A fast liquid chromatographic/mass spectrometric screening method for the simultaneous detection of synthetic glucocorticoids, some stimulants, anti-oestrogen drugs and synthetic anabolic steroids

A fast liquid chromatographic/mass spectrometric (LC/MS/MS) screening method for the detection, in urine, of synthetic glucocorticoids, stimulants (formoterol, modafinil and mesocarb), anti-oestrogens (finasteride, exemestane, anastrozole, letrozole and formestane) and synthetic anabolic steroids (stanozolol, gestrinone and tetrahydrogestrinone) is described. All these drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid extraction step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase, and assayed in 7 min by LC/MS/MS using electrospray ionization in positive ion mode and multiple reaction monitoring as the acquisition mode. All compounds show good reproducibility of both the retention times (CV% <2%) and the relative abundances (CV% <10%). The limits of detection for the anti-oestrogens, glucocorticoids and steroids are in the range of 1-30 ng/mL, and for the stimulants are in the range of 100-200 ng/mL, thus satisfying the minimum required performance limits of the World Anti-Doping Agency.     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

Development of a qualitative liquid chromatography/tandem mass spectrometric method for the detection of narcotics in urine relevant to doping analysis

A new screening procedure for 18 narcotics in urine for anti-doping purposes has been developed using liquid chromatography/triple quadrupole mass spectrometry (LC/MS). Electrospray ionization (ESI) was used as interface. Infusion experiments were performed for all substances to investigate their mass spectrometric behaviour in terms of selecting product specific ions. These product ions were then used to develop a tandem mass spectrometric method using selected reaction monitoring (SRM). For the LC/MS analysis, chromatography was performed on an octadecylsilane column. The total run time of the chromatographic method was 5.5 min. For the sample preparation prior to LC/MS analysis, the urine samples were liquid-liquid extracted at pH 9.5 after overnight enzymatic hydrolysis. Two extraction solvents were evaluated: dichloromethane/methanol 9/1 (v/v), which is currently used for the extraction of narcotics, and diethyl ether, used for the extraction of steroids. With diethyl ether the detection limits for all compounds ranged between 0.5 and 20 ng/mL and with the mixture containing dichloromethane the detection limits ranged between 0.5 and 10 ng/mL. Taking into account the minimum required performance limits of the World Anti-Doping Agency of 200 ng/mL for narcotics, diethyl ether can also be considered as extraction solvent for narcotics. Finally, the described method was applied to the analysis of urine samples previously found to contain narcotics by our routine gas chromatography/mass spectrometry (GC/MS) method.     

A screening method for the detection of synthetic glucocorticosteroids in human urine by liquid chromatography–mass spectrometry based on class-characteristic fragmentation pathways

This paper describes a liquid chromatographic/tandem mass spectrometric (LC/MS–MS) method specifically designed for the screening of synthetic glucocorticosteroids in human urine. The method is designed to recognize a common mass spectral fragment formed from the particular portion of the molecular structure that is common to all synthetic glucocorticosteroids and that is fundamental to their pharmacological activity. As such, the method is also suitable for detecting unknown substances, provided they contain the portion of the molecular structure selected as the analytical target. The effectiveness of this approach was evaluated on seventeen synthetic glucocorticosteroids. Urine samples, including blank urines spiked with one or more synthetic glucocorticosteroids, were treated according to a standard procedure (enzymatic hydrolysis, liquid/liquid extraction and evaporation to dryness) and analyzed using LC/MS-MS with electrospray ionization (ESI). MS–MS acquisition was carried out in a precursor ion scan, and the results were compared with those obtained by a previously developed reference technique based on acquisition in the multiple reaction monitoring (MRM) mode. All of the glucocorticosteroids considered in this study are clearly detectable in urine, with a limit of detection in the concentration range 5–20 ng/mL, depending on the glucocorticosteroid structure. The proposed method is therefore suitable for the detection of glucocorticosteroids in urine samples taken for “in competition” sport anti-doping control tests, matching the requirements of the World Anti-Doping Agency (WADA) for accredited anti-doping laboratories. Figure Structures of the synthetic glucocorticoids considered in this study     

Study of the matrix effect on the determination of nonconjugated xenobiotics in human urine by high-performance liquid chromatography/tandem mass spectrometry

Factors affecting the sensitivity and selectivity of the determination of diuretics, anabolic steroids, central nervous system stimulants, and narcotics in the analysis of human urine extracts by high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure electrospray ionization and recording of positive ions were investigated. Mass spectra were obtained for all of the test compounds; the characteristic ions, retention times, detection limits, degree of ionization suppression by the matrix, the extraction of the analytes from human biological fluids were determined for all analytes; the selectivity and specificity of determination were evaluated.     

