined using the CE-DAD. The analytical procedure was linear for concentrations ranging from 20 to 1000ng/mL, with determination coefficients higher than 0.99 for all the analytes. The validated analytical procedure was then applied to the measurement of antibiotic drugs and their metabolites in human urine samples.

Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis.

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.

Development of an analytical method based on solid-phase extraction and LC-MS/MS for the monitoring of current-use pesticides and their metabolites in human urine

Pyrethroids, organophosphorus pesticides and fipronil have been listed as priority chemicals in human biomonitoring studies because of their wide use and potential health effects in humans. The determination of 13 pesticides, including pyrethroids (deltamethrin, cypermethrin, permethrin, cyfluthrin, bifenthrin), organophosphorus (chlorpyrifos, chlorpyrifos-methyl, and malathion), fipronil, neonicotinoids (imidacloprid, acetamiprid and thiacloprid) and triazole (prothioconazole), together with 13 corresponding metabolites in human urine samples was achieved by solid-phase extraction and analysis by liquid chromatography coupled to tandem mass spectrometry. All targeted compounds, except malathion dicarboxylic acid, were measured with a mean within-accuracy (n = 5) of 71%–114% (RSD: 1%–14%) and between-run (n = 15) accuracy of 80%–118% (RSD: 2%–14%). Limits of quantitation of the targeted analytes ranged from 0.1 to 16 pg/mL. The detection result of urine samples from 25 volunteers indicated that the detection frequencies of 3,5,6-trichloro-2-pyridinol (median: 448 pg/mL), 6-chloropyridine-3-carboxylic acid (median: 193 pg/mL), 2-methyl-3-phenylbenzoic acid (median: 181 pg/mL), 3-phenoxybenzoic acid (median: 99 pg/mL), 2-isopropyl-6-methyl-4-pyrimidinol (median: 77 pg/mL), cyfluthrin (median: 59 pg/mL), cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (cis-DCCA, median: 53 pg/mL), trans-DCCA (median: 25 pg/mL), prothioconazole (median: 21 pg/mL), imidacloprid (median: 7 pg/mL), and prothioconazole-desthio (median: 1 pg/mL) were > 50%. The obtained results show that the validated method is suitable for the human biomonitoring of these current-use pesticides and their metabolites.

Qualitative and quantitative analysis of opiate and related metabolites in human urine samples by UPLC-MS/MS

Although opioids such as morphine are regarded as essential drugs for clinical pain treatment, they are forbidden in athletic events. To maintain the fairness of the competition, rapid detection and quantitative analysis methods are employed. The present study developed a sensitive and robust LC-MS/MS method for the simultaneous quantification of morphine, codeine, and active metabolites, including morphine 3β-glucuronide, morphine 6β-glucuronide, codeine 6β-glucuronide. With a methanol/0.1% formic acid gradient, morphine, codeine, and metabolites were separated on an F5 column. We detected all compounds by multiple reaction mode (MRM) in the positive mode. In addition, the method had a range of reliable concentration-responses of 1.25–100 ng/mL (r2 > 0.995) for morphine, codeine, and metabolites in urine. The selectivity of the method was established in six different human subjects. For accuracy and precision, they were within 15% of the nominal values. There was no carryover, and the matrix effect was within the allowable range. The average storage stability at Low, Mid, and High QC levels met the requirements, respectively. The assay fulfilled all predefined acceptance criteria. A specific, stable, and precise LC-MS/MS method has been validated for morphine, codeine, and active metabolite quantification in human urine. Moreover, this straightforward method is reproducible and robust to support future morphine, codeine, active metabolite-related pharmacokinetics, bioequivalence, and clinical studies.

