title: A rapid method for the separation of vitamin D and its metabolites by ultra-high performance supercritical fluid chromatography-mass spectrometry.
abstract: In this study, a new supercritical fluid chromatography-mass spectrometry (SFC-MS) method has been developed for the separation of nine vitamin D metabolites within less than eight minutes. This is the first study of analysis of vitamin D and its metabolites carried out by SFC-MS. Six columns of orthogonal selectivity were examined, and the best separation was obtained by using a 1-aminoanthracene (1-AA) column. The number and the position of hydroxyl groups in the structure of the studied compounds as well as the number of unsaturated bonds determine the physiochemical properties and, thus the separation of vitamin D metabolites that is achieved on this column. All D2 and the D3 forms were baseline separated with resolution values>1.5. The effects of pressure, temperature, flow rate and different gradient modes were studied. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were compared in positive mode, both by direct infusion and after SFC separation. The results showed that the sensitivity in APCI(+) was higher than in ESI(+) using direct infusion. In contrast, the sensitivity in APCI(+) was 6-fold lower than in ESI(+) after SFC separation. The SFC-MS method was validated between 10 and 500ng/mL for all analytes with coefficient of determination (R(2))≥0.999 for all calibration curves. The limits of detection (LOD) were found to range between 0.39 and 5.98ng/mL for 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1-hydroxyvitamin D2 (1OHD2), respectively. To show its potential, the method was applied to human plasma samples from healthy individuals. Vitamin D3 (D3), 25-hydroxyvitamin D3 (25OHD3) and 24,25(OH)2D3 were determined in plasma samples and the concentrations were 6.6±3.0ng/mL, 23.8±9.2ng/mL and 5.4±2.7ng/mL, respectively.
authors.affiliation: Centre for Analysis and Synthesis, Department of Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden.
authors.forename: Firas
authors.initials: F
authors.lastname: Jumaah
authors.affiliation: Division of Diabetes, Department of Experimental Medical Science, Metabolism and Endocrinology, Lund University, BMC C11, SE-221 84 Lund, Sweden.
authors.forename: Sara
authors.initials: S
authors.lastname: Larsson
authors.affiliation: Centre for Analysis and Synthesis, Department of Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden.
authors.forename: Sofia
authors.initials: S
authors.lastname: Essén
authors.affiliation: Centre for Analysis and Synthesis, Department of Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden.
authors.forename: Larissa P
authors.initials: LP
authors.lastname: Cunico
authors.affiliation: Division of Diabetes, Department of Experimental Medical Science, Metabolism and Endocrinology, Lund University, BMC C11, SE-221 84 Lund, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Holm
authors.affiliation: Centre for Analysis and Synthesis, Department of Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden.
authors.forename: Charlotta
authors.initials: C
authors.lastname: Turner
authors.affiliation: Centre for Analysis and Synthesis, Department of Chemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden. Electronic address: Margareta.Sandahl@chem.lu.se.
authors.forename: Margareta
authors.initials: M
authors.lastname: Sandahl
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D002112:Calcifediol / Q000032:analysis / Q000097:blood; D002138:Calibration; D002762:Cholecalciferol / Q000032:analysis / Q000097:blood; D025924:Chromatography, Supercritical Fluid; D006801:Humans; D057230:Limit of Detection; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D014807:Vitamin D / Q000031:analogs & derivatives / Q000032:analysis* / Q000097:blood / Q000378:metabolism
chemical_list: C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D002762:Cholecalciferol; D002112:Calcifediol
keywords: Mass spectrometry; Method development; Orthogonal column screening; Plasma; Ultra-high performance supercritical fluid chromatography; Vitamin D(2) and D(3) metabolites