Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1533, Issue -, Pages 57-65Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2017.12.012
Keywords
Vitamin D; Vitamin D sulfate; LC-MS/MS; Pentafluorophenyl (PFP) column; Solid phase extraction (SPE)
Funding
- Ministry of Education
- Universiti Teknologi MARA, Malaysia
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The development and validation of a method to simultaneously quantify 12 vitamin D compounds in human serum by LC-MS/MS is described. The main challenge was that of extracting and chromatographing vitamin D compounds with a range of polarities, both lipophilic and hydrophilic, in a single analytical procedure. The extractions of all 12 vitamin D compounds were achieved by an optimised protein precipitation method using acetonitrile as the precipitant, and the separation was accomplished by using a pentafluorophenyl (PEP) column. The sensitivity was increased by minimising matrix effects in MS detector rather than using a lengthy derivatisation procedure; an online solid phase extraction (SPE) using a PFP guard column was used for cleanup. Detection limits for all compounds were in the picomole range when using a 500 mu L sample volume. Recovery percentages ranged from 92% to 99%. LC-MS/MS resolution of all 12 vitamin D compounds, including the chromatographic separation of 25(OH)D-3 from the isomer 3-epi-25(OH)D-3 was achieved. Stable isotope labelled vitamin D compounds were used as internal standards for the quantification of all 12 vitamin D compounds. This is a simple yet accurate, selective, and sensitive method for the quantification of 12 major vitamin D compounds, including the sulfated forms, in human serum. The method is sufficiently robust to offer potential for use in routine analysis in a pathology laboratory setting. (C) 2017 Elsevier B.V. All rights reserved.
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