(M1130-09-51) Development and Validation of an LC-MS/MS Method for Determination of Difelikefalin in Human Plasma

Purpose: Difelikefalin (CR845, Korsuva ^TM) is a selective kappa-opioid receptor agonist, first line treatment option for moderate-to-severe pruritus associated with chronic kidney disease in adults undergoing hemodialysis and manage post-operative pain. A simple, fast and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for determining difelikefalin in human plasma.

Methods: Sample extraction using protein precipitation with acetonitrile was optimized to extract analytes from human plasma with maximum recovery. The chromatographic separation was performed on an Acquity UPLC HSS T3 (2.1 X 30 mm, 1.8 mM) column, maintained at 50°C with chromatographic run of 1 min. Electrospray ionization in positive ion mode and multiple reaction monitoring were used to monitor the ion transition at m/z 680.7®295.1. The method was fully validated according to the FDA guidelines on bioanalytical method validation over concentration ranges.

Results: The assay was linear over a range of 3.9 – 4000 ng/mL (r²>0.99) for difelikefalin with the lower limit of quantification at 3.9 ng/mL. The lower limit of detection (LOD) was determined to be 1.9 ng/mL. The inter-day precision (%CV) and accuracy ranged from 6.79 to 11.5% and 93.3 to 105%, respectively. The intraday precision (%CV) and accuracy ranged from 0.38 to 14.1% and 97.2 to 113%, respectively. No matrix effect and no significant carryover were observed. The mean extraction recovery were >85%. There were 45% - 75% loss of difelikefalin in human plasma after three freeze-thaw cycles. Plasma difelikefalin was stable for about one month when stored at 4°C, -20°C and -80°C.

Conclusion: The HPLC-MS/MS method proved to be specific, selective, robust and highly reproducible for the quantification of difelikefalin.