Gary L Lensmeyer1, Donald A Wiebe, Neil Binkley, Marc K Drezner. 1. 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
Abstract
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.
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.
Authors: Karen W Phinney; Mary Bedner; Susan S-C Tai; Veronica V Vamathevan; Lane C Sander; Katherine E Sharpless; Stephen A Wise; James H Yen; Rosemary L Schleicher; Madhulika Chaudhary-Webb; Christine M Pfeiffer; Joseph M Betz; Paul M Coates; Mary Frances Picciano Journal: Anal Chem Date: 2011-12-28 Impact factor: 6.986
Authors: K E Barbour; J M Zmuda; M J Horwitz; E S Strotmeyer; R Boudreau; R W Evans; K E Ensrud; C L Gordon; M A Petit; A L Patrick; J A Cauley Journal: Osteoporos Int Date: 2010-11-23 Impact factor: 4.507
Authors: Elizabeth A Yetley; Christine M Pfeiffer; Rosemary L Schleicher; Karen W Phinney; David A Lacher; Sylvia Christakos; John H Eckfeldt; James C Fleet; George Howard; Andrew N Hoofnagle; Siu L Hui; Gary L Lensmeyer; Joseph Massaro; Munro Peacock; Bernard Rosner; Donald Wiebe; Regan L Bailey; Paul M Coates; Anne C Looker; Christopher Sempos; Clifford L Johnson; Mary Frances Picciano Journal: J Nutr Date: 2010-09-29 Impact factor: 4.798
Authors: Brunhild M Halm; Jennifer F Lai; Ian Pagano; William Cooney; Reni A Soon; Adrian A Franke Journal: J Am Coll Nutr Date: 2013 Impact factor: 3.169
Authors: N Binkley; D Gemar; J Engelke; R Gangnon; R Ramamurthy; D Krueger; M K Drezner Journal: J Clin Endocrinol Metab Date: 2011-02-02 Impact factor: 5.958
Authors: Debin Wan; Jun Yang; Bogdan Barnych; Sung Hee Hwang; Kin Sing Stephen Lee; Yongliang Cui; Jun Niu; Mitchell A Watsky; Bruce D Hammock Journal: J Lipid Res Date: 2017-02-01 Impact factor: 5.922