Orbitrap defeats the athletes who cheat

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  • Published: Aug 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Orbitrap defeats the athletes who cheat

Doping compounds need flexible detection methods

Detecting the large number of doping compounds used in sport represents a significant challenge to the regulatory authorities, especially since new substances are being introduced all the time

Detecting the large number of doping compounds used in sport represents a significant challenge to the regulatory authorities, especially since new substances are being introduced all the time. LC-MS is often used for the detection of banned substances, but some of these compounds, such as anabolic steroids, suffer from poor ionisation with the ESI (electrospray ionisation) typically used with this technique. The use of GC-MS with EI (electron ionisation) gives more robust ionisation and greater sensitivity. However, derivatisation is required prior to the GC stage, usually by the formation of silylated derivatives. Often GC-MS/MS is employed with selective ion monitoring (SIM) or selective reaction monitoring (SRM), but these methods are geared to only looking at compounds that are specifically tested for, rather than picking up novel substances.

The scientists from Rio and Austin looked at the use of GC with Orbitrap MS using EI. The Orbitrap is a relatively new device, consisting of an outer electrode surrounding a spindle-shaped inner electrode. It has high resolution and is capable of detecting a vast number of compounds when run in full scan mode, including new, unexpected species.

GC-Orbitrap shown to detect a wide range of doping compounds

Urine samples were taken and either run as blanks or spiked with doping compounds. Deuterated testosterone was added as an internal standard. Enzymatic hydrolysis, to remove conjugated sugar groups, etc., was carried out with β-glucuronidase. The pH was adjusted to 9–10 and the samples were extracted with methyl tert-butyl ether (MTBE). The MTBE extracts were evaporated, and the residue was silylated using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and a catalytic mixture of 2-mercaptoethanol and ammonium iodide.

GC was carried out on a Thermo Scientific TRACE 1310 system fitted with an Agilent Ultra 1 100% dimethylpolysiloxane column. The temperature gradient was increased from 140 to 300 °C in a series of ramps. Mass spectrometry employed a Thermo Scientific GC-EI-hybrid quadrupole-Orbitrap instrument, operating in full scan mode. The data were processed using Thermo Scientific TraceFinder 4.1 software. Extracted ion chromatograms (EIC or XIC) were used to examine the compounds of interest; these were generally very ‘clean’ with few interfering peaks, permitting rapid checking of the data.

In all, 81 spiked compounds were detected, including steroids, amphetamine analogues, and various metabolites. Most of these were clearly identified by the Orbitrap system in full scan mode at concentrations below the minimum detection level required by the regulatory authorities. However, for a few compounds the mass spectrometer had to be operated in SIM mode to ensure sufficient sensitivity. The SIM mode can be employed simultaneously with the full scan mode with the Orbitrap system. A few steroids were subject to interference that made quantification difficult. In these cases GC-MS/MS was run in order to achieve clearer detection.

Orbitrap shows promise in sport drug control

The paper clearly shows that the Orbitrap instrument can be used in conjunction with derivatisation and GC to detect a large number of banned doping agents. It has the advantage over standard GC-MS/MS techniques that it is more easily extended to new species and can potentially detect unexpected new compounds in urine samples. Further refinement may lead to conditions in which the few compounds not easily detected by the Orbitrap on full scan mode can be quantified, giving an almost ‘universal’ single injection detection system for non-protein doping agents.

Related Links

Drug Testing and Analysis, 2017, Early View paper. Cavalcanti et al. Non-targeted acquisition strategy for screening doping compounds based on GC-EI-hybrid quadrupole-Orbitrap mass spectrometry: focus on exogenous anabolic steroids.

Wikipedia, Mass Chromatogram

Wikipedia, Orbitrap

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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