Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.     

Phase I and II metabolites of speciogynine, a diastereomer of the main Kratom alkaloid mitragynine, identified in rat and human urine by liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry

Mitragyna speciosa (Kratom in Thai), a Thai medical plant, is misused as herbal drug of abuse. Besides the most abundant alkaloids mitragynine (MG) and paynantheine (PAY), several other alkaloids were isolated from Kratom leaves, among them the third abundant alkaloid is speciogynine (SG), a diastereomer of MG. The aim of this present study was to identify the phase I and II metabolites of SG in rat urine after the administration of a rather high dose of the pure alkaloid and then to confirm these findings using human urine samples after Kratom use. The applied liquid chromatography coupled to low- and high-resolution mass spectrometry (LC-HRMS-MS) provided detailed information on the structure in the MS(n) mode particularly with high resolution. For the analysis of the human samples, the LC separation had to be improved markedly allowing the separation of SG and its metabolites from its diastereomer MG and its metabolites. In analogy to MG, besides SG, nine phase I and eight phase II metabolites could be identified in rat urine, but only three phase I and five phase II metabolites in human urine. These differences may be caused by the lower SG dose applied by the user of Kratom preparations. SG and its metabolites could be differentiated in the human samples from the diastereomeric MG and its metabolites comparing the different retention times determined after application of the single alkaloids to rats. In addition, some differences in MS(2) and/or MS(3) spectra of the corresponding diastereomers were observed.     

Confirmation testing of amphetamines and designer drugs in human urine by capillary electrophoresis-ion trap mass spectrometry

Monitoring of amphetamines and designer drugs in human urine is a timely topic in clinical toxicology, surveillance of drug substitution, forensic science, drug testing at the workplace, and doping control. Confirmation testing of urinary amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and 3,4-methylenedioxyamphetamine (MDA) by capillary electrophoresis (CE) combined with atmospheric pressure electrospray ionization and ion trap mass spectrometry (MS) is described. Using an aqueous pH 4.6 buffer composed of ammonium acetate/acetic acid, CE-MS and CE-MS2 provided data that permitted the unambiguous confirmation of these drugs in external quality control urines. Furthermore, other drugs of abuse present in alkaline urinary extracts, including methadone and morphine, could also be monitored. The data presented illustrate that the sensitivity achieved with the benchtop MS is comparable to that observed by CE with UV absorption detection. CE-MS2 is further shown to be capable of identifying comigrating compounds, including the comigration of amphetamine with nicotine.     

High pressure optical studies of the chirality and phase behaviour of liquid crystalline blue phases

The blue phase (BP) polymorphism of chiral S -(+)-4-(2-methylbutyl)phenyl-4-decyloxy benzoate (CE6) and S -(+)-4-(2-methylbutyl)phenyl-4-dodecyloxy benzoate (CE7) was studied at elevated transition pressures up to 280MPa using optical activity measurements. The increased density causes an increase in phase chirality which in the case of CE6 manifests itself in the disappearance of BP II (BP I-BP II-BP III triple point) and in the case of CE7 in the appearance of BP II. At atmospheric pressure, CE6 exhibits BP I, BP II and BP III, while CE7 only possesses BP I. This pressure behaviour of CE6 and CE7 is contrary to that of cholesteryl nonanoate, which has been investigated previously. This result is particularly interesting when discussed in connection with the effect of elongation of the terminal n alkylcarboxy chain of the cholesteryl n -alkanoates and of the terminal n -alkyloxy chain in the homologous series to which CE6 and CE7 belong. In the first case the phase chirality increases; in the second case it decreases. Since CE6 shows an obvious pretransitional optical activity in its isotropic liquid phase, pretransition and BP phase behaviour could be compared with each other at elevated transition pressures and temperatures. For those transition pressures or temperatures where anomalies in the BP phase behaviour occur, anomalies in the pretransition behaviour are also observed.     

Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection

A method using capillary electrophoresis-mass spectrometry (CE-MS) was developed for the structural elucidation of bupivacaine and metabolites in rat urine. Prior to CE-MS analysis, solid-phase extraction (SPE) was used for sample cleanup and preconcentration purposes. Exact mass and tandem mass spectrometric (MS/MS) experiments were performed to obtain structural information about the unknown metabolites. Two instruments with different mass analyzers were used for mass spectrometric detection. A quadrupole time-of-flight (Q-TOF) and a magnetic sector hybrid instrument were coupled to CE and used for the analysis of urine extracts. Hydroxybupivacaine as well as five other isomerically different metabolites were detected including methoxylated bupivacaine.     

Achiral and chiral determination of tramadol and its metabolites in urine by capillary electrophoresis

An achiral and chiral separation for the determination of tramadol and its main metabolite O-demethyltramadol in urine samples by CE with UV detection was developed. It was possible to separate tramadol and its phase I and phase II metabolites in one single run using a borate buffer. Furthermore, the simultaneous chiral separation of tramadol and the phase I metabolites was achieved using carboxymethyl-beta-cyclodextrin as chiral selector. To reach the required limits of quantification for the analytes, a preconcentration by solid-phase extraction for the achiral assay and by liquid-liquid extraction for the chiral assay was used. The methods were validated and their applicability was shown by the determination of tramadol and O-demethyltramadol in urine samples.     

Automated high-performance liquid chromatographic determination of antipyrine and its main metabolites in plasma, saliva and urine, including 4,4'-dihydroxyantipyrine

A rapid, selective and sensitive method was developed for the determination of antipyrine and its main metabolites in plasma, saliva and urine by an automated high-performance liquid chromatographic system. Using a MOS-Hypersil reversed-phase column with a phosphate buffer--acetonitrile mobile phase, baseline separation of antipyrine, its metabolites 3-hydroxymethylantipyrine, norantipyrine and 4-hydroxyantipyrine, and the internal standard, phenacetin, was achieved within 6 min. Factors regarding the accuracy and precision of the method and the stability of phase I metabolites during sample preparation are discussed, taking into account certain drawbacks of previously published methods. Based on the same chromatographic system a method was developed for the assay of 4,4'-dihydroxyantipyrine in urine. This compound is an important metabolite of antipyrine in the rat, representing 12.6 +/- 1.8% of the administered dose (n = 18).     

2-Methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling “research chemicals.” The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors’ well-established gas chromatography–mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MSⁿ) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography–high-resolution linear ion trap mass spectrometry (LC-HR-MSⁿ) and the phase II metabolites by LC-HR-MSⁿ. The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user’s dose, 2-MPA and its metabolites were identified in rat urine using the authors’ GC-MS and the LC-MSⁿ screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.     

Qualitative studies on the metabolism and the toxicological detection of the fentanyl-derived designer drugs 3-methylfentanyl and isofentanyl in rats using liquid chromatography–linear ion trap–mass spectrometry (LC-MS<Superscript>n</Superscript>)

The opioid 3-methylfentanyl, a designer drug of the fentanyl type, was scheduled by the Controlled Substance Act due to its high potency and abuse potential. To overcome this regulation, isofentanyl, another designer fentanyl, was synthesized in a clandestine laboratory and seized by the German police. The aims of the presented study were to identify the phase I and phase II metabolites of 3-methylfentanyl and isofentanyl in rat urine, to identify the cytochrome P450 (CYP) isoenzymes involved in their initial metabolic steps, and, finally, to test their detectability in urine. Using liquid chromatography (LC)–linear ion trap–mass spectrometry (MSⁿ), nine phase I and five phase II metabolites of 3-methylfentanyl and 11 phase I and four phase II metabolites of isofentanyl could be identified. The following metabolic steps could be postulated for both drugs: N-dealkylation followed by hydroxylation of the alkyl and aryl moiety, hydroxylation of the propanamide side chain followed by oxidation to the corresponding carboxylic acid, and, finally, hydroxylation of the benzyl moiety followed by methylation. In addition, N-oxidation of isofentanyl could also be observed. All hydroxy metabolites were partly excreted as glucuronides. Using recombinant human isoenzymes, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were found to be involved in the initial metabolic steps. Our LC-MSⁿ screening approach allowed the detection of 0.01 mg/L of 3-methylfentanyl and isofentanyl in spiked urine. However, in urine of rats after the administration of suspected recreational doses, the parent drugs could not be detected, but their common nor metabolite, which should therefore be the target for urine screening.     

