Sensitive measurement of serum 1α,25-dihydroxyvitamin D by liquid chromatography/tandem mass spectrometry after removing interference with immunoaffinity extraction

Vitamin D plays important roles in bone health and a variety of other pathophysiological conditions. 1 alpha, 25-Dihydroxyvitamin D is the active form of vitamin D. Quantification of serum 1 alpha,25-dihydroxyvitamin D is useful for evaluation of several diseases including chronic renal failure, hypoparathyroidism, sarcoidosis, and rickets. Measurement of 1 alpha,25-dihydroxyvitamin D is very challenging due to its low circulating concentration and presence of interfering substances in serum. In this report, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantifying serum 1a, 25-dihydroxyvitamin D is described. Lithium adducts of 1 alpha,25-dihydroxyvitamin D were formed prior to mass spectrometry analysis to improve ionization efficiency. We tested a number of different sample preparation procedures and found that immunoaffinity extraction was the method of choice because it completely removed isobaric interferences and matrix effects present in patient serum. Extraction efficiency, expressed as absolute recovery, was greater than 60% in both patient serum and charcoal-stripped serum. This method was linear from 3.4 to 206.2 pg/mL for 1 alpha,25-dihydroxyvitamin D(3) and 3.9 to 212.6 pg/mL for 1 alpha,25-dihydroxyvitamin D(2) with an accuracy of 89.8-98.4% and 97.5-115.7%, respectively. Inter-assay and intra-assay coefficients of variance (CVs) for both analytes at two different concentration levels ranged from 2.5-7.0%. Comparison with a radioimmunoassay for measuring total 1 alpha,25-dihydroxyvitamin D concentration using 40 patient samples showed a Deming regression slope of 0.751, a y-intercept of 0.84 pg/mL, an r value of 0.7909, and a mean percentage difference of -27.1%. Comparison with a reference LC/MS/MS method (n = 20) showed a Deming regression slope of 1.020, a y-intercept of 1.32 pg/mL, an r value of 0.9797, and a mean percentage difference of -2.9%. In conclusion, usage of immunoaffinity extraction enabled a sensitive LC/MS/MS method for quantification of 1 alpha,25-dihydroxyvitamin D in serum. Copyright (C) 2011 John Wiley & Sons, Ltd.