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Objective The objective is to develop a LC-MS/MS method to quantify 1α,25-dihydroxyvitamin D without using antibody-based extraction procedure. Methods Vitamin D is a lipid-soluble micronutrient that regulates calcium and phosphate absorption. 25-Hydroxyvitamin D is the primary form in circulation and 1α,25-dihydroxyvitamin D (DHVD) is the biologically active form. DHVD assay is challenging because of very low concentrations (picomolar) and numerous isomers or epimers present in serum. Solid phase extraction and Cookson-type triazolinedione derivatization of vitamin D increases the analytical sensitivity of DHVD assay. However, presence of isomers and epimers remain to be a challenge. One approach is adding antibody-based purification steps prior to PTAD derivatization. However, antibody-based extraction increases sample preparation time, test turn-around time, and the cost per reportable. In this study, we developed a liquid chromatography method to separate DHVD peak from major interference without using affinity purfication. Specimens were added acetonitrile to precipitate serum proteins. After centrifugation and solid phase extraction, analytes were derivatized with PTAD reagent at ambient temperature. Analytical column was a Kinetex Phenyl-hexyl column, 2.1X100 mm, 1.7 µm particle size. AB Sciex 5500 QTrap mass spectrometer was operated in triple quadrupole mode. MRM transitions and ion source parameters were optimized according to previous report. Quantitation was performed using MultiQuant software with a six-point calibration curve. Results Previous reports showed that 4β,25-dihydroxyvitamin D3 and 3-epi-1α,25-dihydroxyvitamin D3 may be coeluted with 1α,25-dihydroxyvitamin D3. In a method comparison study, we found that this interference could result in a 70% positive bias if using a short LC separation method on a C18 column. On the phenyl-hexyl column, we found that PTAD derivatized 1α,25-dihydroxyvitamin D3 has two epimer peaks and the peak area ratio was 1 to 2. Neat 4β,25-dihydroxyvitamin D3 has only one peak following derivatization. We did a mixing study and found that 4β,25-dihydroxyvitamin D3 peak was eluted between the two epimer peaks of 1α,25-dihydroxyvitamin D3. This is a direct evidence proving 4β,25-dihydroxyvitamin D3 causes positive bias in DHVD3 quantitation. DHVD3 assay based on the dominant epimer peak is the optimal strategy to quantify DHVD3 accurately in serum. The results were within 15% of expected values. We observed similar interference for DHVD2 quantitation, which was likely due to the presence of 4β,25-dihydroxyvitamin D2. Our LC separation method was 9 minute, which is slightly long and but acceptable as a clinical method. The analysis time can be reduced by using a multiplex LC system. Conclusions We developed a LC-MS/MS method for the quantitation of DHVD in serum without using immunoextraction procedure. Results demonstrated that 4β,25-dihydroxyvitamin D3 is a major interference. DHVD assay based on the dominant epimer peak is an optimal method to quantify DHVD3 and DHVD2 levels.

Quantitation of 1α,25-Dihydroxyvitamin D by Liquid Chromatography-Tandem Mass Spectrometry without immunoextraction