Identification and structural characterization of febuxostat metabolites in rat serum and urine samples using UHPLC–QTOF/MS

Febuxostat is a novel nonpurine type of highly selective xanthine oxidoreductase inhibitor. A rapid and sensitive ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry method for simultaneous separation and determination of febuxostat and its metabolites in rat serum and urine was developed at various time points after oral administration to the rats. The febuxostat metabolites were predicted by biotransformation software and transformed to a personal compound database to quickly determine the possible metabolites from the MS1 data. The possibility of the MS/MS fragmentation was calculated by the Molecular Structure Correlator software. As a result, five phase I and two phase II metabolites in rat serum, and seven phase I and three phase II metabolites in rat urine were identified, of which four metabolites (M2, M5, M6, M7) have not been reported before. The metabolite toxicities are predicted, and the results are helpful for the design of new xanthine oxidoreductase inhibitors.     

Studies on the metabolism of the α-pyrrolidinophenone designer drug methylenedioxy-pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-high-resolution MS and its detectability in urine by GC-MS

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples.     

Novel approach for developing urinary nucleosides profile by capillary electrophoresis-mass spectrometry

A simple, rapid and efficient capillary electrophoresis-mass spectrometry (CE-MS) method was developed to analyze urinary nucleosides for the first time. The composition of CE buffer and MS parameters were systematically optimized. The optimum buffer was 150 mM acetic acid containing 15% methanol and 15% ethanol. The optimum MS parameters were: methanol containing 0.5% acetic acid was selected as the sheath liquid and the flow rate was 5 microL/min; the flow rate and temperature of drying gas were 6L/min and 150 degrees C, respectively; the pressure of nebulizing gas was 2 psig; and the fragmentor and ESI voltage were 100 V and 4000 V, respectively. Under the optimum CE-MS conditions, the urinary nucleosides were separated within 18 min. The linearity between the relative peak areas and the corresponding concentration of nine nucleosides markers were excellent. The limits of detection (S/N=3) of markers were 0.00862-3.82 nmol/mL. The optimum CE-MS method was applied to analyze urine from 20 bladder cancer patients and 20 healthy volunteers. Considering the standards of many nucleosides cannot be obtained, it is not the ratios of the concentrations of nucleosides to that of creatinine in the literatures, but the ratios of the relative peak area of nucleosides to the concentration of creatinine that used for pattern recognition. And, the statistical analysis result indicated this method was feasible.     

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.     

Reproducibility Evaluation of Urinary Peptide Detection Using CE-MS

In recent years, capillary electrophoresis coupled to mass spectrometry (CE-MS) has been increasingly applied in clinical research especially in the context of chronic and age-associated diseases, such as chronic kidney disease, heart failure and cancer. Biomarkers identified using this technique are already used for diagnosis, prognosis and monitoring of these complex diseases, as well as patient stratification in clinical trials. CE-MS allows for a comprehensive assessment of small molecular weight proteins and peptides (<20 kDa) through the combination of the high resolution and reproducibility of CE and the distinct sensitivity of MS, in a high-throughput system. In this study we assessed CE-MS analytical performance with regards to its inter- and intra-day reproducibility, variability and efficiency in peptide detection, along with a characterization of the urinary peptidome content. To this end, CE-MS performance was evaluated based on 72 measurements of a standard urine sample (60 for inter- and 12 for intra-day assessment) analyzed during the second quarter of 2021. Analysis was performed per run, per peptide, as well as at the level of biomarker panels. The obtained datasets showed high correlation between the different runs, low variation of the ten highest average individual log2 signal intensities (coefficient of variation, CV < 10%) and very low variation of biomarker panels applied (CV close to 1%). The findings of the study support the analytical performance of CE-MS, underlining its value for clinical application.     

