Recent development in liquid chromatography/mass spectrometry and emerging technologies for metabolite identification

Metabolism studies play a pivotal role in drug discovery and development since the active metabolites is critical to toxicological profile, efficacy and designing new drug candidates. From the instrumentation standpoint, liquid chromatography/mass spectrometry (LC/MS) has secured a central analytical technique for metabolite identification with the continuous developments and improvements in LC and MS technologies. Recently, a wide range of experimental strategies and post acquisition data processing and mining modes have emerged driven by the need to identify and characterize metabolites at ever increasing sensitivity and in ever more complex samples. In this article, the classical and practical mass spectrometry-based techniques, such as low resolution MS (quadruple, ion trap, linear ion trap, etc), high resolution MS (time-of-flight, hybrid time-of-flight instruments, Qrbitrap, Fourier transform ion cyclotron resonance MS, etc) and corresponding post acquisition data processing and mining modes (precursor ion filtering, neutral loss filtering, mass defect filter, isotope-pattern-filtering, etc) are described comprehensively. In addition, this review is also devote to discuss several novel MS technologies (ambient ionization techniques, ion mobility MS, imaging MS, LC/MNR/MS, etc) that hold additional promise for the advancement of metabolism studies.     

Investigating the human metabolism of acetaminophen using UPLC and exact mass oa-TOF MS

The ability to rapidly detect and characterize drug metabolites in biological fluids often relies on a combination of a high quality chromatographic separation and sensitive high resolution mass spectrometry. Here, the performance of two high throughput LC/MS approaches, namely monolith columns and sub-2 microm particle Ultra Performance Liquid Chromatography (UPLC) columns is compared for the detection and identification of the human metabolites of acetaminophen in urine. The UPLC system produced approximately three times the sensitivity and detected more metabolites than the monolithic column approach. The sharp peaks produced by UPLC seem to be particularly advantageous when coupled to electrospray mass spectrometry, apparently reducing ion suppression leading to superior sensitivity and hence lower limits of detection.     

Development of a high sensitivity bioanalytical method for alprazolam using ultra-performance liquid chromatography/tandem mass spectrometry

A rapid, specific, assay was developed for the benzodiapine alprazolam in rat plasma using sub-2 μm particle liquid chromatography (LC) and tandem quadrupole mass spectrometry (MS/MS). The limit of quantification using protein precipitation was determined to 10 pg/mL, whereas the limit of quantification using solid-phase extraction (SPE) was determined to be 1.0 pg/mL. The assay was optimized for throughput and resolution of the analyte of interest from the hydroxy metabolite. During the method development process the plasma matrix signal was monitored, for lipids and other endogenous metabolites, to maximize signal response and minimize ion suppression. This was achieved by using a tandem quadrupole mass spectrometer equipped with a novel collision cell design which allowed for the simultaneous collection of full scan MS and multiple reaction monitoring (MRM) data. The lipid profile from the SPE process was significantly less than obtained with the protein precipitation approach.     

Identification of higenamine and its metabolites in rat by gas chromatography/mass spectrometry

(±)-Higenamine is known as a cardiotonic principle of aconite root (root ofAconitum spp., Ranunculaceae). A simple and sensitive detection method for higenamine was developed by using gas chromatography-mass spectrometry (GC/MS). The recovery of higenamine after extraction and concentration with XAD-2 resin column was around 95% from rat biological fluids such as bile, plasma and urine. The limits of detection of higenamine in these biological fluids were approximately 0.1 ng/ml each. It has well been suggested that tetrahydroisoquinolines possessing catechol moiety such as higenamine should be subjected to the catechol-O-methyl transferase (COMT) activityin vivo. We detected two major peaks of presumed metabolites of higenamine in the total ion chromatogram obtained from the rat urine sample after the oral administration of (±)-higenamine. The scan mass spectrum of one of the metabolites coincided with those obtained from coclaurine (C₆-O-methyl higenamine) and those of the other metabolite are suggestive of isococlaurine (C₇-O-methyl higenamine).     

