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Analysis of vitamin D and its metabolites using thermospray liquid chromatography/mass spectrometry
Author(s) -
Watson David,
Setchell Kenneth D. R.,
Ross Richardus
Publication year - 1991
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130050404
Subject(s) - chemistry , thermospray , mass spectrometry , chromatography , absorbance , ammonium acetate , analytical chemistry (journal) , mass spectrum , protonation , ion , high performance liquid chromatography , selected reaction monitoring , tandem mass spectrometry , organic chemistry
A new method is described for the analysis of vitamin D and its metabolites utilizing thermospray (TSP) mass spectrometry as an on‐line detector for high performance liquid chromatogrpahy. Ionization conditions were optimized for use with isocratic reversed phase chromatography. TSP mass spectrometry was employed in series with a UV absorbance detector to facilitate comparisons between the two methods of detection. Positive ion TSP mass spectra were recorded for vitamin D 2 , vitamin D 3 , 25‐hydroxyvitamin D 3 (25(OH)D 3 ), 1,25‐dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and 24,25‐dihydroxyvitamin D 3 (24,25(OH) 2 D 3 ). The spectra contained protonated molecular ions, ammonium adduct ions and fragment ions due to the loss of one or more molecules of water. A comparison of quantitative precision was made by determining UV absorbance and TSP standard curves for vitamin D 3 using two different methods: (1) External standard method with post‐column (post UV detector) addition of ammonium acetate. (2) As (1) but using the method of internal standards with a closely eluting internal standard (vitamin D 2 ). In each case the quantitative precision (correlation coefficient) for UV absorbance detection was superior owing to intrinsic instability of the TSP ion beam. A stable isotopically labelled internal standard was employed in the development of an assay for 1,25(OH) 2 D 3 . The assay was used to quantify in vitro enzymic conversion of 25(OH)D 3 to 1,25(OH) 2 D 3 in guinea pig and sheep renal mitochondrial incubations. TSP LC/MS was also applied to analysis of an extract of human blood plasma in which D 3 and each of its principal metabolites were identified in a single analysis.