Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography-tandem mass spectrometry

The development and validation of a method to simultaneously quantify 12 vitamin D compounds in human serum by LC-MS/MS is described. The main challenge was that of extracting and chromatographing vitamin D compounds with a range of polarities, both lipophilic and hydrophilic, in a single analytical procedure. The extractions of all 12 vitamin D compounds were achieved by an optimised protein precipitation method using acetonitrile as the precipitant, and the separation was accomplished by using a pentafluorophenyl (PFP) column. The sensitivity was increased by minimising matrix effects in MS detector rather than using a lengthy derivatisation procedure; an online solid phase extraction (SPE) using a PFP guard column was used for cleanup. Detection limits for all compounds were in the picomole range when using a 500μL sample volume. Recovery percentages ranged from 92% to 99%. LC-MS/MS resolution of all 12 vitamin D compounds, including the chromatographic separation of 25(OH)D from the isomer 3-epi-25(OH)D was achieved. Stable isotope labelled vitamin D compounds were used as internal standards for the quantification of all 12 vitamin D compounds. This is a simple yet accurate, selective, and sensitive method for the quantification of 12 major vitamin D compounds, including the sulfated forms, in human serum. The method is sufficiently robust to offer potential for use in routine analysis in a pathology laboratory setting