Simultaneous Determination of Vitamin D and Its Hydroxylated and Esterified Metabolites by Ultrahigh-Performance Supercritical Fluid Chromatography-Tandem Mass Spectrometry

Abstract

In this study, an analytical method has been developed that, for the first time, allows simultaneous determination of vitamin D₂ and vitamin D₃ along with their hydroxylated and esterified forms. A group of 12 vitamin D analogues including vitamin D₂ and vitamin D₃, 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-D₃, vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, 24,25-dihydroxyvitamin D₃, and 1,25-dihydroxyvitamin D₃ could be detected in most samples, while the two latter also were quantified in all analyzed samples.

Figure 1

Normalized UHPSFC-(QqQ)-MS/MS chromatogram of the best separation achieved for all compounds under the chromatographic conditions described in the Experimental Section. Torus 1-aminoanthracene (1-AA) column at 50 °C using a mobile phase consisting of CO₂ (mobile phase A) and MeOH as the co-solvent (mobile phase B). (1) Palmitate-D₃; (IS_1) palmitate-D₃-¹³C₁₆; (2) stearate-D₃; (3) linoleate-D₃; (4) D₃; (5) D₂; (6) 25-OH-D₂; (IS_2) 25-OH-D₃-¹³C₅; (7) 25-OH-D₃; (8) 1-OH-D₃; (9) 1-OH-D₂; (10) 24,25-(OH)₂-D₃; (11) 1,25-(OH)₂-D₂; and (12) 1,25-(OH)₂-D₃.

Figure 2

Contour plots of some representative compounds obtained from the RSM full factorial DoE for the optimization of the ionization source parameters for APCI. The variation of the areas, taking into account the modification of the vaporizer temperature and source gas temperature, around the optimal point (center of the black cross) is represented for each analyte. Conditions are described in the Experimental Section. Fixed conditions: capillary voltage: 3.75 kV; corona current: 5 μA; nebulizer gas pressure (N₂): 30 psi.

Figure 3

Examples of UHPSFC-(QqQ)-MS/MS extracted ion chromatograms of vitamin D₃, D₂, and various metabolites from a blood plasma sample under the chromatographic conditions described in the Experimental Section. Torus 1-aminoanthracene (1-AA) column at 50 °C using a mobile phase consisting of CO₂ (mobile phase A) and MeOH as the co-solvent (mobile phase B).