Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2-20 ng ml(-1) for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses.     

Multivariate approaches for efficient detection of potential metabolites from liquid chromatography/mass spectrometry data

This work describes a novel method for rapid screening of unknown metabolites in urine samples that narrows down the list of potential metabolites. Prior to analysis by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS), urine samples were prepared using solid-phase extraction (SPE). Automatic curve resolution was used for deconvolution of the LC/MS data, followed by peak alignment. Preprocessed data were then used for metabolite pattern recognition using principal component analysis (PCA), parallel factor analysis (PARAFAC), and multilinear partial least squares (N-PLS). This approach enabled the rapid detection of metabolites of citalopram in urine by maximizing the information extracted. The metabolites thus identified were compared with earlier studies on the metabolism of citalopram. In addition, new, unreported metabolites were found and characterized by LC/MS/MS and accurate mass measurements. A combination of data from positive and negative ionization enhanced the identification of metabolites.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry for determination of trace rosiglitazone in urine

This project evaluated solid-phase extraction (SPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the trace amount of rosiglitazone in human urine. The analytical performance of four modes of LC-MS and tandem MS operation (atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), positive and negative ionization) was compared for two mass spectrometers, a triple-quadrupole and a quadrupole ion trap instrument. Rosiglitazone was extracted from urine using a SPE cartridge of 50mg C8 sorbent and acetonitrile used as the eluting solvent. Samples were then separated on a RP18 column interfaced with a tandem mass spectrometer. The recovery of rosiglitazone was greater than 91.2%. The urine assay combining SPE and LC-APCI-MS/MS of triple-quadrupole was proved a very selective and sensitive method for determination of trace rosiglitazone. The assay was linear over a wide range, with a lower limit of quantification of 0.1 ng/mL using 1 mL of urine. The intra- and inter-day precisions were <9.8% and <7.9%, respectively, and the accuracies were in the range 91.0-103.6%. The rosiglitazone concentration profile in human urine was also determined. The results of this study reveal the adequacy of SPE-LC-APCI-MS/MS method for analyzing rosiglitazone from diabetic patients' urines. The concentrations of rosiglitazone were detected to range from 760 to 164 pg/mL.     

Investigation of the in vitro metabolism of the emerging drug candidate S107 for doping-preventive purposes

The metabolic fate of the emerging drug candidate S107, possessing the potential for misuse as performance-enhancing agent in sports, was investigated by in vitro phase I and II experiments with human microsomal and S9 liver enzymes. The metabolites were identified by liquid chromatography-mass spectrometry with electrospray ionisation in positive mode (LC-ESI-MS/MS). Their collision-induced dissociation behaviour was studied by high-resolution/high accuracy Orbitrap MS(n) analysis, supported by stable isotope labelling, H/D-exchange experiments and density functional theory calculations. Monooxygenation accounted for the main phase I metabolic transformation due to N- and S-oxidation of the 1,4-benzothiazepine core, as substantiated by chemical synthesis, selective reduction methods and characteristic APCI in source fragmentation behaviour of the metabolites. Another dominant metabolic pathway was demethylation, yielding the N- and O-demethylated metabolite, respectively. The latter was further conjugated by glucuronidation as well as sulfonation in subsequent phase II metabolic reactions, whereas the N-demethylated metabolite was not amenable to conjugation. The active drug molecule itself was converted to two glucuronic acid conjugates, which are proposed to consist of two quaternary S107-N(+)-glucuronide isomers. All glucuronides were susceptible to enzymatic hydrolysis with β-glucuronidase (Escherichia coli). A comprehensive LC-ESI-MS(/MS)-based detection method for urine was developed and its fitness for purpose was assessed. The assay can serve as a potential screening and/or confirmation method for S107 in clinical drug testing and doping control analysis in the future.     

Characterization and determination of the alkaloid metabolites of Evodiae fructus in rat urine by liquid chromatography-tandem mass spectrometry detection

The lack of authentic standards limits the quantitative analysis of herbal drugs in biological samples. This present work demonstrated a practicable assay of herbs and their metabolites independent of the availability of authentic standards. A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for the qualitative and quantitative determination of the metabolites after oral administration of Evodiae fructus and Zuojinwan preparation in rat urine has been developed. Urine samples extracted with a protein precipitation procedure were separated on a C(18) column using a mixture of water (containing 0.1% formic acid) and acetonitrile (30:70, v/v) as mobile phase. The detection was performed by MS with electrospray ionization interface in positive selected reaction monitoring (SRM) mode. One urine sample after administration was selected as 'standard'. The method validation was carried out according to a conventional method that was calibrated by authentic standards. The fully validated method was applied to the pharmacokinetic study of the metabolites in rat urine. The results could provide evidence to explain the combination of Coptidis rhizoma and Evodiae fructus in terms of elimination.     

