Mark M Kushnir, Affiliation 1
Julie A Ray, Affiliation 1
Alan L Rockwood1, Affiliations 1,2
William L Roberts, Affiliations 1,2
Sonia L La’ulu, Affiliation 1
A Wayne Meikle1, Affiliations 1,2,3
Affiliation 1 - ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;
Affiliation 2 - Department of Pathology, University of Utah, Salt Lake City, UT
Affiliation 3 - Department of Medicine, University of Utah, Salt Lake City, UT |
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| Measurement of 25-hydroxy vitamin D (25OH-vitD) is used to assess vitamin D status. Commercial immunoassays for 25OH-vitD suffer from interference from other hydroxy metabolites of vitamin D. Our goal was to develop a high sensitivity method for 25OH-vitD, and to assess the relationship between concentrations of 25OH-vitD and PTH in healthy adults. Aliquots of 100 uL of serum were spiked with internal standard and proteins were precipitated. The samples were filtered and analyzed by LC-MS/MS using two-dimensional chromatographic separation. A Zorbax Eclipse XDB-CN (Agilent) was used as the first dimension and Gemini C18 (Phenomenex) as the second dimension; the analysis time was 6.5 min. The primary mass transitions were m/z 413 to 337 (25OH-vitD2) and m/z 401 to 365 (25OH-vitD3). The total imprecision for the method was less than 9.8%. The assay was linear up to 1,000 ug/L; the LOD and LOQ were 0.5 and 1 ug/L. The method was used for determining the distribution of concentrations of 25OH-vitD2 and 25OH-vitD3 in serum of healthy adults (location 40° North, 111° West; average elevation 1320 m) collected during winter and summer, and evaluation of the association between 25OH-vitD and PTH. The difference between median concentrations of 25OH-vitD in samples collected during winter and summer was 11 ug/L. Statistically significant differences in concentrations of PTH were observed between groups of samples with 25OH-vitD <11 and 11-15 ug/L; 25-30 and >40 ug/L. Our data support a cutoff of 30 ug/L of 25OH-vitD as the lower limit of adequacy, while also demonstrating a trend for a continuing reduction of PTH for samples with concentrations of 25OH-vitD >30 ug/L. This simple, robust method is suitable for routine measurement of the 25OH-vitD2 and 25OH-vitD3 in human serum and plasma. Among the advantages of this method are its high sensitivity and specificity. |
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