LC‐MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone

The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post‐administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐5β‐androsta‐1,4,13‐triene‐3‐one‐17‐glucuronide. One metabolite was resistant to hydrolysis with β ‐glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC‐MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

A semiquantitative method for the determination of reactive metabolite conjugate levels in vitro utilizing liquid chromatography-tandem mass spectrometry and novel quaternary ammonium glutathione analogues

An in vitro semiquantitative reactive metabolite detection assay is described that incorporates NADPH-supplemented human liver microsomes, a novel quaternary ammonium glutathione analogue conjugating agent (QA-GSH), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection. The assay was developed to have high sample capacity and the potential for high sample throughput. MS/MS detection is selective and sensitive for the QA-GSH conjugating agent and semiquantitation of QA-GSH-reactive metabolite conjugates is performed using QA-GSH standards added to samples prior to analysis [i.e., internal standards (ISs)]. The reactive metabolite trapping capability of the free thiol group in QA-GSH was assessed using model drugs acetaminophen, clozapine, and flutamide, which are bioactivated to afford reactive metabolites. MS signal responses of equimolar amounts of QA-GSH standards were compared to assess the feasibility of using a QA-GSH IS approach to semiquantify reactive metabolite levels in vitro. The full scan Q1 MS response for each standard was within 3.3-fold of one another even though the "parent" moiety structure of each QA-GSH conjugate standard differed significantly. Standard curve analysis using selected reaction monitoring for each QA-GSH standard gave slope values that differed by only 1.5-fold. The QA-GSH IS semiquantitation method was tested by determining the level of QA-GS-acetaminophen conjugate formation at three different concentrations of acetaminophen and comparing the results to those from linear regression of authentic standards. The calculated levels of conjugate formed compared closely with those calculated from linear regression data of authentic standard curves. These results show that the QA-GSH semiquantitation assay described herein is a viable method for semiquantitatively assessing the bioactivation potential in vitro and is well-suited for use in early drug discovery high throughput screening paradigms.     

Application of mass spectrometry for metabolite identification

Metabolism studies play a pivotal role in drug discovery and development. Characterization of metabolic "hot-spots" as well as reactive and pharmacologically active metabolites is critical to designing new drug candidates with improved metabolic stability, toxicological profile and efficacy. Metabolite identification in the preclinical species used for safety evaluation is required in order to determine whether human metabolites have been adequately tested during non-clinical safety assessment. From an instrumental standpoint, high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) dominates all analytical tools used for metabolite identification. The general strategies employed for metabolite identification in both drug discovery and drug development settings together with sample preparation techniques are reviewed herein. These include a discussion of the various ionization methods, mass analyzers, and tandem mass spectrometry (MS/MS) techniques that are used for structural characterization in a modern drug metabolism laboratory. Mass spectrometry-based techniques, such as stable isotope labeling, on-line H/D exchange, accurate mass measurement to enhance metabolite identification and recent improvements in data acquisition and processing for accelerating metabolite identification are also described. Rounding out this review, we offer additional thoughts about the potential of alternative and less frequently used techniques such as LC-NMR/MS, CRIMS and ICPMS.     

Automated optimization of XCMS parameters for improved peak picking of liquid chromatography–mass spectrometry data using the coefficient of variation and parameter sweeping for untargeted metabolomics

Accurate peak picking and further processing is a current challenge in the analysis of untargeted metabolomics using liquid chromatography–mass spectrometry (LC–MS) data. The optimization of these processes is crucial to obtain proper results. This study investigated and optimized the detection of peaks by XCMS, a widely used R package for peak picking and processing of high‐resolution LC–MS metabolomics data by their coefficient of variation using neat standard solutions of drug like compounds. The obtained results were additionally verified by using fortified pooled plasma samples. Settings of the mass spectrometer were optimized by recommendations in literature to enable a reliable detection of the investigated analytes. XCMS parameters were evaluated using a comprehensive parameter sweeping approach. The optimization steps were statistically evaluated and further visualized after principal component analysis (PCA). Concerning the lower concentrated solution in methanol samples, the optimization of both mass spectrometer and XCMS parameters improved the median coefficient of variation from 24% to 7%, retention time fluctuation from 9.3 seconds to 0.54 seconds, and fluctuation of the mass to charge ratio (m/z) from m/z 0.00095 to m/z 0.00028. The number of parent compounds and their related species annotated by CAMERA increased from 88 to 113 while the total amount of features decreased from 3282 to 428. Optimized MS settings such as increased resolution led to a higher specificity of peak picking. PCA supported these findings by showing the best clustering of samples after optimization of both mass spectrometer and XCMS parameters. The results implied that peak picking needs to be individually adapted for the experimental set up. Reducing unwanted variation in the data set was most successful after combining high resolving power with strict peak picking settings.     