LC-MS/MS fast analysis of androgenic steroids in urine

The liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated to detect androgenic steroids: trenbolone, nortestosterone, boldenone, methylboldenone, testosterone, methyltestosterone, 17β-1-testosterone and their metabolites in bovine urine. Sample preparation before LC-MS/MS analysis involved an enzymatic hydrolysis with glucuronidase AS-HP, isolation of free hormones from urine on C(18) SPE column and purification of the extract using liquid-liquid extraction with n-pentane and SPE NH(2) column. For the chromatographic separation of steroids, the Poroshell 120-EC C18 column (150?×?2.1 mm, 2.7 μm) was used. Mass spectrometric measurement was achieved using the API 4000 triple quadrupole (QqQ) instrument with a TurboIon-Spray source operating in positive electrospray ionization mode. The procedure was validated according to the Decision 2002/657/EC. Recovery ranged from 76.5% to 118.9% for all examined compounds. The repeatability was below 20% and reproducibility did not exceed the 25%. The linearity was good for all analytes in the whole range of tested concentrations, as proved by the correlation coefficients greater than 0.99. The decision limit (CCα) ranged from 0.10 to 0.17 μg L(-1) for all analytes, whereas the detection capability (CCβ) ranged from 0.17 to 0.29 μg L(-1). The application of an innovative Poroshell column allowed for very good chromatographic separation of steroids with a much shorter time of analysis.     

Optimizing the extraction and analysis of DHEA sulfate, corticosteroids and androgens in urine: application to a study of the influence of corticosteroid intake on urinary steroid profiles

A method of detecting and quantifying dehydroepiandrosterone (DHEA) sulfate, corticosteroids, and androgens has been developed. All of the compounds were first extracted from urine using solid phase extraction (SPE), enzymatically hydrolyzed, and separated into three samples using a second SPE. A DHEA sulfate sample was acetylated and re-extracted using SPE for purification before analysis. Corticosteroid samples were oxidized and re-extracted using liquid/liquid extraction for analysis. Androgen samples were acetylated and re-extracted using SPE prior to analysis. The extraction and analysis methods were investigated and optimized. Analyses were performed with gas chromatography/mass spectrometry (GC/MS) and gas chromatography/flame ionization detection (GC/FID). The entire procedure was then applied to the study of urine profiles of healthy volunteers and patients treated with corticosteroids. The results showed that the quantities of androgens found in patient urines were lower than in those of healthy volunteers. In addition, other metabolites were detected in patient urines.     

Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.     

高效液相色谱-串联质谱法同时测定人尿液中7种邻苯二甲酸酯代谢物

建立了同时检测人尿液中7种邻苯二甲酸酯代谢物的高效液相色谱-串联三重四极杆质谱法。尿液经酶水解后,采用萃取柱净化,以2%(v/v)甲酸甲醇溶液为洗脱剂,经苯基柱分离,以0.1%(v/v)乙酸水溶液和0.1%(v/v)乙酸乙腈溶液为流动相进行梯度洗脱,采用电喷雾离子源负离子模式和多反应监测模式采集信号,用同位素内标法进行定量分析。尿液中7种邻苯二甲酸酯代谢物在0.2~200.0 μg/L范围内定量离子的相对峰面积比值与质量浓度均呈良好线性关系(r≥0.99976);检出限(LOD)为13.43~80.21 ng/L,定量限为44.77~267.37 ng/L; 3个水平的加标回收率为88.8%~108.9%,日内和日间精密度均不大于17.05%。该方法可同时准确、灵敏、简便地测定人尿液中7种邻苯二甲酸酯代谢物的暴露水平。     

丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中的环己胺

建立丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中环己胺的方法。冷冻样品经解冻、离心后,用丹磺酰氯衍生,固相萃取小柱净化。目标化合物采用 Waters ACQUITY CSHTM C18色谱柱(50 mm×2.1 mm,1.7μm)分离,以甲醇和0.002 mol/L乙酸铵溶液为流动相梯度洗脱,采用电喷雾离子源电离、正离子多反应监测模式质谱检测。环己胺在2.5~200μg/L浓度范围内有较好的线性关系,相关系数大于0.999,回收率为98.7%~102.3%,精密度为3.1%~5.2%,检出限和定量限分别为1.0和3.0μg/L。结果表明,本方法操作简单、准确可靠,可适用于人体尿液中环己胺的定量分析。应用本方法测定200份学生尿液样品,环己胺检出率为34.5%。     

Quantitative analysis of cortisol and 6β‐hydroxycortisol in urine by fully automated SPE and ultra‐performance LC coupled with electrospray and atmospheric pressure chemical ionization (ESCi)‐TOF‐MS

An ultra‐performance LC TOF MS method for quantitative analysis of cortisol and 6β‐hydroxycortisol in urine was developed. The method was used for determination of the ratio between 6β‐hydroxycortisol and cortisol in urine received from autopsy cases and living persons as a measure of cytochrome P450 3A enzyme activity. Urine samples (0.25 mL) were extracted with an in‐house developed fully automated 96‐well SPE system. The compounds were quantified using a Waters ACQUITY UPLC system coupled to a Waters SYNAPT G2. The MS sensitivity was optimized by using negative ionization in sensitivity mode (resolution >10 000 full‐width at half‐maximum), and further optimized by using the enhanced duty cycle around the 410 m/z. ESCi (simultaneous electrospray and atmospheric pressure chemical ionization) mode was used to compensate for the matrix effects of postmortem urine. Finally, the SYNAPT G2 was tested as a quantitative instrument. The developed method has a measurement range from 2.5–300 ng/mL for cortisol to 10–1200 ng/mL for 6β‐hydroxycortisol. Mean overall process efficiencies were 29.4 and 23.0% for cortisol and 6β‐hydroxycortisol, respectively. In 20 forensic reference cases, the range of the 6β‐hydroxycortisol/cortisol ratio was 0.29–14.2 with a median of 3.04.     

