Mass Spectrometry and Illicit Drug Testing: Analytical Challenges

The "Early Days": Detection of Low-molecular Weight Xenobiotics

The first official anti-doping tests began in the late 1960s, when the substances prohibited for their effect on sport performance were limited to those used "in competition". The list of prohibited substances included simply some stimulants and drugs of abuse (see^[2–4] for reviews). These substances could be effectively detected by chromatographic methods, especially by gas chromatography (GC), a technique which, at that time, was rapidly developing as one of the most powerful in analytical chemistry. Viewed through the eyes of an anti-doping analyst of today, those analytical challenges now appear relatively easy for a number of reasons, including the fact that all the abused substances were xenobiotics and therefore not naturally present in the body. In addition to this, the pharmacokinetic characterization of those compounds was sufficiently well known, and the doses to be administered in order to obtain a performance-enhancing effect were high enough to produce a relatively high concentration of drug/metabolite in the urine; in most cases well above the detection limit of the analytical methods. In other words, it was very unlikely for a prohibited substance to be "invisible" (i.e., able to be taken without the risk of being caught positive at the anti-doping test).

In the search by the cheaters for new, more effective and less easily detectable performance-enhancing drugs, a significant step forward was represented by the use of androgenic anabolic steroids (AAS; i.e., the class of steroid hormones whose prototype compound is T, the main male hormone). AAS became extremely popular in the 1970s since they were, in a sense, the "invisible" doping agents of those times. In fact, the laboratory techniques used at that time for the detection of doping agents were not sufficiently selective and sensitive. Unlike the "traditional" doping agents (primarily stimulants and narcotics) which, to be effective, must be administered just before the competition, the efficacy of AAS is maximal if they are used for prolonged periods, so that they can stimulate muscle growth and the development of strength and power in combination with the training sessions. The result of this is that at the time of the test, especially if it is performed after the competition or during a multistage event, the concentration of AAS and/or their metabolites in the athletes' body fluids can be extremely low and thus fall below the detection limits of the analytical method.

Another difference with stimulants, diuretics and several other compounds belonging to different classes of low-molecular-weight doping agents is that the majority of AAS, as well as other steroid hormones, are extensively metabolized and are not excreted free in the urine, but instead are excreted in a conjugated form (mainly as glucuro-conjugates or sulfo-conjugates). This means, once again, that the "traditional" methods used for the detection of other more easily detectable doping agents were ineffective if applied to screen for AAS. A relatively straightforward sample pretreatment procedure (essentially, a liquid/liquid extraction to separate the drugs of interest from the matrix, followed by a preconcentration of the organic extract under nitrogen stream) combined with GC alone was not enough.

Fortunately for the testers, benchtop GC/MS systems were ready to enter the arena of doping control analysis, and the development of more complex and effective pretretament procedures (also including a hydrolysis step, either chemical or enzymatic, and the derivatization of the final extract to yield more thermally stable and volatile derivatives) made the detection of AAS in urine possible, with a window of detectability that for some steroids could be as wide as several weeks from the time of the last administration. The reliable and correct identification of a specific compound was therefore ensured thanks to the development of highly specific methods, also capable of distinguishing between a synthetic steroid and an endogenous steroid, naturally present in the body. The advances in microelectronics and the development of informatic systems for the control of GC/MS instruments (from the set up of all operating parameters to the final data acquisition, processing and presentation) made GC/MS systems the gold standard for doping analysis.

The first official event in which the synthetic AAS were tested was back in 1976, during the Montreal Summer Olympic Games.^[5] At the Atlanta Summer Olympic Games 20 years later, high-resolution MS, still coupled to GC, was first used for anti-doping analysis in an international sport event. This ensured an increased selectivity and sensitivity for the detection of AAS, while also allowing retrospective analyses that, for some compounds, could reveal the presence of substances that had been taken up to 6 months before. In the meantime, other substances were added to the list (including diuretics, β₂-agonists and β-blockers, cannabinoids, glucocorticoids and local anesthetics; this last class of compounds removed from the list in 2004). All of these compounds could effectively be detected, identified and, where so required by the anti-doping rules, quantified, by GC/MS-based techniques (reviewed in ^[6]).

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