NP-HPLC method for determining phytonadione and its impurities

摘要

A normal-phase high-performance liquid chromatography method has been developed to separate vitamin cis-phytonadione (cis-K1), trans-phytonadione (trans-K1), trans-epoxyphytonadione (trans-EK1), menadione (K3), cyclo-menadione (CK3), hydroperoxy-phytonadione (K1OOH), and other impurities on a Zorbax RX-SIL column, characterized by very pure porous silica, low acidity, and low metal content. A dual-mode gradient elution was applied involving simultaneous changes in flow rate and mobile-phase composition. The present method was sensitive enough to detect trans-EK1 (limit of detection, LOD: 1.8 ng), K3 (LOD: 0.6 ng), CK3 (LOD: 1.2 ng), and K1OOH (LOD: 2.4 ng). A linear relationship between peak area and concentration was found for four compounds with correlation coefficient r exceeding 0.9994. The impurities (trans-EK1, K3, and K1OOH) were measured accurately (recovery rate 80-120%) and precisely (relative standard deviation less than 2%). Preparing samples under yellow light (520nm), the K1OOH in the three commercial batches of K1 was no higher than 0.10% (w/w) and was measured with a reproducibility of 3.1% and with an intermediate precision of 15.8%. After validation, the developed method was applied for analyzing the impurities occurring in the commercial samples of synthetic K1 and proved to be suitable for routine quality control.