Identification of porcine relaxin in plasma by liquid chromatography-high resolution mass spectrometry.

Relaxin (RLX) has demonstrated diverse pharmacological effects in various scientific and clinical studies. The emergence of porcine relaxin (pRLX) has raised concerns on the doping potential of pRLX. There have also been speculations in the horseracing industry on its covert use. To control the abuse of pRLX in equine sports, a method to detect pRLX effectively and to provide its unequivocal identification in equine biological samples is required. This paper reports on the detection and confirmation of pRLX in equine plasma by liquid chromatography-high resolution mass spectrometry. pRLX was isolated from equine plasma by immunoaffinity purification using anti-pRLX antibody-coated magnetic beads. Anti-pRLX antibody was generated in-house by purifying antisera from rabbits immunized with pRLX. The isolated pRLX was subjected to reduction of their disulfide bonds to obtain their respective A- and B-chains. The extracts were then further purified and concentrated prior to reversed-phase LC separation and high resolution accurate mass measurement. As detection of the A-chains was far more sensitive than that of the B-chains, the A-chain of pRLX was set as the targets for detection and confirmation. The limit of detection for pRLX was around 0.005 ng/mL (~ 0.86 fM) and the limit of confirmation was around 0.02 ng/mL (~ 3.4 fM). It was observed that method sensitivity was improved at least 5-fold by using an EASY-spray column and emitter in place of the conventional ESI source. The applicability of this method was demonstrated by the identification of pRLX and its metabolites in equine plasma obtained after subcutaneous administration. To our knowledge, this is the first report of a validated mass spectrometry method for the unequivocal confirmation of pRLX in any biological fluid. Copyright © 2016 John Wiley & Sons, Ltd.