A screening method for the simultaneous detection of glucocorticoids,diuretics, stimulants,anti-oestrogens,beta-adrenergic drugs and anabolic steroids in human urine by LC-ESI-MS/MS

This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1–50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50–500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.     

UPLC-MS/MS法快速筛查尿液中30种滥用药物

基于超高液相色谱-串联四极杆/线性离子阱质谱(QTRAP UPLC-MS/MS),建立了尿液中30种滥用药物的筛查方法.采用蛋白沉淀法处理尿液样品,实现对多类别滥用药物的高效提取.采用分段多反应监测(sMRM)联合信息依赖性采集(IDA)与增强离子扫描(EPI)模式,结合EPI谱库检索匹配确证检出物信息,并引入内标辅助...     

A high performance liquid chromatographic method for the screening of 17 diuretics in human urine

A simple and adequate HPLC method was developed for screening of human urine for the following 17 diuretic drugs: acetazolamide, bendrofluazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, clopamide, epitizide, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, mefruside, piretanide, spironolactone, torasemide, and triamterene. The assay involves extraction from two 2 mL urine samples with ethyl acetate at pH = 5, washing with a phosphate buffer at pH = 6 and analysis by HPLC using a reversed phase C18 column and ultraviolet detection with a diode array detector for all drugs (except triamterene) using two eluents consisting of water, triethylamine, phosphoric acid and acetonitrile at different ratios and different pH values. Triamterene is determined by direct injection of diluted urine onto the column and is measured by fluorescence detection. The recoveries of the diuretic drugs were determined at two different concentrations and ranged from 43–110% (median: 87%) which is sufficient to detect abuse of these drugs. The repeatability of the assay ranged from 1–12% (median: 5.5%). Received: 13 July 1998 / Revised: 23 October 1998 / Accepted: 29 October 1998     

A high performance liquid chromatographic method for the screening of 17 diuretics in human urine

A simple and adequate HPLC method was developed for screening of human urine for the following 17 diuretic drugs: acetazolamide, bendrofluazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, clopamide, epitizide, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, mefruside, piretanide, spironolactone, torasemide, and triamterene. The assay involves extraction from two 2 mL urine samples with ethyl acetate at pH = 5, washing with a phosphate buffer at pH = 6 and analysis by HPLC using a reversed phase C18 column and ultraviolet detection with a diode array detector for all drugs (except triamterene) using two eluents consisting of water, triethylamine, phosphoric acid and acetonitrile at different ratios and different pH values. Triamterene is determined by direct injection of diluted urine onto the column and is measured by fluorescence detection. The recoveries of the diuretic drugs were determined at two different concentrations and ranged from 43–110% (median: 87%) which is sufficient to detect abuse of these drugs. The repeatability of the assay ranged from 1–12% (median: 5.5%).     

Fast gas chromatographic/mass spectrometric determination of diuretics and masking agents in human urine: Development and validation of a productive screening protocol for antidoping analysis

An analytical procedure was developed for the fast screening of 16 diuretics (acetazolamide, althiazide, amiloride, bendroflumethiazide, bumetanide, canrenoic acid, chlorthalidone, chlorthiazide, clopamide, ethacrynic acid, furosemide, hydrochlorthiazide, hydroflumethiazide, indapamide, triamterene, trichlormethiazide) and a masking agent (probenecid) in human urine. The whole method involves three analytical steps, including (1) liquid/liquid extraction of the analytes from the matrix, (2) their reaction with methyl iodide at 70 degrees C for 2 h to form methyl derivatives, (3) analysis of the resulting mixture by fast gas chromatography/electron impact mass spectrometry (fast GC/EI-MS). The analytical method was validated by determining selectivity, linearity, accuracy, intra and inter assay precision, extraction efficiencies and signal to noise ratio (S/N) at the lowest calibration level (LCL) for all candidate analytes. The analytical performances of three extraction procedures and five combination of derivatization parameters were compared in order to probe the conditions for speeding up the sample preparation step. Limits of detection (LOD) were evaluated in both EI-MS and ECNI-MS (electron capture negative ionization mass spectrometry) modes, indicating better sensitivity for most of the analytes using the latter ionization technique. The use of short columns and high carrier gas velocity in fast GC/MS produced efficient separation of the analytes in less than 4 min, resulting in a drastic reduction of the analysis time, while a resolution comparable to that obtained from classic GC conditions is maintained. Fast quadrupole MS electronics allows high scan rates and effective data acquisition both in scan and selected ion monitoring modes.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

A fast method for screening and/or quantitation of tetrahydrocannabinol and metabolites in urine by automated SPE/LC/MS/MS

Marijuana is one of the most commonly used illicit substances. The high usage of this substance results in it being commonly encountered in clinical samples throughout the USA and Europe. Due to its wide availability and use, marijuana is also commonly encountered in forensic toxicology laboratories. The proposed method utilized an automated solid phase extraction (SPE) coupled to liquid chromatography/mass spectrometry (LC/MS). The automated SPE procedure was developed using Hysphere C8-EC sorbent, and the high performance liquid chromatography (HPLC) separation was performed using an Xterra MS C₁₈ column with a total runtime of 10 min. The standard curves linearity generally fell between 6 and 500 ng/mL. The limits of detection ranged from 2 to 4 ng/mL, and the limits of quantitation ranged from 8 to 12 ng/mL. The bias and imprecision were determined using a simple analysis of variance (single factor). The results demonstrate bias as <11% and percent imprecision as <12% for all components at four quality control levels. This method has been in use for over 2 years and has been applied to numerous forensic samples. When compared to other published methods, it exceeds others in its simplicity and speed of analysis. This method takes advantage of robotics and automation for a total analysis time of 10 min, including sample preparation, separation, and detection.     

