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.
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.
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).
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.
Comment by VitaminDWiki Recall that there are many photoproducts generated by the skin, not just Vitamin D3