Quantitation of 1α,25-dihydroxyvitamin D by LC-MS/MS using solid-phase extraction and fixed-charge derivitization in comparison to immunoextraction

Background: The objective of this study was to present analysis of 1 alpha,25-dihydroxyvitamin D (DHVD) by solid-phase extraction (SPE) using fixed-charge derivitization extraction to enhance ionization for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in comparison to traditional immunoextraction (IE) techniques. Methods: Full analytical validation of both a SPE and IE LC-MS/MS assay was performed, and included accuracy, intra-and inter-assay precision, limit of detection and limit of quantitation. Performance of these two assays was compared with reference laboratory IE LC-MS/MS testing. Results: This SPE LC-MS/MS assay demonstrated similar performance to the IE LC-MS/MS assay validated simultaneously. Intra-assay precision for low (12 pg/mL), medium (25 pg/mL) and high (60 pg/mL) control samples was 7.2%, 13.7% and 11.3% for DHVD2, respectively, and 9.1%, 5.9% and 8.9% for DHVD3. The inter-assay precision was 11.6%, 10.3% and 3.9% for DHVD2 and 10.6%, 7.0% and 5.6% for DHVD3. The limit of detection was 1.9 and 2.7 pg/mL for DHVD2 and DHVD3, and limit of quantitation was 4 pg/mL for both DHVD2 and DHVD3. Comparison to a reference LC-MS/MS assay showed excellent correlation (slope 0.936, R-2 = 0.996, -0.2% bias). Conclusions: The study demonstrated comparability of the SPE-LC-MS/MS assay for analysis of DHVD and offers an attractive option for assessment of vitamin D status as an alternative to traditional IE techniques.