Bioanalytical determination of unstable endogenous small peptides: RFRP3 and its metabolites in rat blood

BACKGROUND: Targeting the gonadotropin-releasing hormone pathway for the treatment of endometriosis leads to an interest in monitoring for endogenous modulators of this pathway (RFRP3 and kisspeptin) as baseline controls for treatment development. RESULTS: Stabilization of RFRP3 was shown to be extremely difficult in a highly enzymatically active matrix, such as rat blood. Sample denaturing with solvent at collection was necessary due to enzyme inhibition being unsuccessful at stabilization leading to difficulties in sample processing. Monitoring multiple fragments formed in blood can aid in profiling these peptides once in-source conversion is controlled. CONCLUSION: generic high-sensitivity LC-MS/MS assay was developed for RFRP3 and the fragments formed from it in whole blood. Use of 2D chromatography circumvents concentration and retention issues related to small fragments with a normal flow setup, making a more open-access approach feasible.