Gas chromatographic quadrupole time-of-flight full scan high resolution mass spectrometric screening of human urine in antidoping analysis

Abstract

This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World Antidoping Agency (WADA) enlists AAS as prohibited doping agents in sports, and our method has been developed to comply with the qualitative specifications of WADA to be applied for the detection of sports antidoping prohibited substances, mainly for AAS. The method also comprises of the quantitative analysis of the WADA's Athlete Biological Passport (ABP) endogenous steroidal parameters. The applied preparation of urine samples includes enzymatic hydrolysis for the cleavage of the Phase II glucuronide conjugates, generic liquid-liquid extraction and trimethylsilyl (TMS) derivatization steps. Tandem mass spectrometry (MS/MS) acquisition was applied on few selected ions to enhance the specificity and sensitivity of GC/TOF signal of few compounds. The full scan high resolution acquisition of analytical signal, for known and unknown TMS derivatives of AAS provides the antidoping system with a new analytical tool for the detection designer drugs and novel metabolites, which prolongs the AAS detection, after electronic data files' reprocessing. The current method is complementary to the respective liquid chromatography coupled to mass spectrometry (LC/MS) methodology widely used to detect prohibited molecules in sport, which cannot be efficiently ionized with atmospheric pressure ionization interface.

Comparison of gas chromatography/quadrupole time‐of‐flight and quadrupole Orbitrap mass spectrometry in anti‐doping analysis: I. Detection of anabolic‐androgenic steroids

“…The same sample set was analyzed on both analytical platforms within a 2‐week period in order to reduce possible variability due to sample instability. The methods applied herein were based on that of a previous study, with the exception of the GC/Q‐Orbitrap MS method, which is described in section 2. The entire sample preparation procedure was conducted at ADLQ, where the aliquots were subsequently analyzed using the Q‐TOF instrument.…”

“…The sample preparation has been described in detail for a previous study . In brief, it included a liquid–liquid extraction with diethyl ether at pH 9–10 and a desalting step, which is an approach that is commonly used to extract doping substances from the urine matrix.…”

“…In another, more comprehensive study from the same research group, a screening method based on quadrupole‐Orbitrap GC/MS was published for use in the detection of a large number of substances in urine, such as synthetic AAS, β‐agonists, stimulants, narcotics, metabolic modulators and diuretics, that fulfilled the WADA sensitivity specifications . Finally, in another recent study, a routine screening method that used GC/MS with a triple quadrupole instrument was adapted for use with a Q‐TOF GC/MS instrument and subjected to full validation, in which the FS/HR MS acquisition mode was applied to detect and quantify exogenous and endogenous steroids…”

Ultra‐fast retroactive processing of liquid chromatography high‐resolution full‐scan Orbitrap mass spectrometry data in anti‐doping screening of human urine

“…The use of HR‐FS‐MS with LC and GC in anti‐doping has been reported and applied recently . However reprocessing of thousands of data files is also a logistically difficult task, because the size of high‐resolution MS data files per sample is in the range 100–500 MB.…”

“…The use of HR-FS-MS with LC and GC in anti-doping has been reported and applied recently. 2,3,[15][16][17][18][19] However reprocessing of thousands of data files is also a logistically difficult task, because the size of high-resolution MS data files per sample is in the range 100-500 MB. This task requires a strong information technology (IT) infrastructure, which is not usually available to the WADA Accredited Laboratories.…”