On-line SPE sample treatment as a tool for method automatization and detection limits reduction: Quantification of 25-hydroxyvitamin D3/D2

Abstract

The development and approbation of new, automated UHPLC-DAD method for the quantification of 25-hydroxyvitamin D₃/D₂ (25OH-D₃/D₂) metabolites in plasma/serum for the evaluation of patient's vitamin D status are presented. The method was developed on the Ultimate 3000 UHPLC dual gradient system supplied with the on-line SPE-concentration column coupled through six port switching valve to analytical column. This configuration and materials selected enable large volume sample injection (500μL) and on-line sample preconcentration, clean up and subsequent selective metabolites transfer onto the analytical column. The new method abrogates main conventional time consuming and error source off-line steps of analysis and thus simplifies analysis. The large volume injection increases the sensitivity of instrumental analysis by about ten-fold on-line pre-concentration of metabolites. The instrument response is linear (R>0.99) in the investigated concentration range 10-100ngmL^-1 which covers all the possible vitamin D status from serious deficiency (<12ngmL^-1) to excess. The method detection limits (S/N=3) are LOD (25OH-D₃)=0.94ngmL^-1 and LOD (25OH-D₂)=2.4ngmL^-1. The method performance was assessed with the use of certified reference samples and perfect agreement between certified and measured values is demonstrated. The method was applied to human samples previously analyzed for total vitamin D by Competitive Protein-binding assay and findings of the two methods are compared.

Keywords: 25-Hydroxy vitamin D(3)/D(2); Large volume injection; On-line solid phase extraction; Vitamin D.