Simultaneous determination of a broad spectrum of nonconjugated xenobiotics by high-performance liquid chromatography-tandem mass spectrometry

A procedure was developed for screening 23 corticosteroids, 12 anabolic steroids, 23 diuretics, and 49 central nervous system stimulants and narcotics. The sample preparation includes extraction, the evaporation of the organic extract in a nitrogen flow, and the dissolution of the dry residue. The extract was analyzed by high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure electrospray ionization under different recording conditions. Negatively charged ions were recorded to determine corticosteroids and diuretics, while positively charged ions, to determine anabolic steroids, diuretics, stimulants, and narcotics. Mass spectra were obtained for all test compounds; the characteristic ions, retention times, detection limits, degree of ionization suppression by the matrix, and recovery were determined for all analytes.     

Highly sensitive and selective analysis of urinary steroids by comprehensive two-dimensional gas chromatography combined with positive chemical ionization quadrupole mass spectrometry

Comprehensive two dimensional gas chromatography (GC × GC) provides greater separation space than conventional GC. Because of fast peak elution, a time of flight mass spectrometer (TOFMS) is the usual structure-specific detector of choice. The quantitative capabilities of a novel GC × GC fast quadrupole MS were investigated with electron ionization (EI), and CH(4) or NH(3) positive chemical ionization (PCI) for analysis of endogenous urinary steroids targeted in anti-doping tests. Average precisions for steroid quantitative analysis from replicate urine extractions were 6% (RSD) for EI and 8% for PCI-NH(3). The average limits of detection (LODs) calculated by quantification ions for 12 target steroids spiked into steroid-free urine matrix (SFUM) were 2.6 ng mL(-1) for EI, 1.3 ng mL(-1) for PCI-CH(4), and 0.3 ng mL(-1) for PCI-NH(3), all in mass scanning mode. The measured limits of quantification (LOQs) with full mass scan GC × GC-qMS were comparable with the LOQ values measured by one-dimensional GC-MS in selected ion monitoring (SIM) mode. PCI-NH(3) yields fewer fragments and greater (pseudo)molecular ion abundances than EI or PCI-CH(4). These data show that a benchtop GC × GC-qMS system has the sensitivity, specificity, and resolution to analyze urinary steroids at normal urine concentrations, and that PCI-NH(3), not currently available on most GC × GC-TOFMS instruments, is of particular value for generation of structure-specific ions.     

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry: I. Screening analysis

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C₁₈ reversed-phase column in two gradients of 9 min (including two 3 min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500 ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.     

Simultaneous determination of beta-blocking agents and diuretics in doping analysis by liquid chromatography/mass spectrometry with scan-to-scan polarity switching

A previously described method for the screening of 18 diuretics and probenecid was substantially extended with 21 beta-blockers and 8 other diuretics allowing simultaneous determination of diuretics and beta-adrenergic blocking agents in human urine. Analysis was performed using an ion trap instrument with an electrospray ionisation (ESI) interface after liquid/liquid extraction with ethyl acetate. Full-scan MS and full-scan MS2 were applied in combination with scan-to-scan polarity switching. All compounds were separated in less than 22 min. The detection limits for the diuretics were between 5 and 100 ng/mL and for the beta-adrenergic blocking agents were between 5 and 500 ng/mL. The excretion of carvedilol was followed after intake of one tablet of Dimitone. Other doping agents including strychnine, norbuprenorphine and mesocarb hydroxysulfate could also be detected with this method.     

Determination of Diuretics in Urine Using Immobilized Multi‐Walled Carbon Nanotubes in Hollow Fiber Liquid‐Phase Microextraction Combined with Liquid Chromatography‐Tandem Mass Spectrometry

