Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS.

Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170 ± 8%) and low analytical recovery (29 ± 15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro(13)C5,(15)N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1h post administration in urine and identification of a urinary catabolite extended this detection window to 4h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine.

Searching for new markers of endogenous steroid administration in athletes: "looking outside the metabolic box".

A simple means of detecting the abuse of steroids that also occur naturally is a problem facing doping control laboratories. Specific markers are required to allow the detection of the administration of these steroids. These markers are commonly measured using a set of data obtained from the screening of samples by gas chromatography-mass spectrometry (GC-MS). Doping control laboratories further need to confirm identified abuse using techniques such as gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). An interesting urinary species was found while following the pharmacokinetics and changes to the steroid profile from single and multiple oral doses of the International Olympic Committee/World Anti Doping Agency (IOC/WADA) prohibited substance, dehydroepiandrosterone (DHEA). The urine samples collected from the administration studies were subject to GC-MS and GC-C-IRMS steroid analysis following cleanup by solid phase extraction techniques. A useful urinary product of DHEA administration was detected in the urine samples from each of the administration studies and was identified by GC-MS experiments to be 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo). This compound occurs naturally but the concentrations of 3alpha,5-cyclo were elevated following both the single DHEA administration (up to 385 ng/mL) and multiple DHEA administrations (up to 1240 ng/mL), in relation to those observed prior to these administrations (70 and 80 ng/mL, respectively). A reference distribution of urine samples collected from elite athletes (n = 632) enabled the natural concentration range of 3alpha,5-cyclo to be established (0-280 ng/mL), with a mean concentration of 22 ng/mL. Based on this an upper 3alpha,5-cyclo concentration limit of 140 ng/mL is proposed as a GC-MS screening marker of DHEA abuse in athletes. GC-C-IRMS analysis revealed significant 13C depletion of 3alpha,5-cyclo following DHEA administration. In the single administration study, the delta13C value of 3alpha,5-cyclo changed from -24.3 per thousand to a minimum value of -31.1 per thousand at 9 h post-administration, before returning to its original value after 48 h. The multiple administration study had a minimum delta13C 3alpha,5-cyclo of -33.9 per thousand during the administration phase in contrast to the initial value of -24.2 per thousand. Preliminary studies have shown 3alpha,5-cyclo to most likely be produced from DHEA sulfate found at high levels in urine. The complementary use of GC-MS and GC-C-IRMS to identify new markers of steroid abuse and the application of screening criteria incorporating such markers could also be adapted by doping control laboratories to detect metabolites of androstenedione, testosterone and dihydrotestosterone abuse.