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

This research article investigates a method for detecting and quantifying gonadotropin-releasing hormone (GnRH) in horse urine using aptamer-based enrichment and LC-MS/MS analysis, offering a potential alternative to traditional antibody-based means of enrichment for peptides and proteins in anti-doping tests.

Introduction and Purpose

• The primary concern of this research is the changing threats to the integrity of racing, particularly in the form of naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Such molecules can be used unfairly to enhance the performance of racing horses.
• The current tools used for detection of these compounds are typically antibodies, which can purify and concentrate the compound of interest. However, given the fast expanding field of peptide-based therapeutics, new methods of enrichment need to be developed.
• A potential alternative proposed by the authors is the use of aptamers. Once the nucleic acid sequence is known, aptamers can be synthesized chemically, providing a significant benefit over traditional methods.

Methodology

• The study presents a method developed for enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
• This method was compared with previously published antibody-based enrichment methods to ascertain its viability.

Findings

• The method developed rendered comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously known antibody-based enrichment methods.
• In terms of precision, both intra-assay and inter-assay achieved less than 10% variation at both 5 pg/mL and 20 pg/mL, displaying a satisfactory working dynamic range of 2.5-100 pg/mL.
• The authors report significant matrix enhancement (an increase in the signal) and low analytical recovery. Nonetheless, the use of an isotopically heavy labelled GnRH peptide as the internal standard allows for compensation for these limitations.
• GnRH peptides were detectable up to 1 hour after administration in urine, while the identification of a urinary catabolite extended this detection window up to 4 hours.

Conclusion

• Based on these findings, the researchers suggest the use of aptamers as a viable alternative to antibodies for enriching peptide targets from equine urine. It’s regarded as an effective method to potentially combat the illegal use of enhancing biological molecules in horse racing.