A novel LC-MS/MS method combined with derivatization for simultaneous quantification of vitamin D metabolites in human serum with diabetes as well as hyperlipidemia

Abstract

Vitamin D plays an important role in calcium homeostasis. Recent studies indicate that vitamin D deficiency has become a major public health problem. In order to define vitamin D status, many analytical methods were used to quantify 25-hydroxyvitamin D (25OHD), as circulating 25OHD is regarded as the best indicator to evaluate vitamin D status. The current LC-MS/MS technology is internationally recognized as the “gold standard” for the detection of vitamin D and its metabolites. The impediment to the analysis of vitamin D metabolites is the low level of 25OHD and 1,25(OH)₂D. Therefore, it is challenging to achieve the desired sensitivity and accuracy in the determination of trace vitamin D compounds in biological liquids. Here, a method based on liquid–liquid extraction in combination with derivatization, followed by liquid chromatography-electrospray/tandem mass spectrometry was developed for determination of the vitamin D metabolites, including 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₂ and 1α,25-dihydroxyvitamin D₃. The method was simple and rapid, and it was validated with good linearity (R² > 0.998), excellent recovery (average value with 81.66–110.31%) and high precision of intra-day and inter-day (0.06–6.38% and 0.20–6.82%). The values of limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.3 ng mL⁻¹ and 1.0 ng mL⁻¹, respectively. Finally, the developed method was successfully applied to determination of the vitamin D metabolites from the human serum samples of healthy subjects and patients with diabetes as well as hyperlipidemia.