Feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization mass spectrometry in analyzing anabolic steroids in urine samples

We examined the feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization tandem mass spectrometry (capLC/µAPPI‐MS/MS) for the analysis of anabolic steroids in human urine. The urine samples were pretreated by enzymatic hydrolysis (with β‐glucuronidase from Helix pomatia), and the compounds were liquid‐liquid extracted with diethyl ether. After separation the compounds were vaporized by microchip APPI, photoionized by a 10 eV krypton discharge lamp, and detected by selected reaction monitoring. The capLC/µAPPI‐MS/MS method showed good sensitivity with detection limits at the level of 1.0 ng mL^?1, good linearity with correlation coefficients between 0.9954 and 0.9990, and good repeatability with relative standard deviations below 10%. These results demonstrate that microchip APPI combined with capLC/MS/MS provides a new potential method for analyzing non‐polar and neutral compounds in biological samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine

The applicability of in-vial two-phase liquid-phase microextraction (LPME) in porous hollow polypropylene fiber was studied for the sample preparation of unconjugated anabolic steroids in urine. Four different anabolic steroids - metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol - were used as test compounds and methyltestosterone as an internal standard. A standard two-phase LPME method for use with liquid chromatography/mass spectrometry (LC/MS) was set up and the influence of different parameters, including the nature of organic solvent, extraction time, salting-out and temperature, on the LPME process was investigated. Taking advantage of the preliminary studies, a novel two-phase LPME method utilizing simultaneous in-fiber silylation was developed and validated for gas chromatographic/mass spectrometric (GC/MS) analysis of a danazol metabolite in urine. In all, LPME allowed a very straightforward, simple and selective way to prepare urine samples for steroid analysis, being most suitable for hydrophobic steroids. The LPME method with in-fiber derivatization for GC/MS analysis exhibited high sensitivity, repeatability and linearity and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine matrix without any other steps in sample pretreatment.     

Gas chromatography/mass spectrometry of polychlorinated biphenyls using atmospheric pressure chemical ionization and atmospheric pressure photoionization microchips

Gas chromatography (GC) and ion trap mass spectrometry (MS) were combined with microchip atmospheric pressure chemical ionization (microAPCI) and microchip atmospheric pressure photoionization (microAPPI) sources. Selected polychlorinated biphenyls (PCBs, IUPAC Nos. 28, 52, 101, 118, 138, 153 and 180) were analyzed by GC/microAPCI-MS and GC/microAPPI-MS to demonstrate the applicability of the miniaturized ion sources in negative ion mode analysis. The microAPCI and microAPPI methods were evaluated in respect of detection limit, linearity and repeatability. The detection limits for the PCB congeners were somewhat lower with microAPCI than with microAPPI, whereas microAPPI showed slightly wider linear range and better repeatability. With both methods, the best results were obtained for highly chlorinated or non-ortho-chlorinated PCBs, which possess the highest electron affinities. Finally, the suitability of the GC/microAPPI-MS method for the analysis of PCBs in environmental samples was demonstrated by analyzing soil extracts, and by comparing the results with those obtained by gas chromatography with electron capture detection (GC/ECD).     

Comparison of liquid chromatography-microchip/mass spectrometry to conventional liquid chromatography–mass spectrometry for the analysis of steroids

The feasibility of a microfluidic-based liquid chromatography-electrospray ionization/mass spectrometric system (HPLC-Chip/ESI/MS) was studied and compared to a conventional narrow-bore liquid chromatography-electrospray ionization/mass spectrometric (LC-ESI/MS) system for the analysis of steroids. The limits of detection (LODs) for oxime derivatized steroids, expressed as concentrations, were slightly higher with the HPLC-Chip/MS system (50–300 pM) using an injection volume of 0.5 μL than with the conventional LC-ESI/MS (10–150 pM) using an injection volume of 40 μL. However, when the LODs are expressed as injected amounts, the sensitivity of the HPLC-Chip/MS system was about 50 times higher than with the conventional LC-ESI/MS system. The results indicate that the use of HPLC-Chip/MS system is clearly advantageous only in the analysis of low-volume samples. Both methods showed good linearity and good quantitative and chromatographic repeatability. In addition to the instrument comparisons with oxime derivatized steroids, the feasibility of the HPLC-Chip/MS system in the analysis of non-derivatized and oxime derivatized steroids was compared. The HPLC-Chip/MS method developed for non-derivatized steroids was also applied to the quantitative analysis of 15 mouse plasma samples.     