Rapid screening of glutathione-trapped reactive metabolites by linear ion trap mass spectrometry with isotope pattern-dependent scanning and postacquisition data mining

The present study describes a novel integrated approach for rapid analysis of reactive metabolites with a linear ion trap mass spectrometer (LTQ). In this approach, an isotope pattern-dependent scanning method was applied to the data acquisition of glutathione (GSH)-trapped reactive metabolites. Recorded full-scan MS and MS/MS data sets were further processed with neutral loss filtering, product ion filtering, and extracted ion chromatographic analysis to search for protonated molecules and MS/MS spectra of GSH adducts. To evaluate the effectiveness and reliability of the approach, GSH adducts of carbamazepine, diclofenac, 4-ethylphenol, acetaminophen, p-cresol, and omeprazole were analyzed, which were formed in human liver microsome incubations fortified with a mixture of nonlabeled GSH and stable isotope-labeled GSH at a 1:0.8 ratio. Results demonstrate that the combination of the isotope pattern-dependent scanning with the postacquisition data mining was very effective in detecting low levels of GSH adducts, regardless of their fragmentation patterns. As compared to a neutral loss scanning method performed with a triple quadrupole mass spectrometer, the LTQ-based approach had several major advantages, including the superior selectivity and sensitivity in detecting different classes of GSH adducts and the higher throughput capability of the detection and MS/MS spectral acquisition of GSH adducts in a single LC/MS run. Overall, this analytical approach provides a simple and efficient means for screening for reactive metabolites using a linear ion trap LC/MS platform.     

Utility of multivariate analysis in support of in vitro metabolite identification studies: retrospective analysis using the antidepressant drug nefazodone

1. The utility of multivariate analysis in in vitro metabolite identification studies was examined with nefazodone, an antidepressant drug with a well-established metabolic profile.

2. The chromatographic conditions were purposefully chosen to reflect those utilized in high-throughput screening for microsomal stability of new chemical entities.

3. Molecular ion, retention time information on groups of human liver microsomal samples with/without nefazodone was evaluated by principal component analysis (PCA). Resultant scores and loadings plots from the PCA revealed the segregation and the ions of interest that designated the drug and its corresponding metabolites. Subsequent acquisition of tandem mass spectrometry (MS/MS) spectra for targeted ions permitted the interrogation and interpretation of spectra to identify nefazodone and its metabolites.

4. A comparison of nefazodone metabolites identified by PCA versus those found by traditional metabolite identification approaches resulted in very good correlation when utilizing similar analytical methods. Fifteen metabolites of nefazodone were identified in β-nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented human liver microsomal incubations, representing nearly all primary metabolites previously reported.

5. Of the 15 metabolites, eight were derived from the N-dealkylation and N-dephenylation of the N-substituted 3-chlorophenylpiperazine motif in nefazodone, six were derived from mono- and bis-hydroxylation, and one was derived from the Baeyer Villiger oxidation of the ethyltriazolone moiety in nefazodone.

     

液相色谱—电喷雾离子阱质谱法检测体液中士的宁、马钱子碱及其主要代谢物

目的：检测体液中士的宁、马钱子碱及其主要代谢物。方法：采用液相色谱－电喷雾离子阱质谱联用法,对送检样品进行了鉴定。结果：与士的宁、马钱子碱对照品的LC／MS^n分析结果比较,证明送检样品中含有大量士的宁、马钱子碱。并根据色谱和质谱行为,推测士的宁在人体内的主要代谢产物为葡萄粮苷酸结合物,马钱子碱的代谢途径为去甲基化。最低检测限为5ng。结论：本法快速、简捷,尤其适合于毒物检测等相关部门的分析检测用。     

Power of Orbitrap‐based LC‐high resolution‐MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSn or GC–MS procedures

Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC–HR–MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSⁿ or GC–MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on‐spot cleavage followed by liquid extraction (DUSglucE), and one worked‐up by acid hydrolysis, liquid–liquid extraction, and acetylation for GC–MS analysis. The 3 series of LC–HR–MS/MS results were compared among themselves, to corresponding published LC–MSⁿ data, and to screening results obtained by conventional GC–MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC–HR–MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one‐third more hits in comparison to the corresponding published LC–MSⁿ results with ≤49% detected hits. The GC–MS approach identified 56% of all detected hits. In conclusion, LC–HR–MS/MS provided the best screening results after conjugate cleavage and precipitation.     