A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry

A general screening method based on solid phase extraction (SPE) and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and investigated with 124 different doping agents, including stimulants, -blockers, narcotics, -adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids. Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was based on gradient elution on a C18 column. Ionization of the analytes was achieved with electrospray ionization in the positive mode. Identification by LC/TOFMS was based on retention time, accurate mass and isotopic pattern. Validation of the method consisted of analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was attained to 97 doping agents. The extraction recoveries varied between 33 and 98% and the median was 58%. Mass accuracy was always better than 5 ppm, corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for spiked urine samples, expressed as median of relative standard deviations (RSD%) at concentrations of MRPL and 10 times MRPL, were 14% and 9%, respectively. The suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine samples.     

A fast, comprehensive screening method for doping agents in urine by gas chromatography-triple quadrupole mass spectrometry

The use of performance enhancing drugs in sports is prohibited. For the detection of misuse of such substances gas chromatography or liquid chromatography coupled to mass spectrometry are the most frequently used detection techniques. In this work the development and validation of a fast gas chromatography tandem mass spectrometric method for the detection of a wide range of doping agents is described. The method can determine 13 endogenous steroids (the steroid profile), 19-norandrosterone, salbutamol and 11-nor-Δ9-tetrahydrocannabinol.9carboxylic acid in the applicable ranges and to detect qualitatively over 140 substances in accordance with the minimum required performance levels of the World Anti-Doping Agency in 1ml of urine. The classes of substances included in the method are anabolic steroids, β2-agonists, stimulants, narcotics, hormone antagonists and modulators and beta-blockers. Moreover, using a short capillary column and hydrogen as a carrier gas the run time of the method is less than 8min.     

Rapid screening and determination of nerve agent metabolites in human urine by LC-MS/MS

Qualitative screening procedures have been developed for the rapid detection and identification of the metabolites of nerve agents in the urine samples and extracts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The combination of negative electrospray ionization (ESI) using a C₁₈ column and water-methanol mobile phase modified with ammonium formate provides a rapid screening procedure for nerve agent degradation products with limit of detection of 1 ng/mL in the precursor-ion analysis. Also, determination of the alkyl methylphosphonic acids was carried out by the SRM scan mode with the limit of detection of 0.1 ng/mL. These procedures will be applicable to the trace analysis of metabolites of nerve agents in human urine matrices in the Organisation for the Prohibition of Chemical Weapons (OPCW) proficiency test.     

A sensitive and validated LC-MS/MS method for high-throughput determination of pomalidomide in human plasma and pharmacokinetic studies

Pomalidomide is an immunomodulatory agent （IMiD） that has been approved by the US Food and Drug Administration （FDA） for clinical treatment of patients with multiple myeloma.In this work,we developed a sensitive and validated LC-MS/MS method for high-throughput determination of pomalidomide over the range of 1.006–100.6 ng/m L （R²=0.9991） in human plasma and pharmacokinetic studies.A liquid-liquid extraction method using ethyl acetate was applied to extract pomalidomide and afatinib （...     

A high-sensitivity UPLC-MS/MS method for simultaneous determination and confirmation of triptolide in zebrafish embryos

A high‐sensitivity ultra‐performance liquid‐chromatography (UPLC) coupled with tandem mass spectrometric method was developed for simultaneous quantification and confirmation of triptolide in both zebrafish embryos and the aqueous‐exposure solution on a tandem quadrupole mass spectrometer (TQ‐MS). This was achieved by performing quantification using the multiple reaction monitoring (MRM) acquisition with simultaneous characterization of the MRM peak using product ion confirmation (PIC) acquisition as it elutes from the chromatographic system. Separation was achieved on a 1.7 µm C₁₈ UPLC column using 0.1% formic acid water–acetonitrile mobile phase with a cycle time of 6 min. The linear range of 0.115–360 ng/mL, and lower limits of detection of 0.02 ng/mL and quantification of 0.064 ng/mL were established. This method was successfully applied to determine the time course of triptolide absorption by zebrafish embryos and the amount of triptolide remaining in the culture medium after administration of two triptolide dosages at three time points. This coupled MRM with PIC approach could provide both qualitative and quantitative results without the need for repetitive analyses. This resulted in the reduction of further confirmative experiments and analytical time, and ultimately increased laboratory productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

A validated positive chemical ionization GC/MS method for the identification and quantification of amphetamine, opiates, cocaine, and metabolites in human postmortem brain