Detection of Anabolic Steroids By Gc/Sim/Ms With Trifluoroacetylation in Equine Plasma and Urine

ABSTRACT

A method to detect three anabolic steroids (boldenone, nandrolone and mesterolone) is presented. The anabolic steroids are isolated from equine plasma and urine by extraction with diethyl ether and C₁₈ Sep-Pak cartridge adsorption, respectively. The extracts obtained were derivatized with trifluoroacetyl anhydride and analyzed by GC/SIM/MS. The selected ion monitoring (SIM) mode was applied to increase the sensitivity and, when possible, the higher m/z ions were selected to improve identification. Stability of derivatives was good and compounds having hydroxy and conjugated ketone groups produced trifluoroacetyl ester derivatives that were also stable. Repeatability of the chromatographic analysis was evaluated on the basis of area repeatability, and the coefficient of variation obtained was lower than 4.4. The detection limit was 1 and 5 ng/ml for all the anabolic steroids studied in equine plasma and urine, respectively.     

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.     

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.     

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry for analysis of Dechloranes

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) was investigated as an instrumental method for the analysis of the halogenated norbornene flame retardants, Mirex, Dechloranes 602, 603, 604, and Dechlorane Plus (DP). The LC separation was optimized by screening a variety of stationary and mobile phases, resulting in a short LC separation time of 5 min. Different atmospheric pressure ionization approaches were examined including electrospray ionization, atmospheric pressure chemical ionization, and APPI, each with and without post-column addition. APPI without post-column addition was chosen for providing the best ionization response. The optimized LC/APPI-MS/MS approach resulted in instrument detection limits ranging between 25 and 50 pg. Good linearity was also achieved (up to 25.0 ng/μL; R >0.999). The method was applied to extracts of environmental samples including surface water, fish and sediments for screening purposes, and the results agreed well with those obtained by gas chromatography/mass spectrometry.     

Ionspray microchip

An ionspray microchip is introduced. The chip is based on the earlier presented nebulizer microchip that consists of glass and silicon plates bonded together. A liquid inlet channel, nebulizer gas inlet, and nozzle are etched on the silicon plate and a platinum heater is integrated on the glass plate. The nebulizer microchip has been previously used in atmospheric pressure chemical ionization, atmospheric pressure photoionization, sonic spray ionization, and thermospray ionization modes. In this work we show that the microchip can be operated also in ionspray mode by introducing high voltage to the silicon plate of the microchip. The effects of operation parameters (voltage, nebulizer gas pressure, sample solution flow rate, solvent composition, and analyte concentration) on the performance of the ion spray microchip were studied. Under optimized conditions the microchip provides efficient ionization of small and large compounds and good quantitative performance. The feasibility of the ion spray microchip in liquid chromatography/mass spectrometry (LC/MS) was demonstrated by the analysis of tryptic peptides of bovine serum albumin. Copyright © 2010 John Wiley & Sons, Ltd.     

Capillary liquid chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.     

High resolution orbitrap mass spectrometry in comparison with tandem mass spectrometry for confirmation of anabolic steroids in meat

A prominent trend which has been observed in recent years in the analysis of veterinary drugs and growth-promoting agents is the shift from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform (FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS for confirmatory analysis of growth-promoting agents in meat was compared to that of a QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most compounds >0.99) but somewhat inferior sensitivity. Indeed, CC_αs reached from 0.04–0.88 μg kg⁻¹ for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS detection a range of 0.07–2.50 μg kg⁻¹ was observed. Using QqQ-MS adequate precision was obtained since relative standard deviations, associated with the repeatability and intra-laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds (i.e. some estrogens) this threshold was exceeded and thus poor precision was observed, which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity for some of the compounds.     

Optimization of the Extraction and Analysis of Natural Androgen Steroids and Their Metabolites in Urine by GC/MS and GC/FID

A multiresidue method was developed for screening, quantification, and confirmation of nine natural androgen steroids and their metabolites in urine. Steroids were first extracted from urine by solid phase extraction, enzymatically deglucuronated, re-extracted using a liquid/liquid extraction for purification, and finally acetylated for GC/MS and GC/FID analysis. Each step of sample preparation, as well as analysis, was optimized: solid phase extraction, liquid/ liquid extraction, and derivatization reaction … Therefore, a rugged sample preparation procedure was developed leading to extracts of sufficient purity (recoveries >66% and few matrix compounds). The whole methodology allowed reliable detection and quantification of the nine steroids at low concentration levels. Linearity and repeatability were established and were found to be satisfactory (R² > 0.996, RSD < 11%). Finally, the method was applied to quantify compounds of interest in real samples collected from healthy volunteers and patients treated with 4-androstenedione or dehydroepiandrosterone.     