Determination of malotilate and its metabolites in plasma and urine

A method for the determination of malotilate (I), the corresponding monocarboxylic acid (II) and its decarboxylated product (III) in plasma is described. Plasma was extracted with chloroform spiked with internal standard. The residue, dissolved in methanol, was chromatographed on a reversed-phase column with a mobile phase of 60% acetonitrile and 1% acetic acid in water. The sensitivity limit for I, II and III was 50, 25 and 100 ng/ml of plasma, respectively. Compound I in the same plasma extract was also analysed by gas chromatography--electron-impact mass spectrometry. The base peaks m/z 160 for I and m/z 162 for internal standard (IV) were monitored; the sensitivity limit for I was 2.5 ng/ml of plasma. The determination of the metabolites of I, II and its conjugate (V), and isopropyl-hydrogen malonate (VI) in urine by high-performance liquid chromatography is also described. The limit of quantification for VI was 2.0 micrograms/ml, and the overall coefficient of variation of VI was 4.7%. The limit of quantification for II in urine was 0.5 micrograms/ml and that for V was 1.0 micrograms/ml as total II (II + V). The overall precision of the method was satisfactory. The method was used to determine plasma and urine concentrations in four dogs orally dosed with 100, 200 or 400 mg of malotilate.     

In silico and in vitro metabolism studies support identification of designer drugs in human urine by liquid chromatography/quadrupole-time-of-flight mass spectrometry

Human phase I metabolism of four designer drugs, 2-desoxypipradrol (2-DPMP), 3,4-dimethylmethcathinone (3,4-DMMC), α-pyrrolidinovalerophenone (α-PVP), and methiopropamine (MPA), was studied using in silico and in vitro metabolite prediction. The metabolites were identified in drug abusers’ urine samples using liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/Q-TOF/MS). The aim of the study was to evaluate the ability of the in silico and in vitro methods to generate the main urinary metabolites found in vivo. Meteor 14.0.0 software (Lhasa Limited) was used for in silico metabolite prediction, and in vitro metabolites were produced in human liver microsomes (HLMs). 2-DPMP was metabolized by hydroxylation, dehydrogenation, and oxidation, resulting in six phase I metabolites. Six metabolites were identified for 3,4-DMMC formed via N-demethylation, reduction, hydroxylation, and oxidation reactions. α-PVP was found to undergo reduction, hydroxylation, dehydrogenation, and oxidation reactions, as well as degradation of the pyrrolidine ring, and seven phase I metabolites were identified. For MPA, the nor-MPA metabolite was detected. Meteor software predicted the main human urinary phase I metabolites of 3,4-DMMC, α-PVP, and MPA and two of the four main metabolites of 2-DPMP. It assisted in the identification of the previously unreported metabolic reactions for α-PVP. Eight of the 12 most abundant in vivo phase I metabolites were detected in the in vitro HLM experiments. In vitro tests serve as material for exploitation of in silico data when an authentic urine sample is not available. In silico and in vitro designer drug metabolism studies with LC/Q-TOF/MS produced sufficient metabolic information to support identification of the parent compound in vivo.