New sample preparation method to analyse 15 specific and non-specific pesticide metabolites in human urine using LC-MS/MS

This study presents a novel sample preparation method for the determination of both specific and non-specific pesticide metabolites in human urine samples. The method combines a deconjugation step with QuEChERS-based method and solid-phase extraction. In total, 15 pesticide metabolites (diethyl phosphate; diethyl thiophosphate; dimethyl phosphate; diethyl thiophosphate; 2,4-dichlorophenoxyacetic acid; 3-phenoxybenzoic acid; 4-fluoro-3-phenoxybenzoic acid; coumaphos; diethyl dithiophosphate; malathion dicarboxylic acid; p-nitrophenol; cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid; 3,5,6-trichloro-2-pyridinol; N,N-diethyl-3-methylbenzamid and 2-isopropyl-4-methyl-6-hydroxypyrimidine) were separated using liquid chromatography coupled to a mass spectrometer and isotope dilution method for quantitation. The method was validated using recovery tests with recoveries generally ranging from 80 to 120%. Additionally, 20 urine samples collected from South African children were analysed using the presented method. The median levels of pesticide metabolites found in the urine samples ranged from not detected (N,N-diethyl-3-methylbenzamid) to 22.36 µg/g creatinine (dimethyl phosphate). The novel method developed in this study is sensitive, selective, robust and reproducible while also conserving the amount of sample, chemicals, material and time required. Due to the low limits of detection obtained for individual pesticide metabolites, the method is capable of quantifying trace levels of pesticide metabolites in urine, which thus makes it an ideal tool for biomonitoring studies.

A Novel LC-MS Based Targeted Metabolomic Approach to Study the Biomarkers of Food Intake.

In this work, an integrated strategy is developed for rapid discovery, precise identification, and automated quantification for the biomarkers of food intake (BFIs) for specific food exposure using an ultra-high-pressure liquid chromatography-high-resolution mass spectrometry (MS) based targeted metabolomics approach. Using whole grain (WG) wheat intake as an example, the combination of paired mass distance networking and parallel reaction monitoring analysis is applied to selectively extract and identify WG metabolites in human urine samples. As a result, a total of 76 wheat phytochemical-derived metabolites, including 17 alkylresorcinol metabolites, 20 benzoxazinoid derivatives, and 39 phenolic acid metabolites are identified. Subsequently, a MS spectral database consisting of the identified metabolites is created by mzVault. The characteristics of identified metabolites from the database are incorporated into the TraceFinder software to establish a quantification platform. Using a standardized urine sample, the authors are able to simultaneously quantify both free and conjugated (sulfate and glucuronide) WG wheat metabolites in real samples without further enzymatic hydrolysis, which is validated by using authentic standards to quantify these metabolites. This novel strategy opens the window to study the biomarkers of specific food intake and make it feasible to validate the BFIs in large-scale human studies.

Development of a HILIC-MS/MS method for the quantification of histamine and its main metabolites in human urine samples

An LC-MS/MS method was developed enabling the separation and quantification of histamine and its main metabolites (imidazole acetaldehyde, imidazole acetic acid, methyl imidazole acetic acid, methyl histamine, acetyl histamine) in urine samples. A fast separation was achieved in 10 min on two HILIC columns connected in series by adopting a linear gradient followed by an isocratic hold. The sample preparation consisted of a simple dilution step wherein 10 μL of urine was diluted with acetonitrile (ACN) to a final volume comprising 95% ACN. For methyl imidazole acetic acid, an additional dilution step was incorporated due to its high natural levels. Hereafter, the samples were stored at −20 °C and centrifuged prior to injection. Matrix matched calibrators were unavailable due to the endogenous occurrence of the compounds of interest. The occurrence of matrix effects and the lack of labeled internal standards prompted the use of the standard addition method as a viable alternative to solvent calibration. The validation of the method entailed matrix effects, accuracy and precision and was performed in compliance with the recent guidelines on endogenous compounds issued by the International Conference of Harmonization (ICH). The method was then adopted for the quantification of histamine and its metabolites in human urine samples collected from healthy volunteers and patients suffering from gastrointestinal discomfort.

Development of an ELISA for detection of mitragynine and its metabolites in human urine

An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.

