Application of liquid chromatography / time of flight mass spectrometry for the doping control of prohibited substances in athlete's and horse urine

In the current study, new qualitative and quantitative methods were developed andvalidated for the doping control screening analysis, confirmation, and/or quantitativedetermination of prohibited substances using LC/TOF-MS technology. The first part of thiswork presents the use of LC/TOF-MS for the multiple detection of a great number ofprohibited substances in athletes urine. In the second part, the same technology was appliedfor the quantification and identification of 4 threshold substances in horse urine using directinjection analysis.Unification of screening protocols for a wide range of doping agents has become animportant issue for doping control laboratories. In the present study, a high throughputscreening method was developed for the multiple detection of 266 small molecule analytesfrom all categories of prohibited substances included in the WADA List. The proposedmethodology is based on a single-step liquid-liquid extraction of hydrolyzed urine and theuse of a rapid-resolution LC/TOF-MS system acquiring continuous full scan spectral data.Electrospray ionization in the positive mode was applied. The extraction protocol andsample preparation procedure used in this study was applied in the OACA doping controllaboratory for the routine screening analysis of anabolic agents and corticosteroids. Ourapproach on sample preparation was to study the efficiency of this procedure for otherclasses of substances (stimulants, narcotics, diuretics). Validation parameters consisted ofidentification capability, limit of detection, specificity, ion suppression, extraction recovery,repeatability and mass accuracy. Detection criteria were established on the basis ofretention time reproducibility and mass accuracy. The suitability of the methodology fordoping control was demonstrated with positive urine samples. The preventive role of themethod was proved by the case where full scan acquisition with accurate massmeasurement allowed the retrospective reprocessing of acquired data from past dopingcontrol 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 particularsubstance, when, initially, they had been reported as negatives.Hydrocortisone is the primary endogenous glucocorticosteroid and is used in equineveterinary medicine for its anti-inflammatory properties. As an endogenous substance, itscontrol in equine sports is regulated by a threshold value of 1 μg ml-1 free hydrocortisone inurine. Two simple and rapid LC/MS methods with direct injection analysis were developedand validated for the quantification and identification of hydrocortisone in equine urineusing the same sample preparation but different mass spectrometric systems: IT-MS andTOF-MS. The main advantage of the proposed methodology is the minimal samplepreparation procedure, as particle-free diluted urine samples were directly injected intoboth LC/MS systems. Desonide was used as internal standard (IS). The tested linear rangewas 0,25–2,5 μg ml−1 for both methods. Matrix effects were evaluated by preparing andanalyzing calibration curves in water solutions and different horse urine samples. A greatvariation of the signal both for hydrocortisone and the internal standard was observed indifferent matrices. To overcome matrix effects, the unavailability of blank matrix and theexcessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standardadditions approach in a method where neither non isotopic internal standards nor extensivesample preparation is utilized and no blank matrix is available. Validation results on linearity,accuracy and precision for both methods prove their suitability for the doping controlanalysis of hydrocortisone in horse urine. The above method was applied to a real sample,using both LC/MS methods.In equine sport, theobromine is prohibited with a threshold level of 2 μg mL-1 in urine. Twosimple LC/MS methods for the identification andquantification of theobromine weredeveloped and validated using the same sample preparation procedure but different massspectrometric systems (IT-MS and TOF-MS. Particle-free diluted urine samples were directlyinjected 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 μgmL-1. Matrix effects were evaluated analyzing calibration curves in water and differentfortified horse urine samples. A great variation in the signal of theobromine and the internalstandard was observed in different matrices. To overcome matrix effects, a standardadditions calibration method was applied. The linearity, accuracy and precision for bothmethods prove their suitability for the doping control analysis of theobromine in horseurine. The methods were applied to two case samples, demonstrating simplicity, accuracyand selectivity.Salicylates have analgesic, anti-inflammatory, and antipyretic properties. In horses,salicylates may derive both from feeding as well as from medication. In equine sport,salicylic acid is prohibited with a threshold level of 750 μg mL−1 in urine. A simple and rapidLC/MS method was developed and validated for the quantification and identification ofsalicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the LC/QTOF-MS system. Electrosprayionization in negative mode with full scan acquisition mode and product ion scan mode werechosen for the quantification and identification of salicylic acid, respectively. Run time was2.0 min. The tested linear range was 2.5–50 μg mL−1 (after 100-fold sample dilution). Thelinearity, accuracy, and precision results prove the method’s suitability for the dopingcontrol analysis of salicylic acid in horse urine. The area ratios of the diagnostic product ionsof salicylic acid were found to be precise for confirmation purposes. The above method wasapplied to the quantitative and confirmatory analysis of two real samples.The endogenous catecholamine dopamine and its pharmacologically inert precursor, L-dopa,are both available as human pharmaceutical preparations and can be used as performanceenhancing drugs in horses. Urinary 3-methoxytyramine is used as an indicator ofdopaminergic manipulation resulting from dopamine or levodopa administration and isprohibited with a urinary threshold of 4 μg mL−1 (free and conjugated). A simple LC/MSmethod was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis andprotein precipitation. ΗILIC was selected as a separation technique that allows effectiveretention of polar substances like 3-methoxytyramine and efficient separation from matrixcompounds. Electrospray ionization (ESI) in positive mode with product ion scan mode waschosen for the detection of the analytes. Studies on matrix effects showed ion suppressiondepending on the horse urine used. To overcome the variability of the results originatingfrom the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. Thelinearity, accuracy and precision results prove the method’s suitability for the doping controlanalysis of 3-methoxytyramine in horse urine. The above method was applied to thequantitative and confirmatory analysis of three case samples