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

• Aptamer enrichment of GnRH from equine urine enables effective quantitation.
• GnRH is unstable in equine urine if stored at ambient or cold temperatures.
• A protease inhibitor cocktail can minimise the degradation of GnRH in plasma.
• Analysis of the catabolite GnRH (5−10) increases the detection window of GnRH doping.

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 (Pro13C5,15N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1 h post administration in urine and identification of a urinary catabolite extended this detection window to 4 h. 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.

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