A Stable Isotope Dilution Assay for Multi-class Antibiotics in Pregnant Urines by LC–MS/MS

An accurate assay suitable for human biomonitoring of multiclass antibiotics preferred for veterinary use is currently lacking. In this study, we developed a liquid chromatography-triple quadrupole tandem mass spectrometry (LC–QqQ–MS/MS) method for analysis of 41 representative antibiotics (eight categories) and their two metabolites in human urine samples. Additionally, along with matrix-matched calibration and solid-phase extraction (SPE), use of 36 stable isotopically labeled internal standards (SIL-ISs) compensated for matrix effects and helped in the accuracy of quantitation. The absolute matrix effects (MEs), the linearity of limits of detection (LODs) and limits of quantitation (LOQs), stability, accuracy and precision of this method were validated. The validation parameters obtained for the targeted compounds are as follows: MEs (68.0–129.2%), LODs (0.016–1.481 ng mL−1), LOQs (0.050–4.812 ng mL−1), and higher recoveries (80.3–112.5%) except cyadox and cefaclor. The regression coefficients (r2) of targeted compounds at a concentration range of 0.05–100 ng mL−1 ranged from 0.9958 to 0.9998. The intra- and inter-precisions were obtained with a relative standard deviation (RSD%) < 13%. The method was successfully applied to analyze 302 urine samples of pregnant women from the China-Anhui Birth Cohort (C-ABC), and 36 of the selected antibiotics and two metabolites were detected. The total detection frequencies were 52.6% (fluoroquinolones), 40.4% (tetracyclines), 33.1% (sulfonamides), 16.9% (chloramphenicols), 11.6% (macrolides), 10.9% (β-lactams), 2.3% (lincosamides), and 0.3% (quinoxalines), respectively. The developed method has been successfully employed to obtain reliable quantitative results in the urine of pregnant women.

Monitoring of organophosphate and pyrethroid metabolites in human urine samples by an automated method (TurboFlow™) coupled to ultra-high performance liquid chromatography-Orbitrap mass spectrometry

There is a growing concern to pesticide exposure in humans, being the organophosphate (OP), carbamate and pyrethroid the most commonly used insecticides. Therefore, a fast, simple and automated screening method based on ultra-high performance liquid chromatography coupled to Orbitrap high resolution mass spectrometry (UHPLC-Orbitrap-MS) has been developed to determine six OPs metabolites (3,5,6-trichloro-2-pyridinol “TCPy”, 2-isopropyl-6-methyl-4-pyrimidinol “IMPY”, 4-nitrophenol “PNP”, 4-nitrophenol-d4 “PNP-d4″, 3-chloro-7-hydroxy-4-methylcoumarin “CMHC” and 2-diethylamino-6-methylpyrimidin-4ol “DEAMPY”) and three pyrethroid metabolites (3-phenoxybenzoic acid “3-PBA”, 4-fluoro-3-phenoxybenzoic acid “4-F-3-PBA” and 3-(2,2-dichlorovinyl)-2,2-dimethyl-1-cyclopropanecarboxylic acid “cis/trans-DCCA”) in human urine samples. Off-line solid phase extraction (SPE), on-line SPE and turbulent flow chromatography (TurboFlow™) extraction methodologies were compared, obtaining better results when TurboFlow™ was applied, achieving a total analysis time of 13.83 min. Several validation parameters were tested, obtaining suitable results for all studied compounds. Recoveries ranged from 70 to 116%, meanwhile repeatability and reproducibility were lower than or equal to 7% and 13%, respectively. The limits of quantification (LOQs) were set in the range from 1 μg/L to 5 μg/L (except for DCCA at 10 μg/L). Finally, thirty seven urine samples from women and men living near the agricultural areas of Almeria (Spain) were analyzed to know and control the contamination on the human health. PNP, DEAMPY and IMPY were detected in six urine samples at trace levels except IMPY, which was detected at 3.44 μg/L in one of the samples. Additionally, a post-targeted and untargeted analysis were carried out in the samples, identifying eleven insecticides such as bensulide or cinerin II, one insecticide metabolite, fipronil sulfone, one drug metabolite, carboxyibuprofen and two nicotine metabolites, cotinine and 2-(1-pyrrolidinyl)isonicotinic acid, in several samples.

