In this study, an analytical method has been developed that, for the first time, allows simultaneous determination of vitamin D-2 and vitamin D-3 along with their hydroxylated and esterified forms. A group of 12 vitamin D analogues including vitamin D-2 and vitamin D-3, seven hydroxylated metabolites, and three ester forms were separated in a single 8.0 min run using ultrahigh-performance supercritical fluid chromatography coupled with triple quadrupole tandem mass spectrometry. Electrospray ionization and atmospheric pressure chemical ionization were investigated as ion sources, of which the latter showed a higher ionization efficiency. Chromatographic conditions were thoroughly evaluated by a step-by-step method, whereas an experimental design was applied for the optimization of the ionization parameters. Calibration and repeatability studies were carried out to validate the instrumental methodology showing determination coefficients higher than 0.9992 and good intra- and interday precision with relative standard deviations for areas and retention times lower than 10 and 2.1%, respectively, for all target analytes. Limits of quantification were below 3.03 mu g/L for all compounds. The methodology was then validated and applied for the evaluation of human plasma samples in order to demonstrate its applicability to the analysis of vitamin D analogues in biological samples. Samples of five individuals were analyzed. Results show that linoleate-D-3, vitamin D-2, vitamin D-3, 25-hydroxyvitamin D-2, 24,25-dihydroxyvitamin D-3, and 1,25-dihydroxyvitamin D-3 could be detected in most samples, while the two latter also were quantified in all analyzed samples.
