Rapid simultaneous determination of 7 fat-soluble vitamins in human serum by ultra high performance liquid chromatography tandem mass spectrometry

Abstract

Accurate detection of vitamins is critically important for clinical diagnosis, metabolomics and epidemiological studies. However, the amounts of different vitamins vary dramatically in human serum. It is a challenge to achieve simultaneous detection of multiple vitamins rapidly. Herein, we developed and validated a sensitive and specific method using ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for simultaneous quantification of 7 fat-soluble vitamins (FSVs) across their physiological concentrations in serum for the first time, which was subjected to protein precipitation, liquid–liquid extraction to an organic phase, evaporation to dryness and reconstitution with acetonitrile. In the present procedure, retinol (vitamin A), ergocalciferol (25-OH-D2), cholecalciferol (25-OH-D3), α-tocopherol (vitamin E), phylloquinone (vitamin K1), menatetrenone-4 (MK-4), and menaquinone-7 (MK-7) were detected in one analytical procedure for the first time within 5.0 min by triple quadrupole tandem mass spectrometry. The limit of quantification (LOQ) for vitamin A was 10.0 ng mL⁻¹, LOQs for 25-OH-D2 and 25-OH-D3 were 1.0 ng mL⁻¹, LOQ for vitamin E was 100.0 ng mL⁻¹, and LOQs for vitamin K1, MK-4 and MK-7 were 0.10 ng mL⁻¹, respectively, with a correlation (R²) of 0.995–0.999. Recoveries ranged from 80.5% to 118.5% and the intra-day and inter-day coefficients of variance (CVs) were 0.72–8.89% and 3.2–9.0% respectively. The method was validated according to the European Medicines Agency (EMA) and U.S. Food and Drug guidelines and C62-A on bioanalytical methods, and was used for clinical routine determination.