Identification of some benproperine metabolites in humans and investigation of their antitussive effect

AIM: To identify 4 unknown metabolites of benproperine (BPP, 1) in human urine after a po dose, and to investigate the antitussive effect of monohydroxylate metabolites. METHODS: The putative metabolite references were prepared using chemical synthesis. Their structures were identified using 1H and 13C nuclear magnetic resonance, and mass spectrometry. The metabolites in human urine were separated and assayed using liquid chromatography-ion trap mass spectrometry (LC/MS/MS), and further confirmed by comparison of their mass spectra and chromatographic retention times with those of synthesized reference substances. The antitussive effects of metabolites were evaluated on coughs induced by 7.5% citric acid in conscious guinea pigs. RESULTS: 1-[1-Methyl-2-[2-(phenylmethyl)phenoxy]-ethyl]-4-piperidinol (2), 1-[1-methyl-2-[2-(phenylmethyl)phenoxy] ethyl]-3-piperidinol (3) and their glucuronides 4 and 5 were obtained from chemical synthesis. Four urinary metabolites in human urine showed peaks with the same chromatographic retention times and mass spectra in LC/MS/MS as synthetic substances 2, 3, 4 and 5. Phosphates of compounds 2 and 3 prolonged the latency of cough and reduced the number of coughs during the 3 min test using citric acid, but did not reduce the number of coughs during the 5 min immediately after the test in conscious guinea pigs. CONCLUSION: Compounds 2, 3, 4, and 5 were identified as the metabolites of BPP in human urine. Among them, compounds 2 and 3 are inactive in the antitussive effect.     

Identification of phase I and phase II metabolites of Guanfu base A hydrochloride in human urine.

Guanfu base A is a novel arrhythmic drug candidate isolated from the tuber of a traditional Chinese herb. Phase I and Phase II metabolites of Guanfu base A (GFA) Hydrochloride were studied in human urine by means of liquid chromatography mass spectrometry (LC/MSD) and tandem mass spectrometry (MS/MS). For phase I metabolites, Guanfu base I (GFI) was separated by HPLC and identified by comparison with authentic reference for their retention times, molecular ion peaks, fragment ions, and UV spectra. GFA oxide was also indicated to exist in human urine. For phase II metabolites, after human urine was treated either with glucuronidase or sulfatase, GFA occured in the chromatograms. It was suggested that there were GFA glucuronide and GFA sulfate in human urine. Further more, positive molecular ions, m/z 606 and m/z 510, of the two conjugates were detected in human urine by LC/MSD. In addition, characteristic ion of m/z 606 was identified as the precursor ion of m/z 177 [Glucuronic acid+H]+ by using MS/MS. Characteristic ion of m/z 430 [GFA+H]+ was also identified as a product ion of m/z 606 [GFA glucuronide+H]+. It was concluded that there were GFI. GFA oxide, GFA glucuronide and GFA sulfate in human urine.     

Screening for detection of new antidepressants, neuroleptics, hypnotics, and their metabolites in urine by GC-MS developed using rat liver microsomes

A gas chromatography-mass spectrometry (GC-MS) procedure for the detection of new antidepressants, neuroleptics, hypnotics, and their metabolites in urine is presented. The metabolites were first identified in rat liver microsome preparations by GC-MS after isolation and derivatization. Using these GC-MS data, a GC-MS screening was developed for urine as part of the authors' modified systematic toxicologic analysis procedure. After acid hydrolysis of a 2.5-mL aliquot of urine, a further aliquot was added. The mixture was then liquid-liquid extracted at pH 8-9, acetylated, and GC separated. Using mass chromatography with the ions m/z 58, 100, 120, 182, 195, 235, 261, 276, 284. and 293, the presence of new antidepressants, neuroleptics, hypnotics, and their metabolites could be indicated. Positive peaks could be identified by library search using the reference mass spectra recorded during the microsome studies. The intake of therapeutic doses of the following drugs could be monitored in urine: dosulepin, mirtazapine, moclobemide, nefazodone, trazodone, venlafaxine, and zolpidem. Olanzapine and zotepine were detectable in human urine only under steady-state conditions, and low-dose zopiclone was detectable only in overdose. The detection limit was less than 100 ng/mL (signal-to-noise ratio = 3) for the parent drugs.     

