Analysis of retinol, α-tocopherol, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC-MS/MS method.

Fat-soluble vitamins play a pivotal role in the progression of atherosclerosis and the development of cardiovascular disease. Therefore, plasma monitoring of their concentrations may be useful in the diagnosis of these disorders as well as in the process of treatment. The study aimed to develop and validate an HPLC-MS/MS method for determination of retinol, α-tocopherol, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in plasma of patients with cardiovascular disease. The analytes were separated on an HPLC Kinetex F5 column via gradient elution with water and methanol, both containing 0.1% (v/v) formic acid. Detection of the analytes was performed on a triple-quadrupole MS with multiple reaction monitoring via electrospray ionization. The analytes were isolated from plasma samples with liquid-liquid extraction using hexane. Linearity of the analyte calibration curves was confirmed in the ranges 0.02-2 μg/mL for retinol, 0.5-20 μg/mL for α-tocopherol, 5-100 ng/mL for 25-hydroxyvitamin D2 and 2-100 ng/mL for 25-hydroxyvitamin D3. Intra- and inter-assay precision and accuracy of the method were satisfactory. Short- and long-term stabilities of the analytes were determined. The HPLC-MS/MS method was applied for the determination of the above fat-soluble vitamin concentrations in patient plasma as potential markers of the cardiovascular disease progression.