C. Georgakopoulos

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.

Generic sample preparation combined with high-resolution liquid chromatography–time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories

A unification of doping-control screening procedures of prohibited small molecule substances—including stimulants, narcotics, steroids, β2-agonists and diuretics—is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography–time-of-flight mass spectrometry method and one liquid chromatography–time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, β2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples.

Determination of ephedrines in urine by gas chromatography-mass spectrometry.

A selective gas-liquid chromatographic method with mass spectrometry (GC-MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines--one derivative was formed for each ephedrine--were prepared and analyzed by GC-MS, after alkaline extraction of urine and evaporation of the organic phase, using d3-ephedrine as internal standard. Calibration curves, with r2>0.98, ranged from 3.0 to 50 microg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.

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.

Doping control analysis: the 6th World Championships of Athletics, Athens, Greece

Abstract The first official report on the organization, analytical methodologies, and the results of the doping control analysis performed by the International Olympic Committee (IOC)-accredited Doping Control Laboratory of Athens, Greece, during the 6th World Championships of Athletics, held in Athens on 1–10 August 1997, is presented. The significance of the various analytical parameters of the doping control is shown. The results include 20 positive cases, of which two were detected in screening procedures and confirmed exclusively by high resolution mass spectrometry. The screening results and analytical data on the excretion of a new black-market doping agent, phenylpiracetam or carfedon, which was unknown in the literature, are also presented.

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.

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 750u2009µgxa0mL−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 2.0xa0min. The tested linear range was 2.5–50u2009µgxa0mL−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.

Excretion study of the β2-agonist reproterol in human urine

Abstract An excretion study of the β2-agonist 7-[3-[(β-3,5-trihydroxyphenethyl)amino]-propyl]theophylline (reproterol) in human urine, which is reportedly misused by athletes and horses as a doping agent, is presented. The study was performed after an oral administration of 20 mg of reproterol hydrochloride. The collected urine samples were prepared using the standard anabolic steroid extraction procedure and analyzed by gas chromatography coupled with quadrupole mass spectrometry and, also, with high-resolution mass spectrometry (HRMS). The main reproterol metabolite was found, whereas unchanged reproterol was not detected. The structure of the main metabolite was confirmed by an accurate HRMS measurement of diagnostic ions. Finally, an excretion urine profile of the main metabolite is presented. The mass spectrum of another possible unidentified reproterol metabolite is also reported.