In vitro identification of metabolites of verapamil in rat liver microsomes

AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS^n), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eightmetabolites, including two novel metabolites (M4 and MS), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (MS-MT), and N, O-didemethyl-verapamil (MS). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.     

In vitro metabolic study of temsirolimus: preparation, isolation, and identification of the metabolites.

The in vitro metabolism of temsirolimus, (rapamycin-42-[2,2-bis-(hydroxymethyl)]-propionate), an antineoplastic agent, was studied using human liver microsomes as well as recombinant human cytochrome P450s, namely CYP3A4, 1A2, 2A6, 2C8, 2C9, 2C19, and 2E1. Fifteen metabolites were detected by liquid chromatography (LC)-tandem mass spectrometry (MS/MS or MS/MS/MS). CYP3A4 was identified as the main enzyme responsible for the metabolism of the compound. Incubation of temsirolimus with recombinant CYP3A4 produced most of the metabolites detected from incubation with human liver microsomes, which was used for large-scale preparation of the metabolites. By silica gel chromatography followed by semipreparative reverse-phase high-performance liquid chromatography, individual metabolites were separated and purified for structural elucidation and bioactivity studies. The minor metabolites (peaks 1-7) were identified as hydroxylated or desmethylated macrolide ring-opened temsirolimus derivatives by both positive and negative mass spectrometry (MS) and MS/MS spectroscopic methods. Because these compounds were unstable and only present in trace amounts, no further investigations were conducted. Six major metabolites were identified as 36-hydroxyl temsirolimus (M8), 35-hydroxyl temsirolimus (M9), 11-hydroxyl temsirolimus with an opened hemiketal ring (M10 and M11), N- oxide temsirolimus (M12), and 32-O-desmethyl temsirolimus (M13) using combined LC-MS, MS/MS, MS/MS/MS, and NMR techniques. Compared with the parent compound, these metabolites showed dramatically decreased activity against LNCaP cellular proliferation.     

Identification of domperidone metabolites in plasma and urine of gastroparesis patients with LC–ESI-MS/MS

Abstract

1. Domperidone is a prokinetic agent used to treat gastroparesis. Previous studies reported oxidative metabolites of domperidone, detected by radiometric high-performance liquid chromatography or single quadrupole mass spectrometric techniques. Our aim was to identify domperidone Phase I and Phase II metabolites using liquid chromatography combined with electrospray ionization-enabled tandem mass spectrometry.

2. Domperidone metabolites were identified in the plasma and urine of 11 gastroparesis patients currently being treated with domperidone. In addition, oxidative and conjugative metabolites of domperidone were characterized in human liver subcellular fractions.

3. Seven metabolites were detected in vivo. Domperidone was metabolized to two mono-hydroxylated metabolites (M1 and M2), a de-alkylated metabolite (M5) and a di-hydroxylated metabolite (M7). The mono-hydroxylated metabolites were further glucuronidated to M8, M9 and sulfated to M11. To the best of our knowledge, M7, M8, M9 and M11 have not been reported previously. Five additional metabolites were identified in vitro in human subcellular fractions which comprise two additional mono-hydroxylated metabolites (M3 and M4), an alcohol metabolite (M6) possibly formed from an aldehyde intermediate, and other conjugative metabolites (M10 and M12). M6, M10 and M12 have not been characterized previously.

4. In total, 12 domperidone metabolites including 7 new metabolites were identified in the present study. These results allow a better understanding of domperidone disposition in humans.     

