Markers of mesterolone abuse in sulfate fraction for doping control in human urine.

This manuscript describes the direct detection of mesteroloe sulfo-conjugated metabolites by liquid chromatography/quadrupole/time of flight mass spectrometry (LC/Q/TOFMS) with special focus on evaluation of their retrospective detectability and their structure elucidation. A comparison of their long-term detectability, with the mesterolone main metabolite (1α-methyl-5α-androstan-3α-ol-17-one) excreted in glucuronide fraction and detected by gas chromatography/high resolution mass spectrometry (GC/HRMS), is also presented. Studies on mesterolone were performed with samples obtained from two excretion studies after single oral administration of Proviron© by healthy volunteers. Potential sulfate metabolites were detected in post administration samples after liquid-liquid extraction (LLE) with ethyl acetate and LC/TOFMS analysis, in negative mode. Twelve mesterolone sulfate metabolites from the first excretion study and nine from the second were subsequently confirmed by LC/Q/TOFMS. Finally, six mesterolone sulfate metabolites were considered important taking into account their abundance and long-term detectability, encoded as M1, M2, M4, M5, M6 and M7. The proposed mesterolone sulfate metabolites M1, M2, M4 and M5 (excreted as sulfates) have the same retrospectivity with the main mesterolone metabolite, excreted in glucuronide fraction. For metabolite characterization, LC fractionation was performed. The metabolites were identified and characterized by GC/MS, after solvolysis and derivatization. Combined mass spectra data from trimethyl-silyl (TMS), TMS-enolTMS and methoxime-TMS derivatives were taken into account for the characterization of these metabolites. It was concluded that M1 is 1α-methyl-5α-androstan-3ß-ol-17 one, M2 is 1α-methyl-5α-androstan-3α-ol-17 one, M4 is 1α-methyl-5a-androstan-3ß, 16z-diol-17-one, M5 is 1α-methyl-5α-androstan-17z,4ξ-diol-3one, M6 is 1α-methyl-5α-androstan-3z,6z-diol-17-one and M7 is 4z-hydroxy-1α-methyl-5α-androstan-3,17-dione.

Comparison of sulfo-conjugated and gluco-conjugated urinary metabolites for detection of methenolone misuse in doping control by LC-HRMS, GC-MS and GC-HRMS.

Methenolone (17ß-hydroxy-1-methyl-5α-androst-1-en-3-one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1-methylene-5α-androstan-3α-ol-17-one) excreted conjugated with glucuronic acid using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for the parent molecule, after hydrolysis with ß-glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC-high resolution (HR)MS and the estimation of the long-term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC-HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti-doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC-HRMS using electrospray ionization in negative mode searching for [M-H](-) ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1-methylene-5α-androstan-3α-ol-17-one, 3z-hydroxy-1ß-methyl-5α-androstan-17-one and 16ß-hydroxy-1-methyl-5α-androst-1-ene-3,17-dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z-hydroxy-1ß-methyl-5α-androstan-17-one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC-HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC-MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction.

Two-step derivatization procedures for the ionization enhancement of anabolic steroids in LC-ESI-MS for doping control analysis.

BACKGROUND: Two-step derivatization procedures were developed for the enhancement of the positive ESI in LC-MS detection of anabolic androgenic steroids, a class of prohibited substances with limited ionization efficiency in atmospheric pressure interfaces. The developed procedures are based on the esterification of hydroxyl groups of anabolic steroids with picolinic acid, followed by conversion of carbonyl groups to Schiff bases by either Girard's reagent T or 2-hydrazino pyridin. RESULTS: Ionization efficiency for the model derivatized compounds 19-norandrosterone (nandrolone main metabolite) and methasterone was higher by almost two orders of magnitude compared with the respective efficiency of the underivatized compounds. CONCLUSION: The obtained derivatives provided a significant improvement in the ESI sensitivity, compared with those of underivatized molecules in positive LC-ESI-ion trap-MS full-scan mode.

Preventive doping control screening analysis of prohibited substances in human urine using rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometry.

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, beta(2)-agonists, beta-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.

Direct injection horse urine analysis for the quantification and identification of threshold substances for doping control. III. Determination of salicylic acid by liquid chromatography/quadrupole time-of-flight mass spectrometry.

