Characterization of unknown impurities in Coenzyme Q10 using LC–MS and NMR

Abstract

During studies on related substances in coenzyme Q10 (CoQ10) active pharmaceutical ingredient (API) and capsules, two impurities (Impurity 1 and Impurity 2) with each content more than 0.1% attracted our attention. Accelerated testing (40 °C /75%RH) and forced degradation of CoQ10 API and drug products in different stress conditions (base and heat) gave rise to the two unknown impurities. These unknown impurities were separated by reverse phase ultra-performance liquid chromatography (UPLC), where they were eluted at 0.77 (Impurity 1) and 1.10 (Impurity 2) relative retention times to CoQ10 peak. Impurity 2 was separated into two peaks (Impurity 2-1 and Impurity 2-2) by normal phase high performance liquid chromatography (NP-HPLC). In this study, an ultra-performance liquid chromatography complied with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF/MS) via atmospheric pressure chemical ionization (APCI) was used to identify the proposed structure of the three impurities (Impurity 1, Impurity 2-1 and Impurity 2-2). We got the three impurities through preparative high performance liquid chromatography and their structures were further confirmed by 1D and 2D nuclear magnetic resonance (NMR). Based on the significance of these results, Impurity 1 was the impurity E referred in British Pharmacopoeia (BP), Impurity 2-1 and Impurity 2-2 were a pair of isomers which had not been reported before.