High‐throughput screening for various classes of doping agents using a new ‘dilute‐and‐shoot’ liquid chromatography‐tandem mass spectrometry multi‐target approach

Abstract

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.

</title></titles> <contributors> <person_name sequence='first' contributor_role='author'> <given_name>Mike</given_name> <surname>Earl</surname> </person_name> <person_name sequence='additional' contributor_role='author'> <given_name>Marc</given_name> <surname>Vouillamoz</surname> </person_name> <person_name sequence='additional' contributor_role='author'> <given_name>Dorota</given_name> <surname&g

“…Since the method is for screening purposes, one of these ions can be monitored in the first instance to reveal suspicious samples, similarly as described in other high-throughput screening analyses [10]. In case a potential positive sample is detected, retrospective “post-targeted” data evaluation using multiple diagnostic ions is suggested before the necessary confirmatory analysis by partially methylated alditol acetates (PMAA) [24] or enzymatically derived isomaltose [8].…”

“…Aside from colorimetric microtitre plate methods [2, 7, 18], used for screening due to the low cost and limited sample work up, detection of HES and dextran in doping analysis can be accomplished by mass spectrometric techniques including gas chromatography–mass spectrometry (GC-MS) [15, 16, 21, 23], liquid chromatography–mass spectrometry (LC-MS) [4, 9, 10, 13] and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) [17]. Due to the polar nature and high molecular weight of HES and dextran, analysis by GC-MS requires an acidic hydrolysis step and a time-consuming derivatization step [15].…”

Human sports drug testing by mass spectrometry

“…The majority of preventive doping research projects conducted in Germany and dedicated to other anabolic agents has been focused on the comprehensive class of SARMs (Table ) (Thevis & Schänzer, ), including the elucidation of dissociation pathways of active (intact) drugs (Thevis et al, , , ,c,d), and the characterization of in vitro (human liver microsomes, fungal incubations) (Thevis et al, ; Rydevik et al, ), in vivo (Thevis et al, , , ), and electrochemically generated (Thevis et al, ) metabolites, followed by the development of specific and sensitive detection assays. Despite the considerably diverse structural features of SARMs, modern LC‐MS/MS‐based test methods are expected to allow for their comprehensive coverage as exemplified with selected approaches (Guddat et al, ; Thomas et al, , , ). All of these assays, nevertheless, require information on commonalities of the target analytes such as specifics on conserved core structures or, preferably, on distinct metabolites.…”

Analysis of Glycerol with Isolation of Endogenous Interferences using "Dilute and Shoot" Strategy and High-Resolution Mass Spectrometry in Human Urine for Antidoping Testing

“…8 This strategy was successfully demonstrated for selected compounds in diuretics, stimulants, narcotics, plasma volume expander, and beta-2-agonists. [9][10][11][12] Although "dilute and shoot" method can provide various advantages, some problems persist concerning an ionization suppression 13 or a spectral congestion.…”