The use of mass spectrometry in the study of chemically-reactive drug metabolites. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

Strategies for characterization of drug metabolites using liquid chromatography-tandem mass spectrometry in conjunction with chemical derivatization and on-line H/D exchange approaches

Strategies using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with techniques such as chemical derivatization and on-line hydrogen/deuterium (H/D) exchange for structural elucidation of drug metabolites in crude samples are reviewed. Useful mass spectrometric scan techniques discussed include product ion scan, constant neutral-loss scan, precursor ion scan, multistage MS(n), and accurate mass measurements. In biological systems, xenobiotics are transformed into metabolites, which usually involves introduction of one or more polar functional groups or removal or blockage of such structural moieties. Therefore, chemical derivatization strategies for determination of functional groups and on-line H/D exchange approaches for probing number of exchangeable hydrogens are powerful tools for structural elucidation of drug metabolites in drug metabolism studies. More importantly, these experiments can be carried out on crude samples in microscale, providing sufficient material for LC-MS/MS analysis. Therefore, labor intensive and technically challenging purification of low levels of drug metabolites from complex biological matrices can be avoided. It is the authors' conclusion that strategies such as chemical derivatization and on-line H/D exchange should be used more routinely in drug metabolism studies in order to facilitate metabolite identification.     

胶体金法与液相色谱-质谱法MDMA检测比较

目的：通过胶体金法对药物滥用患者尿液中3,4-亚甲基二氧基甲基苯丙胺（MDMA）及代谢产物进行检测,并与液相色谱一质谱法检测结果进行比较以验证其检测效果。方法：选取广州市脑科医院物质依赖科收治的药物滥用患者,分别采用胶体金法和液相色谱一质谱法检测其尿液中MDMA及相关成分,计算分析胶体金法检测试剂的灵敏度、特异度、Youden指数与总符合率。结果：在以液相色谱-质谱法为金标准的检测结果对比中,胶体金法检测试剂的灵敏度100．00％,特异度98．88％,Youden指数0．9888,样本检测总符合率98．97％,Kappa=0．936,P=0．00。结论：与液相色谱-质谱法相比较,胶体金法具有操作简便、直观、快速、省时的特点,其特异性、敏感性较高,具有良好的检测效果。     

Introduction to early in vitro identification of metabolites of new chemical entities in drug discovery and development

The introduction of combinatorial chemistry and robotics for high throughput screening has changed the way drugs are discovered today compared with 10-15 years ago when fewer compounds were tested in animal or organ models. The introduction of new analytical techniques, especially liquid chromatography/mass spectrometry (LC/MS) has made it possible to characterize the chemical properties, permeability, metabolic stability and metabolic fate of a large number of screening hits for further development in a funnel-like manner. The purpose of this contribution is to discuss principles and recent strategies for metabolite identification and to give an introduction to biotransformation studies. Metabolites are experimentally generated with the use of animal and human recombinant expressed enzymes, and different liver and other tissue fractions like microsomes and slices. For separation and identification of structurally diverse metabolites, LC/MS and tandem mass spectrometry (LC/MS/MS) techniques are commonly used. The LC/MS and LC/MS/MS techniques are rapid, sensitive, easy to automate and robust, and therefore, they are the methods of choice for these studies. The outcome of the metabolite identification studies is detection of metabolites that could be pharmacologically active and contribute to the efficacy of a new chemical entity (NCE), and elimination of compounds that form reactive intermediates and/or toxic metabolites that could cause adverse effects of NCE. If such information is available at an early stage during the drug discovery process, the chemical structure of the compound may be modified to reduce the risk of idiosyncratic and/or adverse drug reactions during clinical development.     

METLIN: a metabolite mass spectral database

Endogenous metabolites have gained increasing interest over the past 5 years largely for their implications in diagnostic and pharmaceutical biomarker discovery. METLIN (http://metlin.scripps.edu), a freely accessible web-based data repository, has been developed to assist in a broad array of metabolite research and to facilitate metabolite identification through mass analysis. METLINincludes an annotated list of known metabolite structural information that is easily cross-correlated with its catalogue of high-resolution Fourier transform mass spectrometry (FTMS) spectra, tandem mass spectrometry (MS/MS) spectra, and LC/MS data.     

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography–high resolution mass spectrometry

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography–high‐resolution tandem mass spectrometry (LC–HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC–HRMS/MS system consisted of a YMC‐UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β‐glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono‐hydroxy metabolites, and mono‐hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono‐hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2–19‐fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.     

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) – a group of new psychoactive substances (NPS) – is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography—tandem mass spectrometry (LC?MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography?high resolution tandem mass spectrometry (LC?HRMS/MS) and finally included to the MS/MS spectra in‐house library. Sample preparation was performed using a cheap‐and‐easy salting‐out liquid–liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05 ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.