Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling.
Clin Biochem. 2012 Jul 5.
Baecher S, Leinenbach A, Wright JA, Pongratz S, Kobold U, Thiele R.
OBJECTIVES:
For quantification of 25-hydroxyvitamin D(3) (25OH-D(3)), 25-hydroxyvitamin D(2) (25OH-D(2)), 3-epi-25-hydroxyvitamin D(3) (3-epi-25OH-D(3)) and 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)-D(3)) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated.
DESIGN AND METHODS:
After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi reaction monitoring is used for analyte detection.
RESULTS:
Baseline separation of the analytes is achieved below ten minutes.
The method is linear over the range
- 4.0 - 265.3nmol/L for 25OH-D(3),
- 3.9 - 183.6nmol/L for 25OH-D(2),
- 2.0 - 133.8nmol/L for 3-epi-25OH-D(3) and
- 2.8 - 129.9nmol/L for 24R,25(OH)(2)-D(3) (r(2) >0.998).
The limit of quantification is 4.0nmol/L for 25OH-D(3), 3.9nmol/L for 25OH-D(2), 2.0nmol/L for 3-epi-25OH-D(3) and 2.8nmol/L for 24R,25(OH)(2)-D(3). The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D(3) and 24R,25(OH)(2)-D(3).
CONCLUSIONS:
This highly specific HPLC-MS/MS method is suitable for vitD profiling.
There is a correlation between 25OH-D(3) and 24R,25(OH)(2)-D(3).
Serum concentration of 24R,25(OH)(2)-D(3) increases disproportionally with increasing concentration of 25OH-D(3).
Copyright © 2012. Published by Elsevier Inc.
PMID: 22771503
Comment by VitaminDWiki Recall that there are many photoproducts generated by the skin, not just Vitamin D3