A sensitive and specific method for the simultaneous detection and quantification of amphetamine, opiates, and cocaine and metabolites in human postmortem brain was developed and validated. Analytes of interest included amphetamine, morphine, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, and anhydroecgonine methyl ester. The method employed ultrasonic homogenization of brain tissue in pH 4.0 sodium acetate buffer and solid phase extraction. Extracts were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide and N,O-bis(trimethylsilyl) trifluoroacetamide. Separation and quantification were accomplished on a bench-top positive chemical ionization capillary gas chromatograph/mass spectrometer with selected ion monitoring. Eight deuterated analogs were used as internal standards. Limits of quantification were 50 ng/g of brain. Calibration curves were linear to 1000 ng/g for anhydroecgonine methyl ester and 6-acetylmorphine, and to 2000 ng/g for all other analytes. Accuracy across the linear range of the assay ranged from 90.2 to 112.2%, and precision, as percent relative standard deviation, was less than 16.6%. Quantification of drug concentrations in brain is a useful research tool in neurobiology and in forensic and postmortem toxicology, identifying the type, relative magnitude, and recency of abused drug exposure. This method will be employed to quantify drug concentrations in human postmortem brain in support of basic and clinical research on the physiologic, biochemical, and behavioral effects of drugs in humans.     

A fast gas chromatography/mass spectrometry method for the determination of stimulants and narcotics in urine

A fast method has been developed for the simultaneous determination of 52 stimulants and narcotics excreted unconjugated in urine by gas chromatography/mass spectrometry (GC/MS). The procedure involves the liquid/liquid extraction of the analytes from urine at strong alkaline pH and the injection of the extract into a GC/MS instrument with a fast GC column (10 m × 0.18 mm i.d.); the short column allows the complete separation of the 52 analytes in a chromatographic run of 8 min. The method has been fully validated giving lower limits of detection (LLODs) satisfactory for its application to antidoping analysis as well as to forensic toxicology. The repeatability of the concentrations and the retention times are good both for intra‐ and for inter‐day experiments (%CV of concentrations always lower than 15 and %CV of retention times lower than 0.6). In addition, the analytical bias is satisfactory (A% always >15%). The method proposed here would be particularly useful whenever there are time constraints and the analyses have to be completed in the shortest possible time. Copyright © 2010 John Wiley & Sons, Ltd.     

Validation of QuEChERS-based UPLC-MS/MS method for determination of quinoid niclosamide (LDS) residue in water,soil and rice samples

A sensitive, rapid and easy analytical method was validated for the determination of quinoid niclosamide (LDS) molluscicide in water, rice and soil using a QuEChERS extraction procedure and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection. The LDS was extracted by using acetonitrile and then cleaned up by using dispersive solid-phase extraction with florisil and C18 sorbents. The determination of the target compound was achieved in less than 3 min using an electrospray ionisation source in negative mode. The overall average recoveries for this method in water, rice and soil matrix at three fortified levels ranged from 82.54 to 99.9%, with relative standard deviations in the range of 1.51 to 4.86% (n = 5). The calculated limits of detection were lower than 0.1 µg kg^?1 and quantification was 5 µg kg^?1; these values were much lower than the maximum residue levels established by the Australian standard (0.01 mg kg^?1). The results of the method validation confirmed that this proposed method is convenient and reliable for the determination of LDS molluscicide in water, rice and soil samples.     

Development of a UPLC-MS/MS method for the determination of ten anticancer drugs in hospital and urban wastewaters, and its application for the screening of human metabolites assisted by information-dependent acquisition tool (IDA) in sewage samples

In the present work, the development, optimization, and validation (including a whole stability study) of a fast, reliable, and comprehensive method for the analysis of ten anticancer drugs in hospital and urban wastewater is described. Extraction of these pharmaceutical compounds was performed using automated off-line solid-phase extraction followed by their determination by ultra-performance liquid chromatography coupled to a triple quadrupole–linear ion trap mass spectrometer. Target compounds include nine cytotoxic agents: cyclophosphamide, ifosfamide, docetaxel, paclitaxel, etoposide, vincristine, tamoxifen, methotrexate, and azathioprine; and the cytotoxic quinolone, ciprofloxacin. Method detection limits (MDL) ranged from 0.8 to 24 ng/L. Levels found of cytostatic agents in the hospital and wastewater influents did not differ significantly, and therefore, hospitals cannot be considered as the primary source of this type of contaminants. All the target compounds were detected in at least one of the influent samples analyzed: Ciprofloxacin, cyclophosphamide, tamoxifen, and azathioprine were found in most of them and achieving maximum levels of 14.725, 0.201, 0.133, and 0.188 μg/L, respectively. The rest of target cancer drugs were less frequently detected and at values ranging between MDL and 0.406 μg/L. Furthermore, a feasible, useful, and advantageous approach based on information acquisition tool (information-dependent acquisition) was used for the screening of human metabolites in hospital effluents, where the hydroxy tamoxifen, endoxifen, and carboxyphosphamide were detected.