Development and validation of an LC-MS/MS method for detection and quantification of in vivo derived metabolites of [Pyr1]apelin-13 in humans.
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[Pyr1]apelin-13 is the predominant apelin peptide isoform in the human cardiovascular system and plasma. To date, few studies have investigated [Pyr1]apelin-13 metabolism in vivo in rats with no studies examining its stability in humans. We therefore aimed to develop an LC-MS/MS method for detection and quantification of intact [Pyr1]apelin-13 and have used this method to identify the metabolites generated in vivo in humans. [Pyr1]apelin-13 (135 nmol/min) was infused into six healthy human volunteers for 120 minutes and blood collected at time 0 and 120 minutes after infusion. Plasma was extracted in the presence of guanidine hydrochloride and analysed by LC-MS/MS. Here we report a highly sensitive, robust and reproducible method for quantification of intact [Pyr1]apelin-13 and its metabolites in human plasma. Using this method, we showed that the circulating concentration of intact peptide was 58.3 ± 10.5 ng/ml after 120 minutes infusion. We demonstrated for the first time that in humans, [Pyr1]apelin-13 was cleaved from both termini but the C-terminal was more susceptible to cleavage. Consequently, of the metabolites identified, [Pyr1]apelin-13(1-12), [Pyr1]apelin-13(1-10) and [Pyr1]apelin-13(1-6) were the most abundant. These data suggest that apelin peptides designed for use as cardiovascular therapeutics, should include modifications that minimise C-terminal cleavage.
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2045-2322
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Wellcome Trust (107715/Z/15/Z)
Wellcome Trust (106262/Z/14/Z)
Wellcome Trust (203814/Z/16/Z)
Medical Research Council (MR/M009041/1)
Medical Research Council (MC_UU_12012/3)
Medical Research Council (MR/M024873/1)
Medical Research Council (MC_UU_12012/5)
MRC (MC_UU_00014/3)
MRC (MC_UU_00014/5)
Medical Research Council (MC_PC_12012)