A novel technique utilizing the adsorptive potential of immobilized multi‐walled carbon nanotubes (I‐MWCNT) in hollow fiber liquid‐phase microextraction (HF‐LPME) was developed for the determination of diuretics in urine. In this study, the potential of carbon nanotubes as a sorbent for three‐phase liquid‐phase microextraction of diuretics from urine samples was evaluated. Analysis was performed using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). A novel method was applied to detect acetazolamide (AAA), chlorothiazide (CTA), hydrochlorothiazide (HCT), hydroflumethiazide (HFT), clopamide (CA), trichlormethiazide (TCM), althiazide (AT) and bendroflumethiazide (BFT) in urine. Two‐step extractions using different times and temperatures for each step were adopted. Parameters influencing the extraction efficiency, including the extraction solvent, sample pH, salt concentration, extraction time and extraction temperature were systematically optimized. Under the resulting optimal extraction conditions, this method showed good linearity over an analytes concentration range of 1 to 1000 ng/mL, high extraction repeatability with relative standard deviations of less than 6%, and low detection limits (0.09 to 0.51 ng/mL). The application of the methods to the determination of diuretics in real samples was tested by analyzing urine samples of patient.     

Qualitative detection of diuretics and acidic metabolites of other doping agents in human urine by high-performance liquid chromatography–tandem mass spectrometry: Comparison between liquid–liquid extraction and direct injection

Direct injection of urine has gained interest in the field of analytical toxicology, including doping control analysis. However, implementation of a direct urinalysis method for the LC–MS/MS detection of 34 diuretics and 9 other doping agents yielded several analytical problems, which were not observed using a traditional liquid–liquid extraction. Therefore a comparative study was made between liquid–liquid extraction and direct injection. Comparison of validation results showed that the liquid–liquid extraction at pH 7 allows to analyze samples without major drawbacks regarding matrix effects. Hence, good sensitivity was observed and detection limits ranged between 1 and 250 ng/mL for all compounds. In the direct injection approach shifted retention times were observed for several acidic and basic compounds due to unwanted matrix effects. This shift was reduced by a 25-fold dilution of the urine samples. Besides the improved retention time stability the diluted samples also exhibited lower ion suppression than the undiluted ones. After 25-fold dilution, detection limits ranged between 10 and 250 ng/mL for all compounds. Since these detection limits are at or below the minimum required performance level, imposed by the World Anti-Doping Agency, the method could be applied to routine anti-doping analysis. Samples, previously declared positive, were reanalysed using both the liquid–liquid extraction and direct injection. With both techniques all 26 samples were found to be positive, showing the applicability of direct injection for the analysis of diuretics.     

Relationships between structure,ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I–IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H]⁺ or [M + H–nH₂O]⁺ in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05–20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05–1, 2–5 and 10–20 ng/mL, respectively. Steroids including the conjugated keto‐functional group at C3 showed good proton affinity and stability, and generated the [M + H]⁺ ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H]⁺ ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H ? H₂O]⁺ or [M + H ? 2H₂O]⁺ ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC‐MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I–V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.     

Phytoestrogen biomonitoring: an extractionless LC-MS/MS method for measuring urinary isoflavones and lignans by use of atmospheric pressure photoionization (APPI)

We present here a high-performance liquid chromatography−tandem mass spectrometry (LC-MS/MS) method for quantifying phytoestrogenic isoflavones (daidzein, equol, genistein, and O-desmethylangolensin) and lignans (enterodiol and enterolactone) in urine without the use of extraction or the preconcentration techniques inherent in existing methods. The development of this concept was made possible by use of atmospheric pressure photoionization (APPI); an ionization technique that we found to improve analyte sensitivity relative to electrospray ionization and atmospheric pressure chemical ionization for this particular group of compounds. The analytical performance of this method was equal to or exceeded that of comparable methods. Between-run coefficients of variation (CVs) across three quality control (QC) pool levels analyzed in duplicate over 20 days were 3.1–5.8% CV; within-run CVs were 2.3–6.0%. Accuracy, as determined by average spike recovery in QC pools, was generally within ±10% of being quantitative (100%). Relative limits of detection were 0.04–0.4 ng/mL urine, with absolute detection limits as low as 0.1 pg. This method was applied to the analysis of >2,500 urine specimens for the 2005–2006 Centers for Disease Control and Prevention’s National Health and Nutrition Examination Survey (NHANES). The method was capable of quantifying these compounds in 95–100% of study samples. This work is the first ever report of using APPI for the LC-MS/MS determination of these compounds in urine. It is also the first method of its kind to do so without any need for analyte extraction or preconcentration prior to analysis.     

Fast and sensitive supercritical fluid chromatography – tandem mass spectrometry multi-class screening method for the determination of doping agents in urine

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48–68% of the compounds and higher than 50% for 83–87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites.     

