LC-MS/MS quantification of vitamin K1 after simple precipitation of protein from low volume of serum

Abstract

Quantification of vitamin K1 in serum is still quite a challenge for most analytical laboratories. For long, protein precipitation (PP) coupled with liquid–liquid extraction (LLE) or solid phase extraction (SPE) is the only choice for sample preparation before LC-MS/MS analysis. The objective of this study is to establish a novel method for the simple and rapid quantification of K1. To this end, three issues are illuminated in the first place: (i) the release efficiency of K1 is influenced by the type of precipitator and the volume ratio of serum to precipitator during PP; (ii) the absolute recovery of K1 will be compromised by the partition effect during extraction; (iii) the volume of injected serum per run should be proper rather than high to prevent the potential ion suppression effect. Benefitting from these findings, a single-step PP method is developed for K1 quantification for the first time. For pretreatment, isopropanol (100 μL), instead of the frequently used methanol, ethanol and acetonitrile, is utilized to release K1 from serum (25 μL), which realize the excellent release efficiency (>90%) and dilution effect (1:4) simultaneously. After validation, the method can provide favorable accuracy (91.5–112.2%) and precision (less than13.5%) in the wide concentration range (0.1–2.5 ng/mL). As a result, such method may provide a feasible and high-throughput alternative for evaluating K1 status in practice.