The Future of Liquid Chromatographic Separations Should Include Post Column Derivatisations: The perspective from the analysis of vitamin D

Abstract

The coupling of high performance liquid chromatography (HPLC) with post column derivatisation (HPLC-PCD) will increase the sensitivity of an instrument to the target molecule, improve specificity by selective derivatisation to remove potential interferences from the matrix, and be cost-effective; all these factors may well prove that PCD is an alternative to existing techniques for routine analysis. Calcidiol (25(OH)D) concentrations are used in the determination of vitamin D deficiency in humans, and there is growing interest and demand in its analysis with some controversy surrounding the sensitivity and specificity of its detection. 25(OH)D analysis is currently performed using either competitive binding assays or HPLC analysis, coupled with detection by a variety of means, such as, UV–Vis absorption, electrochemical or mass spectroscopy (MS) detection. HPLC coupled with MS (HPLC-MS) detection is the gold standard for 25(OH)D analysis due to its sensitivity and selectivity compared to all other techniques, however, its utility is limited by its high cost. This article reviews the current status of HPLC based techniques in the detection of 25(OH)D (and its metabolites), and further explores the use of HPLC-PCD.