Identification and determination of fat-soluble vitamins and metabolites in human serum by liquid chromatography/triple quadrupole mass spectrometry with multiple reaction monitoring

Abstract

A method for determination of fat-soluble vitamins K1, K 3, A, D2, D3 and E (as α- and δ-tocopherol) and metabolites 25-hydroxyvitamin D2 and D 3 and 1,25-dihydroxyvitamin D3 in human serum by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in positive mode is proposed. Highly selective identification of the target compounds in serum was confirmed by the most representative transitions from precursor ion to product ion. Quantitative MS/ MS analysis was carried out by multiple reaction monitoring optimizing the most sensitive transition for each analyte in order to achieve low detection limits (from 0.012 to 0.3 ng/mL estimated with serum). The analysis was performed with 1 mL of serum, which was subjected to protein precipitation, liquid-liquid extraction to an organic phase, evaporation to dryness and reconstitution with methanol. The precision of the overall method ranged from 3.17-6.76% as intra-day variability and from 5.07-11.53% as inter-day variability. The method, validated by the standard addition method, provides complete information on the fat-soluble vitamins profile, which is of interest in clinical and metabolomics studies. Copyright © 2007 John Wiley & Sons, Ltd.