人胆汁中罗红霉素代谢产物的研究

目的：研究人胆汁中罗红霉素的代谢转化产物。方法：采用高效液相色谱-离子阱质谱法,对患者po罗红霉素后的胆汁样品进行了分析。结果：发现了13个代谢产物,分别为罗红霉素的(9Z)-异构体及其(9E)-,(9Z)-N-去甲基和N-双去甲基衍生物和(9E)-及(9Z)-脱克拉定糖衍生物,(9E)-和(9Z)-红霉素肟及其(9E)-和(9Z)-N-去甲基及N-双去甲基衍生物,其中9种为新发现的代谢物。结论：罗红霉素及其代谢物均可在体内发生几何异构化。     

Structural elucidation of in vivo and in vitro metabolites of anisodine by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMSn) was employed to investigate the in vivo and in vitro metabolism of anisodine. Feces, urine and plasma samples were collected after ingestion of 20 mg anisodine to healthy rats. Feces and urine samples were cleaned up by liquid-liquid extraction and solid-phase extraction procedures (C18 cartridges), respectively. Methanol was added to plasma samples to precipitate plasma proteins. Anisodine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively, followed by extraction with ethyl acetate. LC-MSn was used for the separation and identification of the metabolites using C18 column with mobile phase of methanol/0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v). The results revealed that five metabolites (norscopine, scopine, alpha-hydroxytropic acid, noranisodine and hydroxyanisodine) and the parent drug existed in feces. Three new metabolites (dimethoxyanisodine, tetrahydroxyanisodine and trihydroxy-methoxyanisodine) were identified in urine. Four metabolites (norscopine, scopine, hydroxyanisodine and anisodine N-oxide) and the parent drug were detected in plasma. Two hydrolyzed metabolites (scopine and alpha-hydroxytropic acid) were found in rat intestinal flora incubation mixture, and two metabolites (aponoranisodine and anisodine N-oxide) were identified in homogenized liver incubation mixture.     

Qualitative and quantitative determination of ten alkaloids in traditional Chinese medicine Corydalis yanhusuo W.T. Wang by LC-MS/MS and LC-DAD

An analytical method incorporating high-performance liquid chromatography (HPLC) with MS and UV-detection was developed for the qualitative and quantitative determination of alkaloids in Corydalis yanhusuo. Ten alkaloids, including seven tertiary alkaloids and three quaternary alkaloids, were identified by comparing their retention times, UV and MS spectra with those of authentic compounds. Furthermore, the collision-induced dissociations of the [M+H](+) and [M](+) ions were studied to clarify the MS behavior of the different types of alkaloids. In positive ion electrospray ionization tandem mass spectrometry (ESI-MS) all the tertiary alkaloids yielded prominent [M+H](+) ions and quaternary alkaloids yielded prominent [M](+) ions in the first order mass spectra. Fragments involving losses of H, CH(3), CO, H(2)O and OCH(3) were observed in the MS/MS spectra. In addition, quantification of the 10 alkaloids in Corydalis yanhusuo from methanol and ethyl acetate extract of different origins were performed by this method, which provides a new tool for the assessment of quality of Corydalis yanhusuo preparations. The method provides the best sensitivity and specificity for characterization and quantitative determination of the alkaloids in Corydalis yanhusuo so far.     

Development and validation of a highly sensitive UPLC-MS/MS method for simultaneous determination of aconitine, mesaconitine, hypaconitine, and five of their metabolites in rat blood and its application to a pharmacokinetics study of aconitine, mesaconitine, and hypaconitine

A rapid, specific and sensitive method was developed for the simultaneous determination of eight Aconitum alkaloids: aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine and mesaconine in rat blood by ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS). The UPLC-MS/MS system coupled with an electrospray ionization (ESI) source was operated in a positive mode via multiple-reaction monitoring (MRM). Samples were treated with methanol to remove protein prior to analysis by UPLC-MS/MS. The analytes were separated with a Waters C18 column (1.7 μm, 50?×?2.1?mm) and a gradient elution using acetonitrile and 0.1% formic acid-water as the mobile phases. The linear response range was from 0.125 to 1000 nmol/L for these eight alkaloids and the correlation coefficients (r(2) values) were all higher than 0.997. The method was validated with respect to precision, accuracy, recovery, matrix effect, carryover effect and sample stability, and found to be within the acceptable limits. The developed and validated method was successfully applied to simultaneously determine the eight Aconitum alkaloids in rats blood after intravenous administration of a mixture of AC, MA and HA.     

