DEVELOPMENT OF VITAMIN D₃ HPLC METHOD AND ITS APPLICATION IN BLOOD SERUM ANALYSIS OF WORKERS OF RADIATION AREA

Abstract

An improved reverse phase HPLC method was developed for the estimation of cholecalciferol in blood serum followed by a preliminary study for the assessment of vitamin D levels in workers in a radiation area, in order to investigate its radiation protection effect. Acetonitrile and methanol was used for extraction of vitamin D from serum samples, allowing a convenient separation of fat soluble fractions from proteins. Primarily, the experimental conditions were optimized, such as a temperature of 28°C, the mobile phase consisting of acetonitrile–methanol (20:80, v/v), λ ≈ 285 nm, and flow rate set as 1.5 mL/min using reverse phase C₁₈ column (5 µm, 250, 4.6 mm). The LOD was 5 ng/mL, while LOQ was reported as 45 ng/mL. The linearity of the method was excellent (r² = 0.9976) over the concentration range of 2–45 ng/mL at λ ≈ 285 nm. Instrument precision determined by four successive injections of the standard prepared showed (RSD) value of 0.76. The studies revealed that the method is specific, accurate, precise, and robust over the concentration range investigated.