A liquid chromatography/tandem mass spectrometry method for detecting UGT‐mediated bioactivation of drugs as their N‐acetylcysteine adducts in human liver microsomes

The detection of the reactive metabolites of drugs has recently been gaining increasing importance. In vitro trapping studies using trapping agents such as glutathione are usually conducted for the detection of reactive metabolites, especially those of cytochrome P450‐mediated metabolism. In order to detect the UDP‐glucuronosyltransferase (UGT)‐mediated bioactivation of drugs, an in vitro trapping method using N‐acetylcysteine (NAC) as a trapping agent followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed in this study. After the test compounds (diclofenac and ketoprofen) had been incubated in human liver microsomes with uridine diphosphoglucuronic acid (UDPGA) and NAC, the NAC adducts formed through their acyl glucuronides were analyzed using LC/MS/MS with electrospray ionization (ESI). The NAC adduct showed a mass shift of 145 units as compared to its parent, and the characteristic ion fragmentations reflected the parent. This is a concise and high‐throughput method for evaluating reactive metabolites by UGT‐mediated bioactivation. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Determination of selected natural hormones and endocrine disrupting compounds in domestic wastewater treatment plants by liquid chromatography electrospray ionization tandem mass spectrometry after solid phase extraction

A new analytical method for the simultaneous determination of two natural hormones (progesterone and estrone) and two selected endocrine disrupter compounds (EDCs) (diltiazem and carbamazepine (Cbz)) was developed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) after pre-concentration with solid phase extraction (SPE). Influent and effluent samples taken from five different wastewater treatment plants throughout Turkey namely Hurma/Antalya, Lara/Antalya, Kemer-1 and Kemer-2 and METU/Ankara were analyzed for their EDCs contents under the optimum conditions. All of the parameters in the pre-concentration step were optimized and the best recoveries for all compounds of interest were achieved at pH 7 (about 100%). Progesterone was not detected in any of the treatment plants while diltiazem was found in all samples with the exception of Lara effluent.     

Direct determination of beta-blockers and their metabolites in urine by liquid chromatography coupled with tandem mass spectrometry

A method is proposed for the detection and confirmation of the presence of beta-blockers and their metabolites in fivefold diluted human urine samples by ultra performance liquid chromatography coupled with electrospay ionization tandem mass spectrometry. The limits of detection for most of compounds are 5–10 ng/mL. A substantial effect of ionization suppression was observed. The determination of metabolites and glucuronides of beta-blockers without additional derivatization and extraction is described for the first time.     

Mass spectrometric and tandem mass spectrometric behavior of nitrocatechol glucuronides: A comparison of atmospheric pressure chemical ionization and electrospray ionization

The mass spectrometric (MS) and tandem mass spectrometric (MS/MS) behavior of six nitrocatechol-type glucuronides using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was systematically studied, and the effect of operation parameters on the fragmentations are presented. The positive ion APCI- and ESI-MS spectra showed an intense protonated molecule and the respective negative ion spectra a deprotonated molecule with minimal fragmentation. The main fragment ions in the MS/MS spectra of the protonated and deprotonated molecules were [M + H - Glu]+ and [M - H - Glu]-, respectively, formed by the loss of the glucuronide moiety. The measured limits of detection indicated that ESI is a significantly more efficient ionization method than APCI in the negative and positive ion modes for the compounds studied. MS/MS was found to be less sensitive, but more reliable and simple than MS due to the absence of chemical noise.     

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography–tandem mass spectrometry with chiral derivatization

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey’s reagent, N ^α- (5-fluoro-2,4-dinitrophenyl)-d-leucinamide, without extraction. The diastereomers of the N ^α-(2,4-dinitrophenyl)-d-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03?μg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20?μg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.     

Systematic screening and characterization of the major bioactive components of Poria cocos and their metabolites in rats by LC-ESI-MS(n)

Poria cocos is a well-known medicinal plant widely used in China and other East Asian countries owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of Poria cocos and their metabolites in vivo are still unclear to date. The aim of the present study was to develop a practical method based on the combined use of the liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS(n) ) for the comprehensive and systematic separation and characterization of the bioactive constituents of Poria cocos extract and their metabolites in rats. Based on the proposed strategy, a total of 34 compounds were characterized from the extract of Poria cocos. Among them, eight were unambiguously identified by comparing their retention times and mass spectra with those of reference standards, and 26 were tentatively identified on the basis of their MS(n) fragmentation behaviors and molecular weight information from literatures. In vivo, seven compounds were successfully detected in rat urine whereas one was found in rat plasma. This study proposed a series of potential bioactive components and provided helpful chemical information for further research on the action mechanism of traditional Chinese medicine.