Measurement of plasma 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in population of patients with cardiovascular disease by UPLC-MS/MS method

Vitamin D has a potential role in protecting against cardiovascular disease (CVD). Serum 25-hydroxyvitamin D (25D) is the most widely used indicator of vitamin D status in the human body. 25D is estimated as total of 25-hydroxyvitamin D2 (25D2) and 25-hydroxyvitamin D3 (25D3). However, the presence of 3-epi-25-hydroxyvitamin D3 (3epi25D3) can affect 25D measurement. In this research a novel validated UPLC-MS/MS technique was developed to measure three vitamin D metabolites, 25D2, 25D3 and 3epi25D3 in human plasma. A liquid-liquid extraction using hexane was applied for isolation of the analytes from the samples. A chromatographic separation was achieved in a Kinetex F5 analytical column with isocratic elution (water and methanol with 0.1% methanoic acid, 20:80 v/v). Mass spectrometry detection of the metabolites was performed in a triple quadruple tandem mass spectrometer under positive ion mode. Concentrations of the analytes were estimated in plasma samples of 54 patients. Validation parameters of the UPLC-MS/MS method, including linearity, precision, accuracy, and stability, fulfilled the requirements for bioanalytical assays. The deficient concentration of 25D (< 20 ng/mL) was stated in over 60% of patients. 3epi25D3 was present in 78% of samples and its relative amount ranged from 0 to 54.1% of 25D concentration. The analysis of 25D2, 25D3 and 3epi25D3 by the validated UPLC-MS/MS method in plasma of patients with CVD permitted the classification of the patients with insufficient levels of 25D. 3epi25D3 might be relevant in the classification of vitamin D status.