Automated method for the determination of vitamin�D3 hydroxymetabolites in serum

Abstract

An almost automated method for the determination of hydroxymetabolites of vitamin D(3) (cholecalciferol) in human serum is reported. The method consists of three steps: 1) a batch liquid-liquid extraction step with 2-propanol and hexane, and drying of the extract and reconstitution with phosphate buffer. 2) A cleanup and preconcentration step based on solid-phase extraction using Prospekt equipment, with CN group cartridges and elution with the chromatographic mobile phase. 3) A chromatographic step for individual separation of the target analytes starting with a 90:10 methanol-water mixture, then a linear gradient to obtain 100% methanol; followed by photometric detection. The method provides a linear range between 1.0 and 100 ng mL(-1) for 24,25-(OH)(2) vitamin D(3) and for 25-(OH)(2) vitamin D(3), and between 1.5 and 100 ng mL(-1) for 1,25-(OH) vitamin D(3), with correlation coefficients ranging between 0.993 and 0.987, repeatability between 1.9% and 4.8% and within-laboratory reproducibility between 2.8% and 8.8%.

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

“…Additionally it is not present in human plasma, its chromatographic behavior is similar and retention time is longer [1].…”

“…Chromatography methods can effectively separate 25(OH)D 3 and other vitamin D metabolites. The determination of 25(OH)D 3 in serum or plasma has been carried out by HPLC with UV [1,2,8,16,17], coulometric [9], and MS detection [3,10,12,18]. Some HPLC methods lack an internal standard, require gradient elution, or analyze crude sample extracts that, when injected on an HPLC column, wind down resolution and column life.…”