Advances in Technology for Targeted and Non-Targeted Equine Drug Screening

With the 2012 Olympics upon us, the need for stringent drug testing is once again a prevalent topic for discussion. In Beijing in 2008, no fewer than 18 athletes were disqualified for substance abuse and five were stripped of their medals. Of those 18, six were competing in the equestrian events and disqualified when their horses tested positive for banned substances (including capsaicin, nonivamide and felbinac). The elimination of one of their riders even caused the Norwegian team to forfeit their bronze medal.

Just as London 2012 intends, Beijing operated a ‘zero tolerance’ approach to banned substances, so that even trace amounts of any performance enhancing, therapeutic or recreational drug found in the system resulted in disqualification. While doping in Olympic equestrian sports has never been as prolific as in horse racing, the Olympic committee and the International Equestrian Federation (FEI) still need to have the best possible technology behind them in order to successfully screen for, quantify and confidently identify drugs in urine samples.

The first ban on ‘stimulating substances’ was not enforced for human athletes until 1928 [Savulescu 2004], but a decree banning the administration of substances designed to improve racehorse performance was introduced in 1666, and carried a maximum punishment of the death penalty up until the late 18th century. The first successful attempts to detect doping agents using bioassays and analytical chemistry were also introduced in horse racing rather than human sport [Thevis 2010].

In 1903, equine drug testing consisted of monitoring the behaviour of frogs when they were injected with horse saliva. Since then, and particularly in the past few decades, equine analytical chemistry has changed substantially. By the early 1960s thin-layer chromatography (TLC) had been introduced by Maynard and Smith [Maynard & Smith 1962] as a primary screening technique in which a mobile phase moves by capillary action across a uniform thin layer of finely divided stationary phase bonded to a plate. Any drugs present when the sample is applied will move across the plate at different rates, depending on their individual chemical characteristics. Despite providing good drug resolution and permitting the simultaneous identification of a wide range of substances in a single analysis, the process is extremely laborious, time-consuming and demands a high level of expertise.

In 1988, Enzyme Linked Immuno-Sorbent Assay (ELISA) testing of equine urine was introduced by the University of Kentucky [Tobin 1988], in part as a response to the prolific use of high potency narcotics, stimulants, bronchodilators and tranquilizers in horse racing. The one-step ELISA is started by adding a urine sample, followed by the drug-enzyme conjugate, to the wells of a microtitre plate previously coated with antibody. Any drug specific to the ELISA employed will bind to the antibody already present. ELISA tests are extremely sensitive and specific (limits of detection in the range of 1.0 ng/ml are typical in comparison to typical limits of detection in the range of 25-2000 in TLC screening), but are limited by the narrow range of drug assays available to laboratories, despite the common misconception that equine testing laboratories have an ELISA for every drug.

Over the past two decades there has been considerable progress in the development of instrumental analytical techniques, which have replaced TLC and ELISA testing in equine analytical chemistry. Most recently, extremely high sensitivity and high specificity technology has become widely available through mass spectrometry, notably liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this method the sample is separated and molecules are isolated and broken into a series of fragments, as well as having their precise mass measured. Both the mass and relative proportions of these fragments (the fragmentation pattern) are specific for the given drug, and can then be matched with known certified reference standards run through the mass spectrometer, in parallel with the test samples.

LC-MS/MS technology allows the unequivocal identification and quantification of substances down to low pictogram per millilitre and low part per trillion concentrations, in relatively small sample volumes, but such sensitivity is limited to targeted screening only. When performed on an LC-MS/MS system, general unknown compound (GUC) screening methods typically employ full-scan MS experiments, for detection of unexpected pharmaceuticals. This results in a slight compromise in the level of detection, primarily due to a reduction in selectivity when performing single-MS, rather than MS/MS experiments. With new performance-enhancing substances in constant development, it is crucial that drug screening technology continues to attempt to stay ahead of the game in terms of confidently identifying unknown compounds within a sample.

During London 2012, more than 6,000 blood and urine samples will be tested for banned substances – the greatest number ever attempted at the Olympics – with the promise of a 24 hour turn around for all negative tests. Working at that speed and with that volume of samples would be almost impossible were it not for recent improvements in technology. With the development of new performance enhancing drugs and masking agents on the rise, demand is increasing for retrospective and non-targeted analyses, and full scan mass analyzers, combined with software that can confidently identify unknowns, are gaining popularity.

Scientists have recently developed a novel system for performing targeted and non-targeted screening in a single LC-MS/MS run, increasing speed of screening and reducing the possibility of missing the detection of compounds [Tai et al. 2012]. The approach was developed on a new hybrid quadrupole/time-of-flight (QqTOF) LC/MS/MS instrument.

As well as achieving quantification of targeted compounds, the speed of the system allows 20-30 Information Dependent Acquisition (IDA) MS/MS spectra to be collected for compound identification based on MS/MS library searching and structural elucidation determinations. The acquired full-scan MS and MS/MS data can further be used to mine data retrospectively for non-targeted compounds.

In order to ensure that both targeted and non-targeted data processing could be achieved, PeakView software was used in conjunction with XIC Manager. The XIC Manager manages large lists of compounds and performs extracted ion chromatogram (XIC) calculations, library searching, and targeted and non-targeted peak finding operations. Using the features of the PeakView software, information such as the accurate mass molecular ion, isotope pattern and detected fragment ions can be used to characterize the structure of unexpected compounds and confidently identify unknowns.