Simultaneous determination of phytoestrogens and key metabolites in breast cancer patients’ urine by liquid chromatography–tandem mass spectrometry

A novel, selective and sensitive liquid chromatography–tandem mass spectrometry method has been developed and validated for the simultaneous determination of phytoestrogens and their key metabolites in human urine in this study. This method includes internal standard (IS) screening, analytical sample preparation procedure establishment, and linear range investigation. The analytical sample was extracted by liquid–liquid extraction from urine sample. The phytoestrogens and related key metabolites were separated with Agilent Zorbax Eclipse XDB-C18 chromatographic column using methanol and water as mobile phase. The Quattro premier MICROMASS mass spectrometer in negative ion selected reaction monitoring (SRM) mode using electrospray ionization was applied to detect the phytoestrogens and key metabolites. To validate the developed liquid chromatography–tandem mass spectrometry method, the intra- and inter-day precisions, specificity, sensitivity, reproducibility, and sample detective concentration range were evaluated. This is the first reported phytoestrogens analysis and validation study that demonstrates the feasibility of using liquid chromatography–electrospray ionization mass spectrometry to simultaneously analyze ten analytes including both phytoestrogens and their key metabolites in urine samples collected for epidemiological studies in human.     

Comparative study on the metabolism of the ergot alkaloids ergocristine,ergocryptine, ergotamine,and ergovaline in equine and human S9 fractions and equine liver preparations

Abstract

1. Ergopeptine alkaloids like ergovaline and ergotamine are suspected to be associated with fescue toxicosis and ergotism in horses. Information on the metabolism of ergot alkaloids is scarce, especially in horses, but needed for toxicological analysis of these drugs in urine/feces of affected horses. The aim of this study was to investigate the metabolism of ergovaline, ergotamine, ergocristine, and ergocryptine in horses and comparison to humans.

2. Supernatants of alkaloid incubations with equine and human liver S9 fractions were analyzed by reversed-phase liquid-chromatography coupled to high-resolution tandem mass spectrometry with full scan and MS² acquisition. Metabolite structures were postulated based on their MS² spectra in comparison to those of the parent alkaloids. All compounds were extensively metabolized yielding nor-, N-oxide, hydroxy and dihydro-diole metabolites with largely overlapping patterns in equine and human liver S9 fractions. However, some metabolic steps e.g. the formation of 8′-hydroxy metabolites were unique for human metabolism, while formation of the 13/14-hydroxy and 13,14-dihydro-diol metabolites were unique for equine metabolism. Incubations with equine whole liver preparations yielded less metabolites than the S9 fractions.

3. The acquired data can be used to develop metabolite-based screenings for these alkaloids, which will likely extend their detection windows in urine/feces from affected horses.     

淫羊藿苷在大鼠体内的代谢

目的：为阐明淫羊藿苷在体内的作用形式,对淫羊藿苷的代谢产物及活性进行研究。方法：将淫羊藿苷给大鼠ig后,分析、分离并鉴定了其在小肠、尿、胆汁中的主要代谢产物。结果：在小肠及尿中鉴定有淫羊藿次苷II(icariside II)及淫羊藿素(icaritin),胆汁中有淫羊藿素3-O-α-L-鼠李吡喃糖基-7-O-β-D-葡萄吡喃糖醛酸苷(icaritin 3-O-α-L-rhamnopyranosyl-7-O-β-D-glucopyranuronoside)和淫羊藿次苷II,并对淫羊藿苷在大鼠体内主要代谢途径进行了探讨。结论：淫羊藿苷在吸收之前已发生代谢,在体内主要是以其代谢产物的形式存在。     

兔体内乌头碱代谢产物研究

目的鉴定乌头碱在家兔尿中主要代谢产物。方法ig给药(5 mg·kg^-1)后,收集给药后♂家兔尿液,固相萃取柱富集、液相色谱-电喷雾离子阱质谱法分离鉴定乌头碱代谢物。结果在给药后尿样中发现乌头碱原形和4种代谢物M1～M4,分别测得准分子离子峰［M+H］+及其各级碎片离子。结论M1～M4为乌头碱的4个代谢物,推测分别为16-O-去甲基乌头碱、乌头次碱、16-O-去甲基乌头次碱、乌头原碱。     

