Analytical challenges in the mavacamten impurity analysis: Exploring various cutting edge analytical methods to achieve mass balance for the accurate quantification of analytes

Abstract

Mavacamten, a cardiac myosin inhibitor, is used to treat hypertrophic cardiomyopathy (HCM). Previous studies identified two drug related impurities (DRIs) of mavacamten under acidic hydrolytic stress conditions: 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione (Imp 1) and 1-phenylethanamine (Imp 2). Imp 2 has a weakly absorbing chromophore, making high performance liquid chromatography (HPLC) quantification challenging due to baseline drifts at lower wavelengths effecting the mass balance parameter. Given the importance of impurity identification and determination for quality control and consumer safety, an alternative analytical method was required. The present study aimed to develop an analytical method for the concurrent determination of mavacamten and its DRIs. HPLC with evaporative light scattering detector (ELSD), LC – triple quadrupole mass spectrometry (LC-QqQ-MS) as well as nuclear magnetic resonance (NMR) spectroscopy techniques were explored. Out of three advanced analytical approaches, NMR proved to be advantageous in detecting both the impurities. A robust method using ¹H quantitative nuclear magnetic resonance (¹H qNMR) spectroscopy for the simultaneous determination of mavacamten and its two degradation products (DPs) was developed. Method validation followed ICH Q2(R2) guidelines. The NMR method showed excellent linearity (R² > 0.99) over a concentration range of 0.1–5 mg/mL for all the compounds. Accuracy was validated with desired recovery rates across multiple concentrations. Precision tests yielded low %RSD values, and robustness studies indicated negligible effects from deliberate parameter changes. The validated method was applied to degradation kinetics studies of mavacamten under acidic conditions, accurately quantifying the drug and its DPs meeting mass balance criteria. This method provides a valuable alternative to HPLC, particularly for compounds lacking chromophores, and can be routinely used in stability studies and impurity profiling in the pharmaceutical industry.