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 μg/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.