Separation and identification of zaleplon metabolites in human urine using capillary electrophoresis with laser-induced fluorescence detection and liquid chromatography-mass spectrometry

A capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection for the determination of the hypnotic drug zaleplon and its metabolites in human urine could be developed using carboxymethyl-beta-cyclodextrin as a charged carrier. By the help of a complementary HPLC method coupled to mass spectrometry, three metabolites present in human urine could be identified as 5-oxozaleplon, 5-oxo-N-deethylzaleplon and 5-oxozaleplon glucuronide. N-Deethylzaleplon, a previously described zaleplon metabolite, as well as zaleplon itself could not be detected in human urine by the CE-LIF assay. The results were confirmed by spiking with reference compounds of the phase I metabolites. The metabolites differed very much concerning their fluorescence intensities, thus the 5-oxo metabolites present as lactam tautomer fluoresced tenfold lower than the unchanged drug zaleplon and its N-deethylated metabolite. The glucuronide of the 5-oxozaleplon, however, showed high fluorescence due to its lactim structure. Limits of quantification yielded by the CE-LIF assay including a ten-fold preconcentration step by solid-phase extraction were 10 ng/ml for zaleplon and N-deethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo-N-deethylzaleplon.     

Identification of some new clemastine metabolites in dog, horse, and human urine with liquid chromatography/tandem mass spectrometry

The metabolism of clemastine was studied in dogs, horses, and humans after a single dose of Tavegyl. The urine collected was extracted by solid-phase extraction or hydrolyzed with beta-glucuronidase and then extracted by liquid-liquid extraction, prior to analysis for unchanged drug and phase I and II metabolites by liquid chromatography/tandem mass spectrometry. The metabolites were identified by their molecular mass and interpretation of the product ion spectra, since no standard substances were available. Unchanged drug was recovered in urine samples from dogs and humans, but not from horses. In dogs and humans, the phase I metabolite, norclemastine, was identified, and clemastine metabolites with one and two additional oxygens were found in all three species. In horses and dogs monohydroxylation on one of the aromatic rings or the adjacent methyl group was favored while, in humans, the additional oxygen was positioned on either the aromatic or the aliphatic part of the structure, and the aliphatic reaction seemed to result in at least three isomers. In the metabolites with two additional oxygens, both the oxygens were found on the aliphatic fragment in humans and dogs, whereas they were situated on the aromatic part of the structure in horses. In human patients, glucuronidated monohydroxyclemastine was recovered, and in urine from horses both mono- and dihydroxyclemastine glucuronides were identified, while phase II metabolites could not be recovered from the dog urine. Clemastine metabolism in dogs and horses has, to our knowledge, not been studied before, and new metabolites from humans are presented in this article. Thus, the metabolites described in the present work have not been previously reported in the literature.     

Utility of membrane preconcentration-capillary electrophoresis-mass spectrometry in overcoming limited sample loading for analysis of biologically derived drug metabolites, peptides, and proteins

The limited loading of capillary electrophoresis (CE) leads to relatively poor concentration limits of detection. In this work a unique method for analyte preconcentration with capillary electrophoresis-mass spectrometry (CE-MS) is described. A cartridge containing an impregnated membrane is installed at the inlet of the CE capillary, and we term this approach membrane preconcentration capillary electrophoresis-mass spectrometry (mPC-CE-MS). The analysis of in vivo derived metabolites, peptides, and proteins is described showing the wide applicability of the technology in the analysis of numerous compound classes ranging in molecular weight from 200–60,000 u. In particular, we describe the direct mPC-CE-MS analysis of urine obtained from a patient receiving the neuroleptic drug haloperidol. Three metabolites were found in the urine, and two of them are implicated in the Parkinsonian-like side effects caused by taking this drug. The technique is also applied to the analysis of major histocompatibility complex class I peptides obtained from EG-7 cells. Furthermore, the clinical potential of this approach is described by the direct analysis of urine from a patient suffering from multiple myeloma, as well as aqueous humor derived from a patient undergoing surgery. Finally we show that the use of mPC-ME-MS in conjunction with either analyte stacking (small organic molecules such as metabolites) or moving-boundary transient isotachophoresis (peptides and proteins) after analytes have been eluted from the adsorptive membrane affords optimal performance and no compromise in CE mass spectrometry performance.     

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MS n , and LC–HR-MS n

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)–mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS ⁿ ). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors’ standard urine screening approaches by GC–MS and LC–MS ⁿ . Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.