HPLC method for 25-hydroxyvitamin D measurement: comparison with contemporary assays.
BACKGROUND
The concentration of 25-hydroxyvitamin D [25(OH)D] in serum has been designated the functional indicator of vitamin D (VitD) nutritional status. Unfortunately, variability among 25(OH)D assays limits clinician ability to monitor VitD status, supplementation, and toxicity.


METHODS
We developed an HPLC method that selectively measures 25-hydroxyvitamin D2 [25(OH)D2] and D3 [25(OH)D3] and compared this assay with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, a competitive protein-binding assay (CPBA) on the Nichols Advantage platform, and an RIA from Diasorin.


RESULTS
For the new HPLC assay, between-run CVs were 2.6%-4.9% for 25(OH)D3 and 3.2%-13% for 25(OH)D2; recoveries were 95%-102%; and the assay was linear from 5 microg/L to at least 200 microg/L. Comparison data were as follows: for HPLC vs LC-MS/MS, y = 1.01x - 4.82 microg/L (Sy/x = 4.93 microg/L; r = 0.996) for 25(OH)D3, and y = 0.902x - 0.566 microg/L (Sy/x = 2.56 microg/L; r = 0.9965 for 25(OH)D2; for HPLC vs Diasorin RIA, y = 0.709x - 5.86 microg/L (Sy/x = 7.35 microg/L; r = 0.7509); and for HPLC vs Nichols Advantage CPBA, y = 1.00x - 3.60 microg/L (Sy/x = 32.7 microg/L; r = 0.6823).


CONCLUSIONS
The new HPLC method is reliable, robust, and has advantages compared with the Nichols Advantage CPBA and the Diasorin RIA. The Nichols Advantage CPBA overestimated or underestimated 25(OH)D concentrations predicated on the prevailing metabolite present in patients' sera.
Clinical Toxicology Laboratory, University of Wisconsin Hospital and Clinics, Department of Pathology, University of Wisconsin-Madison, Madison, WI 53792, USA. g.lensmeyer@hosp.wisc.edu
Gary L
GL
Lensmeyer
Donald A
DA
Wiebe
Neil
N
Binkley
Marc K
MK
Drezner
D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D002112:Calcifediol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013058:Mass Spectrometry; D011863:Radioimmunoassay
D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol