Routine Supercritical Fluid Chromatography Tandem Mass Spectrometry Method for Determination of Vitamin K1 Extracted from Serum with a 96-Well Solid-Phase Extraction Method.

Background: The concentration of vitamin K1 in serum or plasma is the most common index for assessing vitamin K status. The aim of this study was to develop and validate a rapid and reliable routine method for quantifying vitamin K1 above 0.1 ng/mL. Semi-automation of a simple sample preparation with fast analysis by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) was exploited.

Methods: Vitamin K1 was extracted from 250-μL serum samples by the use of protein precipitation and reversed-phase solid-phase extraction (SPE) in 96-well plates and quantified by SFC on a 2.1 × 100 mm Torus 1-Aminoanthracene (1-AA) column in 3.8 min with electrospray ionization-tandem mass spectrometry (MS/MS) detection.

Results: This method shows good linearity in the concentration range of 0.1-50 ng/mL with a correlation coefficient of R2 >0.999. Imprecision was satisfactory, with repeatability and reproducibility <10% CV. The lower limit of the measuring interval was 0.1 ng/mL, and no systematic bias was observed for the method, which used vitamin K1-d7 as internal standard. Recovery of vitamin K1 in external quality controls was satisfactory compared to other laboratories participating in the external quality assurance scheme. The method is currently in routine use for analysis of serum samples.

Conclusions: The method allows high-throughput reliable determination of vitamin K1 in serum in the range 0.1-50 ng/mL.