Update on metabolism of abemaciclib: In silico,in vitro,and in vivo metabolite identification and characterization using high resolution mass spectrometry

Abemaciclib was approved by the US Food and Drug Administration in 2015 as an advanced treatment for metastatic breast cancer. Identification and characterization of limited numbers of abemaciclib metabolites have been reported in the literature. Therefore, the current study focused on the investigation of the in vitro and in vivo metabolic fate of abemaciclib using high resolution mass spectrometry. Initially, a vulnerable site of metabolism was predicted by the Xenosite web predictor tool. Later, in vitro metabolites were identified from pooled rat liver microsomes, rat S9 fractions, and human liver microsomes. Finally, in vivo metabolites have been detected in plasma, urine, and feces matrix of male Sprague–Dawley rats. A total of 12 putative metabolites (11 phase I and 1 phase II) of abemaciclib and their metabolic pathways were proposed by considering accurate mass, mass fragmentation pattern, nitrogen rule, and ring double bonds of the detected metabolites. Abemaciclib was metabolized via hydroxylation, N‐oxidation, N‐dealkylation, oxidative deamination followed by reduction and sulfate conjugation. In the human liver microsomes, maximum numbers of metabolites (11 metabolites) were observed, from which M7, M8, M9, and M11 were human specific.     

Structural elucidation of human oxidative metabolites of muraglitazar: use of microbial bioreactors in the biosynthesis of metabolite standards.

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor activator, is currently in clinical development for treatment of type 2 diabetes. This study describes the structural elucidation of the human oxidative metabolites of muraglitazar through the use of a combination of microbial bioreactors, NMR and accurate mass analyses, and organic synthesis. Plasma, urine, and feces were collected from six healthy subjects following oral administration of 14C-labeled muraglitazar (10 mg, 100 microCi) and pooled samples were analyzed. Approximately 96% of the recovered radioactive dose was found in the feces and 3.5% in the urine. The parent compound represented >85% of the radioactivity in plasma. The fecal radioactivity was distributed among 16 metabolites (M1-M12, M14-M16, and M8a) and the parent drug, of which hydroxylation and O-demethylation metabolites (M5, M10, M11, M14, and M15) represented the prominent human metabolites. The urinary radioactivity was distributed into several peaks including muraglitazar glucuronide (M13) and the parent drug. Low concentrations of metabolites in human samples prevented direct identification of metabolites beyond liquid chromatographic (LC)-mass spectrometric analysis. Microbial strains Cunninghamella elegans and Saccharopolyspora hirsuta produced muraglitazar metabolites that had the same high performance liquid chromatography retention times and the same tandem mass spectrometric (MS/MS) properties as the corresponding human metabolites. The microbial metabolites M9, M10, M11, M14, M15, and M16 were isolated and analyzed by NMR. Based on these LC-MS/MS and NMR analyses, and organic synthesis, the structures of 16 human oxidative metabolites were identified. The oxidative metabolism of muraglitazar was characterized by hydroxylation, O-demethylation, oxazolering opening, and O-demethylation/hydroxylation, as well as O-dealkylation and carboxylic acid formation. This study demonstrated the utility of microbial bioreactors for the identification of metabolites.     

液相色谱-串联电喷雾离子阱质谱法研究人尿中埃坡霉素D的主要代谢产物

目的：研究埃坡霉素D在人尿中的主要代谢产物。方法：受试者6名静脉滴注埃坡霉素D,收集0～72h尿样,经SPE固相萃取小柱分离纯化后,采用液相色谱-串联电喷雾离子阱质谱法（LC—MS^n）对样品进行分析,通过比较空白尿样和给药后的尿样的总离子流色谱图和选择离子扫描色谱图,分析可能的代谢产物。代谢产物的结构鉴定主要依据代谢产物的各级质谱图与原药的各级质谱图的相关性推断所得。结果：在人尿中共检测到11种主要的代谢产物,根据所提供的代谢产物标准品鉴定出3种代谢产物的结构,代谢方式主要有氧化、还原、水解和结合反应。结论：本方法选择性好,为深入研究埃坡霉素类药物在人体内的代谢规律提供了可靠的方法和可参考的依据。     

