Improved sample preparation method for fast LC-MS/MS analysis of vitamin D metabolites in serum

Despite the fact that more than 90% of vitamin D analysis are performed using immuno-enzymatic techniques, it is liquid chromatography coupled with tandem mass spectrometry that is currently the reference method. It allows for specific and selective analysis of all relevant vitamin D metabolites from a variety of biological materials, including serum or a dried blood spot. This paper presents development of a fast, cheap and high-throughput method of serum sample preparation using protein precipitation. For this purpose, organic solvent is used. Several substances were tested, including acetonitrile, methanol and their mixtures with zinc sulfate. However, the highest recovery values for the vitamin D metabolites were obtained for acetonitrile, with an organic solvent to serum ratio of 8:1. The preparation of a sample is carried out in 96-well plates and takes an hour and a half, together with a derivatization reaction using Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione. Due to the fact that vitamin D metabolites are bound to proteins, the relationship between the content of organic solvent in the sample preparation process and their release from the protein complex was examined. The results indicate that the organic solvent content should be 30-70% in order to completely release the tested compounds from the proteins. In addition, the developed chromatographic method has eliminated false positive signals for the 24,25(OH)₂D₃ metabolite. Total analysis time is 5.5 min., while maintaining resolution necessary to separate the analyzed compounds.