Application of fast gas chromatography/mass spectrometry for the rapid screening of synthetic anabolic steroids and other drugs in anti-doping analysis

This paper describes a fast gas chromatographic/mass spectrometric (GC/MS) screening method for the detection, in urine, of 36 xenobiotics (30 synthetic anabolic steroids, four narcotics, one diuretic and one stimulant) excreted free or as glucuro-conjugates in urine and detectable as trimethylsilyl (TMS) derivatives. These drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase and then assayed as TMS derivatives by GC/MS using electron ionisation (EI) and single ion monitoring (SIM) acquisition mode. The total time needed for the GC run is less than 8 min. Good reproducibility of the retention times (CV% <1) and the relative abundances of the diagnostic fragment ions (CV% <10) was observed for all target analytes. The sensitivity of the method is sufficient to match the requirements of the World Anti-Doping Agency (WADA) for the accredited laboratories, with limits of detection (LODs) that are lower than the corresponding WADA minimum required performance limits (MRPLs) for all target compounds.     

Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

Analysis of anabolic steroids in hair by GC/MS/MS

A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.     

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.     

Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.     

气质联用法同时测定猪尿中的20种同化激素

采用安捷仑5975GC/MS对猪尿中20种同化激素进行同时检测.对于10.0 g尿液样品,采用β-葡糖苷酸酶(来自Helix pomatia)对提取物水解后,加入过饱和NaCl,经乙腈提取,用过C18小柱和NH2小柱净化后七氟丁酸酐对其进行衍生,最后利用GC/MS进行检测.采用外标法定量,以S/N≥3确定的,检出限均能达到0.25 μg/kg.通过对3个添加水平的回收实验表明,平均回收率为77.0%～111.0%.     

Comparison of atmospheric pressure photoionization and ESI for CZE-MS of drugs

The performance of atmospheric pressure photoionization (APPI) and ESI for CZE was compared using a set of seven drugs (basic amines, quaternary amines and steroids) and four different BGEs. The influence of volatile and nonvolatile BGEs of acidic and neutral pH on the MS responses of test compounds was evaluated by infusion of test solutions into the respective ion sources, and by actual CZE-MS experiments. The infusion experiments indicate that sodium phosphate buffers cause ionization suppression in ESI-MS, although for the amines the suppression was modest (25-60% signal reduction). By contrast, APPI-MS responses were not affected by nonvolatile BGEs. With phosphate buffers, ESI-MS responses for the basic amines were still a factor 3-13 higher than the APPI-MS signals, whereas the steroids yielded similar responses in ESI-MS and APPI-MS. The quaternary amines could readily be detected in ESI-MS, but detection in APPI-MS required specific interface conditions. Using typical CZE-APPI-MS settings, quaternary amines remained undetected. Remarkably, the S/Ns observed in CZE-ESI-MS for the test compounds, were generally similar when using volatile and nonvolatile BGEs. For basic compounds, the S/Ns obtained in CZE-ESI-MS were a factor 2-5 higher than in CZE-APPI-MS, whereas steroids yielded equal S/Ns in both methods. Overall, it is concluded that when using relatively low BGE concentrations, the sensitivity of ESI-MS detection in CZE is more favorable than APPI-MS detection, even when nonvolatile BGEs are employed.     

Multivariate optimization of a derivatisation procedure for the simultaneous determination of nine anabolic steroids by gas chromatography coupled with mass spectrometry

The medical commission of the International Olympic Committee forbids the use of anabolic androgenic steroids to improve sporting performances. Nine anabolic steroids (androsterone (A), nandrolone, estradiol, testosterone propionate, nandrolone-17 propionate, dydrogesterone, testosterone, epitestosterone, boldenone) and alpha-cholestane as internal standard were studied by gas chromatography coupled with mass spectrometry (GC/MS). The derivatisation reagent employed for the derivatisation of anabolic steroids was a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and 2-mercaptoethanol (1000:2:6, v/w/v). Trimethylsilyl (TMS) derivatives were obtained. Anabolic steroids can be derivatised into one or two forms, mainly for androsterone into A-monoTMS and A-diTMS. The aim of this study was to research the optimization conditions of the derivatisation process (maximum yield of silylation reaction) of each anabolic steroid into only one form. A two-level factorial Doelhert design was used to determine the influence of different parameters and their interactions on each compound, thanks to response surface methodology. The parameters to be optimized were the reaction time and the temperature. The interaction "temperature-reaction time" is significant and has a positive effect on the improvement of the effectiveness of the derivatisation. Considering the large amount of information, often not convergent, a global desirability function was applied for multi-responses optimization. Thus, the optimized temperature and the reaction time of silylation were 85 degrees C and 24 min, respectively. Several GC/MS analytical parameters were also studied: linearity (regression coefficient upper than 0.99 for each compound, sensibility (range of concentration 0.05-0.30 microg/ml). Confirmatory experiments were applied to check the predicted values and to validate the model. The confirmatory assay responses are relatively close to the responses predicted. We observed satisfactory resolutions by GC/MS and a run lower than 12 min.