Liquid chromatography/mass spectrometry-based structural analysis of new platycoside metabolites transformed by human intestinal bacteria

Platycosides, the main active constituents of Platycodi Radix, have been thoroughly studied for the characterization of their potent biological activities. However, metabolism of platycosides has not yet been characterized. A HPLC electrospray ionization-tandem mass spectrometry (LC/ESI-MSⁿ) approach was applied to new complex platycoside metabolites transformed by human intestinal bacteria to identify their structures and determine metabolic pathway. The molecular weights of metabolites were identified by LC/ESI-MS analysis in both positive and negative modes. Structures for the platycoside metabolites were proposed by the molecular weights and the expected enzymatic activity of intestinal microbes on platycoside. In the second step, successive LC–MSⁿ analysis was used to demonstrate the proposed structures. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M+Na]⁺ ions exclusively showed signals, consistent with the cleavage of glycoside bonds, rearrangement and some cross-ring cleavage, thus allowing the rapid identification of platycoside metabolites. The metabolites identified in the time-dependent metabolism experiments enable us to propose several microbial pathways for platycosides. Even though the metabolites of some platycosides may have unknown structures and low levels, the analytical tools presented in this study made it possible to obtain a rapid and complete characterization of new metabolites and their metabolism pathway in human intestinal bacteria.     

Characterization of the Phase II metabolites of rutaecarpine in rat by liquid chromatography-electrospray ionization-tandem mass spectrometry

From the authors' previous studies on the Phase I metabolism of rutaecarpine, nine metabolites formed were identified as products of hydroxylation on the aromatic rings in rat liver microsomes. In order to determine the possible metabolic fate of rutaecarpine, the Phase II metabolites of rutaecarpine were characterized in the present study by using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS). When male Sprague-Dawley rats were treated intravenously with 4 mg kg(-1) rutaecarpine, 16 different Phase I and II metabolites were identified in urine including four sulfate and four glucuronide conjugates. Phase I metabolites of rutaecarpine were identified as four mono-hydroxylated metabolites (M2-5) and four isobaric di-hydroxylated metabolites (M6-9). These metabolites were identical to the in vitro metabolites except one, which was hydroxylated in the aliphatic moiety. In addition, Phase II metabolites were identified as conjugated with sulfate (S1-4) and glucuronide (G1-4). In faeces, 11 different metabolites were identified. The metabolites M8 and glucuronide conjugated (G1-4) were not detected. Structures of all metabolites were confirmed with CID fragmentation spectra of MS(2), MS(3) and retention times by LC/ESI-MS.     

Metabolic profiling of deschloro-N-ethyl-ketamine and identification of new target metabolites in urine and hair using human liver microsomes and high-resolution accurate mass spectrometry

The aim of this study was to identify new markers of deschloro-N-ethyl-ketamine (O-PCE), a ketamine analogue that has been involved in acute intoxications with severe outcomes including death and whose metabolism has never been studied before. In vitro study after 2-h incubation with pooled human liver microsomes (HLMs) cross-checked by the analysis of urine and hair from a 43-year-old O-PCE user (male) were performed by liquid chromatography–high resolution mass spectrometry (LC-HRMS). Acquired data were processed by the Compound Discoverer® software, and a full metabolic profile of O-PCE was proposed. In total, 15 metabolites were identified, 10 were detected in vitro (HLMs) and confirmed in vivo (urine and/or hair), two were present only in HLMs, and the remaining three metabolites were identified only in biological specimens. While O-PCE was no longer detected in urine, nine metabolites were identified allowing to increase its detection window. In descending order of metabolites abundance, we suggest using 2-en-PCA-N-Glu (34%, first), M3 (16%, second), O-PCA-N-Glu (15.4%, third), OH-O-PCE (15%, fourth) and OH-PCE (11.9%, fifth) as target metabolites to increase the detection window of O-PCE in urine. In hair, nine metabolites were identified. OH-PCA was the major compound (78%) with a relevant metabolite to parent drug ratio (=6) showing its good integration into hair and making it the best marker for long-term monitoring of O-PCE exposure.