A new multi-target approach based on liquid chromatography--electrospray ionization tandem mass spectrometry (LC-(ESI)-MS/MS) is presented to screen for various classes of prohibited substances using direct injection of urine specimens. With a highly sensitive new generation hybrid mass spectrometer classic groups of drugs--for example, diuretics, beta2-agonists--stimulants and narcotics are detectable at concentration levels far below the required limits. Additionally, more challenging and various new target compounds could be implemented. Model compounds of stimulant conjugates were studied to investigate a possible screening without complex sample preparation. As a main achievement, the integration of the plasma volume expanders dextran and hydroxyethyl starch (HES), commonly analyzed in time-consuming, stand-alone procedures, is accomplished. To screen for relatively new prohibited compounds, a common metabolite of the selective androgen receptor modulator (SARMs) andarine, a metabolite of growth hormone releasing peptide (GHRP-2), and 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) are analyzed. Following a completely new approach, conjugates of di(2-ethylhexyl) phthalate (DEHP) metabolites are monitored to detect abnormally high levels of plasticizers indicating for illicit blood transfusion. The assay was fully validated for qualitative purposes considering the parameters specificity, intra- (3.2-16.6%) and inter-day precision (0.4-19.9%) at low, medium and high concentration, robustness, limit of detection (1-70 ng/ml, dextran: 30 µg/ml, HES: 10 µg/ml) and ion suppression/enhancement effects. The analyses of post-administration and routine doping control samples demonstrates the applicability of the method for sports drug testing. This straightforward and reliable approach accomplishes the combination of different screening procedures resulting in a high-throughput method that increases the efficiency of the labs daily work.

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In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT < 2.0%); linearity (R > 0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 μg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects.

Since January 2014, the anti-anginal drug trimetazidine [1-(2,3,4-trimethoxybenzyl)-piperazine] has been classified as prohibited substance by the World Anti-Doping Agency (WADA), necessitating specific and robust detection methods in sports drug testing laboratories. In the present study, the implementation of the intact therapeutic agent into two different initial testing procedures based on gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) is reported, along with the characterization of urinary metabolites by electrospray ionization-high resolution/high accuracy (tandem) mass spectrometry. For GC-MS analyses, urine samples were subjected to liquid-liquid extraction sample preparation, while LC-MS/MS analyses were conducted by established 'dilute-and-inject' approaches. Both screening methods were validated for trimetazidine concerning specificity, limits of detection (0.5-50 ng/mL), intra-day and inter-day imprecision (<20%), and recovery (41%) in case of the GC-MS-based method. In addition, major metabolites such as the desmethylated trimetazidine and the corresponding sulfoconjugate, oxo-trimetazidine, and trimetazidine-N-oxide as identified in doping control samples were used to complement the LC-MS/MS-based assay, although intact trimetazidine was found at highest abundance of the relevant trimetazidine-related analytes in all tested sports drug testing samples. Retrospective data mining regarding doping control analyses conducted between 1999 and 2013 at the Cologne Doping Control Laboratory concerning trimetazidine revealed a considerable prevalence of the drug particularly in endurance and strength sports accounting for up to 39 findings per year.