Development and validation of an HPLC-MS/MS method for the quantification of Aficamten in human plasma and urine and application to a clinical pharmacokinetic study.
TL;DR
An innovative, rapid, and highly sensitive HPLC-MS/MS approach for quantifying aficamten concentrations in human plasma and urine is established and critical validation parameters including matrix effects, extraction recovery, carryover, and stability were assessed.
Abstract
Aficamten (CK-3773274) is a next-in-class selective cardiac myosin inhibitor now approved for use for chronic oral therapy in hypertrophic cardiomyopathy (HCM). The present study established an innovative, rapid, and highly sensitive HPLC-MS/MS approach for quantifying aficamten concentrations in human plasma and urine. Samples were prepared using protein precipitation with acetonitrile containing CK-3942912 (D5-labelled aficamten) as the internal standard (IS). Sample separation was achieved using a C18 analytical column, with a mobile phase comprising acetonitrile and ammonium acetate. A Xevo TQS tandem mass spectrometer performed the detection in electrospray ionization (ESI) positive mode. The following MRM ion transitions were monitored: m/z 338.2 → 213.2 for CK-3773274 and m/z 343.2 → 218.2 for IS. This method was validated with excellent selectivity and sensitivity. Linear calibration curves (1-500 ng/mL) demonstrated excellent accuracy (RE% ±15%) and precision (RSD% ≤15%) for both intra- and inter-run analyses. Furthermore, critical validation parameters including matrix effects, extraction recovery, carryover, and stability were also assessed. The validated method was subsequently utilized to quantify aficamten concentrations in a clinical pharmacokinetic study involving healthy Chinese adults.