液相色谱-串联质谱法检测血清中15种甾体激素的动态变化

建立了用液相色谱-串联质谱检测血清中15种甾体激素的方法。在血清样品中加入含内标的乙腈沉淀蛋白质后,用100 mmol/L盐酸羟胺衍生,经Agilent-C18柱(50 mm×3.0 mm,2.7μm)分离,内标法定量。质谱分析采用电喷雾电离(ESI)正离子扫描,多反应监测(MRM)模式检测。数据显示,13种激素在0.05~20 ng/mL范围内线性关系良好(相关系数(R2)不小于0.995 6),检出限不大于1 ng/mL;皮质醇及脱氢表雄酮硫酸酯在50~2 000ng/mL范围内线性关系良好(R2不小于0.997 9),检出限不大于0.5 ng/mL。将该方法应用于女性月经周期激素变化规律的研究,结果表明,15种甾体激素在一个完整的月经周期内有不同特征的动态变化,其中雄激素在滤泡期的后期达到最高值,孕(雌)激素在黄体期达到最高值,而皮质激素则波动不明显。该方法灵敏度高、重复性好,为临床诊疗提供了参考。     

Liquid-phase microextration combined with liquid chromatography-electrospray tandem mass spectrometry for detecting diuretics in urine

Trace amounts of diuretics were determined in human urine by hollow fiber liquid-phase microextraction (LPME) combined with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in this study. Chromatography was performed on a C(8) reversed-phase column. A 25 microL n-octanol was used to extract analytes in urine. Extraction was optimized using a pH 2 solution spiked with 0.15 g/mL NaCl for 40 min at 40 degrees C with 1010 rpm stirring. The limits of detection of diuretics in urine were 0.3-6.8 ng/mL, and linearity range was 1-1000 ng/mL. Recoveries of spiked 50 ng/mL diuretics were 97.7-102.5%. The intra-day precision and inter-day precision were 3-18% and 4-21%, respectively. The diuretics concentration profiles in patient urine were also determined. The results of this study reveal the adequacy of LPME-LC-MS/MS method for analyzing diuretics in urine and quantification limits exceed World Anti-Doping Agency requirements.     

Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the detection of the free anabolic steroid fraction in human urine was examined. Electrospray ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization methods were optimized regarding eluent composition, ion source parameters and fragmentation. The methods were compared with respect to specificity and detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water gradient including 5 mM ammonium acetate and 0.01% acetic acid was found best for the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the detection of free steroids and may be a suitable technique for screening for the abuse of anabolic steroids in sports.     

Simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene using high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

A simple and rapid method using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene in human urine specimens and standard solutions is described. A hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer was compared for the determination of metabolite of aromatic solvents in urine samples. The metabolites selected were: trans,trans-muconic acid, hippuric acid, o-, m- and p-methylhippuric acid and phenylglyoxylic acid. The compounds were well separated from each other on narrow-bore 1-mm i.d. reversed-phase LC C-18 columns. Average recoveries for loading 100 microL of urine samples varied from 88-110% and the quantification limits were less than 30 ng/mL for each analyte (3 ng/mL for trans,trans-muconic acid). The qualitative information obtained (mass accuracy, resolution and full-scan spectra) with the QqTOF mass spectrometer allows a secure identification of analytes in biological matrices.     

Contribution of microextraction in packed sorbent for the analysis of cotinine in human urine by GC–MS

A simple, rapid, sensitive, and non-consuming solvent method for the determination of cotinine in urine was developed, based on sample preparation by the relatively new technique microextraction in packed sorbent (MEPS) and analysis by GC–MS. This optimized method was compared with conventional solid-phase extraction/liquid–liquid extraction method used as reference. The wide linear range (5–5,000 ng/mL) and high sensitivity of the MEPS method (limit of detection 0.8 ng/mL) allow application to analysis of urine from smokers as well as non-smokers susceptible to passive smoking.     

Development and validation of an open screening method for diuretics,stimulants and selected compounds in human urine by UHPLC–HRMS for doping control

A new doping control screening method for the analysis of diuretics and stimulants using ultra high pressure liquid chromatography–high resolution Orbitrap mass spectrometry has been developed. The screening was performed in full scan MS with scan-to-scan polarity switching which allowed to detect more than 120 target analytes. Sample preparation was limited to 10-fold dilution of the urine into the internal standard solution followed by injection. Total run time per sample was 10 min. Validation of the method yielded detection limits for diuretics between 25 and 250 ng mL⁻¹ and for stimulants between 5 and 500 ng mL⁻¹. The screening method has been implemented in routine doping control.