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 K 1 , K 3 , A, D 2 , D 3 and E (as α ‐ and δ ‐tocopherol) and metabolites 25‐hydroxyvitamin D 2 and D 3 and 1,25‐dihydroxyvitamin D 3 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.