A Novel Enzyme Immunoassay for the Detection of Buprenorphine, Norbuprenorphine and Their Glucuronides in Urine.

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.

A novel extraction method for simultaneous determination of neonicotinoid insecticides and their metabolites in human urine

An LLME-LC-MS/MS method for the detection of trace levels of six neonicotinoid insecticides and their metabolites in human urine samples.

Rapid and sensitive determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in human urine samples using UHPLC-MS/MS

Bisphenol analogues, amphenicol antibiotics, and phthalate have widely aroused public concerns due to their adverse effects on human health. In this study, a rapid and sensitive method for determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in the urine based on ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry was developed and validated. The sample pretreatment condition on the base of mixed-mode anion-exchange (Oasis MAX) SPE was optimized to separate bisphenol analogues and amphenicol antibiotics from phthalate metabolites: the former were detected with a mobile phase of 0.1% ammonium water solution/methanol containing 0.1% ammonium water solution in negative mode, whereas the latter were determined with a mobile phase of 0.1% acetic acid solution/acetonitrile containing 0.1% acetic acid in negative mode. The limits of detection were less than 0.26ng/mL for bisphenol analogues, 0.12ng/mL for amphenicol antibiotics, and 0.14ng/mL for phathalate metabolites. The recoveries of all target analytes in three fortification levels ranged from 72.02 to 117.64% with the relative standard deviations of no larger than 14.51%. The matrix effect was adjusted by isotopically labeled internal standards. This proposed method was successfully applied to analyze 40 actual urines and 13 out of 18 studied compounds were detected. Graphical abstract Simultaneous determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in human urine samples.

Sensitive quantification of 5F-PB-22 and its three metabolites 5F-PB-22 3-carboxyindole, B-22 N-5-hydroxypentyl and PB-22 N-pentanoic acid in authentic urine specimens obtained from four individuals by liquid chromatography–tandem mass spectrometry

Urine is the most suitable specimen to collect from individuals because of noninvasiveness and relatively large volumes obtainable. In authentic urine specimens, however, synthetic cannabinoids having the structures of quinolinyl ester indoles, such as 5F-PB-22, PB-22 and BB-22, in unchanged forms as well as their metabolites, have not been quantified yet. Therefore, the aim of this study was to establish a sensitive analytical method for the quantification of 5F-PB-22 and its three metabolites 5F-PB-22 3-carboxyindole, PB-22 N-5-hydroxypentyl and PB-22 N-pentanoic acid in authentic urine samples in four cases. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine via liquid-liquid extraction. The identification and quantification were performed using the QTRAP type of a liquid chromatography–tandem mass spectrometer. The limits of detection were 3–30 pg/mL and their summed quantitation range was 10–10,000 pg/mL. The devised method was applied to quantify these compounds in authentic urine specimens obtained from four individuals. The levels of 5F-PB-22 were 5.1, 13.6, 94.7 and 470 pg/mL; those of 5F-PB-22 3-carboxyindole were 8.25, 3.39, 23.2 and 880 ng/mL; and those of PB-22 N-pentanoic acid were 12.0, 57.4, 959 and 2090 pg/mL, respectively, in four unhydrolyzed urine samples, and the levels of PB-22 N-5-hydroxypentyl could be quantified as 29.9 and 131 pg/mL in two unhydrolyzed urine samples. The 5F-PB-22 and its metabolites PB-22 N-5-hydroxypentyl have been detected from authentic human urine samples for the first time. Also, this is the first report dealing with the quantification of the three metabolites in human urine samples.