Tiia Kuuranne

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography–electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.

Annual banned-substance review: analytical approaches in human sports drug testing

The timely update of the list of prohibited substances and methods of doping (as issued by the World Anti-Doping Agency) is an essential aspect of international anti-doping efforts and represents consensual agreement by expert panels regarding substances and the methods of performance manipulation in sports. The annual banned-substance review for human doping controls critically summarizes recent innovations in analytical approaches; its purpose is to improve the quality of doping controls by reporting emerging and advancing methods that focus on detecting known and recently outlawed substances. This review surveys new and/or enhanced procedures and techniques of doping analysis together with information relevant to doping control that has been published in the literature between October 2009 and September 2010.

Rapid analysis of metabolites and drugs of abuse from urine samples by desorption electrospray ionization-mass spectrometry

Urine samples obtained from drug abusers were screened for drugs of abuse and their metabolites using DESI-MS and the results obtained were compared to results obtained from GC-MS experiments. The detected analyte classes included amphetamines, opiates, cannabinoids and benzodiazepines. The compounds detected were codeine, morphine, oxymorphone, 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol, Delta(9)-tetrahydrocannabinol, alprazolam, temazepam, oxazepam, N-desmethyldiazepam (nordiazepam) and hydroxytemazepam. Identities of all the analytes were confirmed by tandem mass spectrometry, matching MS/MS spectra with authentic standard compounds. The concentrations of the analytes in the samples were obtained from semi-quantitative GC-MS studies and were in the range of 270-22,000 ng mL(-1). The analytes could be detected by DESI even after a hundred-fold dilution indicating that the sensitivity of DESI was more than adequate for this study. Selectivity in the DESI-MS measurements for different kinds of analytes could be increased further by optimizing the spray solvent composition: the use of an entirely aqueous solvent enhanced the signal of polar analytes, such as the benzodiazepines, whereas the use of a spray solvent with a high organic content increased the signal of less polar analytes, such as codeine and morphine.

Electrospray and Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometric Behavior of Eight Anabolic Steroid Glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5α-estran-3α-ol-17-one (5α-NG), 5β-estran-3α-ol-17-one (5β-NG), 17α-methyl-5α-androstane-3α,17β-diol (5α-MTG), and 17α-methyl-5β-androstane-3α,17β-diol (5β-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M − H]−. Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H − Glu]+, [M + H − Glu − H2O]+, and [M + H − Glu − 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5α-NG and 5β-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.

Aryl-Propionamide-Derived Selective Androgen Receptor Modulators: Liquid Chromatography-Tandem Mass Spectrometry Characterization of the in Vitro Synthesized Metabolites for Doping Control Purposes

Selective androgen receptor modulators (SARM) are a prominent group of compounds for being misused in sports owing to their advantageous anabolic properties and reduced side effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM, an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and the structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, monohydroxylation, and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization and detected as deprotonated species [M-H]-. The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control.

Generic sample preparation and dual polarity liquid chromatography-time-of-flight mass spectrometry for high-throughput screening in doping analysis.

The requirements on initial testing in doping control are getting tighter regarding efficiency and speed while the scope of analytes is getting more diverse and, consequently, the need for high-throughput methods is apparent. In this study, a comprehensive screening method for doping agents in human urine is presented, based on solid phase extraction (SPE) and liquid chromatography-time-of-flight mass spectrometry (LCTOFMS). The method covers most of the compound groups in the list of prohibited substances by World Anti-Doping Agency (WADA). Mixed-mode SPE on two types of sorbent and the use of negative ionization mode besides the commonly used positive mode in electrospray ionization (ESI) allowed detection of acidic compounds, such as sulpho-conjugated metabolites. A run time of 8 minutes for each of the two ESI polarities was achieved. The method was validated regarding relative ionization efficiency, selectivity and signal to noise at the WADAs minimum required performance limit (MRPL) level, resulting in the acceptance of 197 compounds. A selection of 20 compounds was submitted for a more thorough validation, including extraction recovery, repeatability and linearity. Recovery and linearity (R(2)) varied mainly between 83-115% and 0.78-0.99, respectively. Median values for repeatability at the MRPL and 10 x MRPL levels were below 20%. A mean and median mass accuracy of 1.2 and 0.80 mDa, respectively, was achieved. The present method represents at the moment the widest coverage of low molecular weight prohibited substances for the screening in sports, providing an approach for further rationalisation of the analytical work-flow in the doping control laboratories.

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.

Enzyme-assisted synthesis and structure characterization of glucuronide conjugates of eleven anabolic steroid metabolites

Enzyme-assisted in vitro synthesis of eleven glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites was performed using biphenyl-induced rat liver microsomal enzymes. The substrates within the study were the main compounds and metabolites detected in human urine after dosing of, e.g. metandienone, metenolone, methyltestosterone, nandrolone, and testosterone. Yields of glucuronidation reactions were 13-28% for most compounds, but significantly higher (77-78%) for the substrates with 4-ene-3-one double bond system of the steroid A-ring. Characterization of glucuronide-conjugated AAS structures was based on nuclear magnetic resonance spectroscopy ((1)H NMR) and on liquid chromatographic-mass spectrometric (LC-MS) and tandem mass spectrometric (LC-MS/MS) analyses in positive and negative ion mode electrospray ionization (ESI). Only minor differences were observed in optimal synthesis conditions between various substrates, which offer a potential to apply this in vitro assay as a default method for glucuronidation of new AAS substrates. The method allowed for a rapid production pathway of stereochemically pure AAS glucuronides in milligram amount, such as needed, e.g. in the development of analytical methods in forensic or pharmaceutical sciences, as well as in doping control.

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry.

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS-MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.

Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine.

A hydrophilic interaction liquid chromatography-time-of-flight mass spectrometry (HILIC-TOFMS) method for the quantification and confirmation of morphine (M), codeine (C), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine-6-glucuronide (C6G) is presented. The method was validated in terms of specificity, selectivity, extraction recovery, accuracy, repeatability, linearity and matrix effect. After a straightforward sample preparation by solid phase extraction (SPE) the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The HILIC technique provided good chromatographic separation which was critical for isomers M3G and M6G. The analytes were detected after electrospray ionization (ESI) in positive mode with mass accuracies below 2 mDa using a 5-mDa window. A measurement range of 50-5000 ng/ml was applied for calibration using deuterated analogs as internal standards. The precision of the method was 5.7% and 10.2% (RSD) within and between days, respectively. The applicability of the method was demonstrated with authentic urine samples known to contain codeine and/or morphine and their intact glucuronide conjugates. Identification of the analytes was based on in-source collision induced dissociation (ISCID), applying three diagnostic ions with accurate mass.