Selective extraction and determination of vitamin B12 in urine by ionic liquid-based aqueous two-phase system prior to high-performance liquid chromatography

A rapid and simple extraction technique based on aqueous two-phase system (ATPS) was developed for separation and enrichment of vitamin B12 in urine samples. The proposed ATPS-based method involves the application of the hydrophilic ionic liquid (IL) 1-hexyl-3-methylimidazolium chloride and K2HPO4. After the extraction procedure, the vitamin B12-enriched IL upper phase was directly injected into the high performance liquid chromatography (HPLC) system for analysis. All variables influencing the IL-based ATPS approach (e.g., the composition of ATPS, pH and temperature values) were evaluated. The average extraction efficiency was 97% under optimum conditions. Only 5.0 mL of sample and a single hydrolysis/deproteinization/extraction step were required, followed by direct injection of the IL-rich upper phase into HPLC system for vitamin B12 determination. A detection limit of 0.09 μg mL−1, a relative standard deviation (RSD) of 4.50% (n=10) and a linear range of 0.40–8.00 μg mL−1 were obtained. The proposed green analytical procedure was satisfactorily applied to the analysis of samples with highly complex matrices, such as urine. Finally, the IL-ATPS technique could be considered as an efficient tool for the water-soluble vitamin B12 extraction.