Analytical approach
Dope is generally administered at or near the therapeutic dose, which results in relatively low concentrations in biological fluids. Any drug used in human treatment or in veterinary practice may be found. Thus, screening procedures must be both sensitive and of wide coverage, and they differ in detail from other analytical schemes. However, the sports chemist enjoys the advantage of examining relatively constant material, usually in fairly fresh condition. He or she thus has a clearer picture of a normal sample than does the forensic chemist who may be required to examine a wide variety of materials in various states of decomposition.
Any sample that fails a screening test is invariably submitted to rigorous confirmatory testing before an adverse report is issued. Although the parent drug is the entity normally described in the rules, screening procedures rely upon the detection of either the unchanged drug or its metabolites. The identification of the corresponding metabolites is often useful supplementary evidence to support the identification of the parent drug. Examples may include the detection of 3- and 4-hydroxy mepivacaine, major metabolites of the local anaesthetic mepivacaine, or nordiazepam after diazepam administration.
Conversely, the absence of any expected metabolites is possible evidence that a sample has been contaminated; this should be refutable by proper chain of custody. Occasionally, the parent drug is not excreted in urine at a detectable concentration, so knowledge of the metabolic pathways in the particular species is essential. An example of this is the identification of 19-norandrosterone and 19-noretio cholanolone in the urine of humans as evidence of the administration of the anabolic steroid nandrolone or a 19-norsteroid precursor. After administration of cocaine, primarily benzoylecgonine and ecgonine methyl ester are detected in urine unless a huge dose of cocaine was given. In that case parent cocaine can also be detected in urine. Some drugs are notable for being excreted in urine almost entirely in conjugated form as, for instance, morphine, apomorphine, fentanyl, nefopam and pentazocine in the horse. When the presence of these drugs is suspected, hydrolysis before extraction is essential because most analytical methods are designed to detect drugs and metabolites in their nonconjugated form. If a hydrolysis procedure is not employed, and the analytical method used is not designed to detect conjugates, the drug may not be detected even though it was admin istered. LC-MS/MS methods are being designed to detect drugs in their conjugated form, thereby obviating the need for hydrolysis procedures. Drugs can be used either to improve or to impair athletic performance, though in human sport the latter category of drug is unlikely to be used knowingly. In sports such as greyhound racing and horseracing, however, decreasing an animal’s speed can be a profitable exercise, so it is important to monitor sedative drugs in these events. Thus, in horse and dog samples it is essential that all groups be covered. No single analytical scheme will suffice to cover so many different types of compounds, and various approaches have evolved in racing chemistry and human doping laboratories to address this challenge. Liquid–liquid extractions designed for group separation of drugs followed by thin-layer chromatography (TLC) was used almost universally for many years and is still used widely in North American and some other racing chemistry laboratories. Alternatively, drugs were extracted on styrene–divinylbenzene copolymer XAD-2 resin. The development of solid-phase extraction (SPE) in the cartridge format in the late 1970s and the rapid advances made in the technology associated with the technique have provided an attractive alternative to liquid–liquid extraction in many drug-screening programmes. Immunochemical methods that cover glucocorticosteroids were first introduced into equine drug-screening programmes in the 1970s. However, in the late 1980s, ELISAs were developed specifically for equine drug-testing programmes and are now widely used to support TLC and instrumental screening programmes.
Human sports drug-testing laboratories have not employed TLC typically and do not use ELISA extensively, but used XAD-2 and now, more commonly, C8 and C18 SPE. Liquid–liquid extraction is still commonly employed in laboratories for some drug classes. Instrumental methods (GC using nitrogen–phosphorus detection (GC-NPD), high-performance liquid chromatography using ultraviolet detection (HPLC-UV), GC-MS and LC-MS(/MS)) are the predominant analytical methods in most animal and human sports drug-testing laboratories. Even if not routinely used for screening, some sort of TLC technology is sporadically used by many laboratories to isolate or purify a compound of interest followed by the analysis of so called scrape from the TLC plate by GC-MS, LC-MS, etc.