In equine sport, salicylic acid is prohibited with a threshold level of 750 microg mL(-1) in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization in negative mode with full scan acquisition mode and product ion scan mode were chosen for the quantification and identification of salicylic acid, respectively. Run time was 2.0 min. The tested linear range was 2.5-50 microg mL(-1) (after 100-fold sample dilution). The relative standard deviations of intra- and inter-assay analysis of salicylic acid in horse urine were lower than 2.5% and 2.8%, respectively. Overall accuracy (relative percentage error) was less than 3.3%. Method was applied to two real samples found to be positive for salicylic acid, demonstrating simplicity, accuracy, and selectivity.

Direct injection liquid chromatography/electrospray ionization mass spectrometric horse urine analysis for the quantification and confirmation of threshold substances for doping control. II. Determination of theobromine.

In equine sport, theobromine is prohibited with a threshold level of 2 microg mL(-1) in urine, hence doping control laboratories have to establish quantitative and qualitative methods for its determination. Two simple liquid chromatography/mass spectrometry (LC/MS) methods for the identification and quantification of theobromine were developed and validated using the same sample preparation procedure but different mass spectrometric systems: ion trap mass spectrometry (ITMS) and time-of-flight mass spectrometry (TOFMS). Particle-free diluted urine samples were directly injected into the LC/MS systems, avoiding the time-consuming extraction step. 3-Propylxanthine was used as the internal standard. The tested linear range was 0.75-15 microg mL(-1). Matrix effects were evaluated analyzing calibration curves in water and different fortified horse urine samples. A great variation in the signal of theobromine and the internal standard was observed in different matrices. To overcome matrix effects, a standard additions calibration method was applied. The relative standard deviations of intra- and inter-day analysis were lower than 8.6 and 7.2%, respectively, for the LC/ITMS method and lower than 5.7 and 5.8%, respectively, for the LC/TOFMS method. The bias was less than 8.7% for both methods. The methods were applied to two case samples, demonstrating simplicity, accuracy and selectivity.

Two-step silylation procedure for the unified analysis of 190 doping control substances in human urine samples by GC-MS.

BACKGROUND: While a number of different derivatization procedures for screening GC-MS analysis of prohibited substances are followed by doping control laboratories, a unified derivatization procedure for the GC-MS analysis of 190 different doping agents was developed. RESULTS: Following preliminary experiments, a two-step derivatization procedure was selected. The evaluation of various silylation parameters, such as reagent composition, reaction time, reaction temperature, catalysts and microwave oven reaction time, for this procedure was carried out. CONCLUSION: The suitability of the developed procedure was demonstrated through application on urine samples at concentration levels of the minimum required performance limit for all tested substances. This new derivatization procedure, which significantly decreases time and cost, is suitable for a routine basis application.

Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3-methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time-of-flight mass spectrometry.

Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 microg mL(-1) (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chromatography (HILIC) was selected as a separation technique that allows effective retention of polar substances like 3-methoxytyramine and efficient separation from matrix compounds. Electrospray ionization (ESI) in positive mode with product ion scan mode was chosen for the detection of the analytes. Quantification of 3-methoxytyramine was performed with fragmentation at low collision energy, resulting in one product ion, while a second run at high collision energy was performed for confirmation (at least three abundant ions). Studies on matrix effects showed ion suppression depending on the horse urine used. To overcome the variability of the results originating from the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. The tested linear range was 1-20 microg mL(-1). The relative standard deviations of intra- and inter- assay analysis of 3-methoxytyramine in horse urine were lower than 4.2% and 3.2%, respectively. Overall accuracy (relative percentage error) was less than 6.2%. The method was applied to case samples, demonstrating simplicity, accuracy and selectivity.

Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone.

Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4%, respectively, for the LC/IT-MS method and lower than 8.4 and 4.4%, respectively, for the LC/TOF-MS method. Accuracy (bias percentage) was less than 9.7% for both methods.

Determination of xylazine and its metabolites by GC-MS in equine urine for doping analysis.

Xylazine and its main metabolites were detected in equine urine after a single-dose intravenous administration of 0.98 and 1.01 mg/kg body weight xylazine, respectively, in two horses, in order to be used for equine doping control routine analysis. The urine levels of the parent drug and its metabolites were determined using gas chromatography-mass spectrometry (GC-MS). Xylazine is metabolised rapidly, down to a concentration level of about 1.0 microg/ml after 1-3h administration. Seven metabolites were identified in urine. 4-Hydroxy-xylazine, the major metabolite, could be traced for 25 h and it is regarded as the long-term metabolite of xylazine in horse. 2,6-Dimethylaniline was, for the first time, reported as metabolite in equine.