Optimization and validation of a high performance liquid chromatography method for the simultaneous determination of vitamins A and E in human serum ... [An article from: Analytica Chimica Acta]

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Description:
In this study a novel, simple and rapid reversed-phase high performance liquid chromatography (HPLC) procedure for simultaneous determination of vitamins A and E (retinol and alpha-tocopherol) in blood serum has been developed and validated using monolithic column and diode-array detection (DAD). The monolithic column Chromolith Performance RP-18e (100mmx4.6mm) was operated at ambient temperature. One hundred percent methanol at flow rate 2.5mlmin^-^1 was used as a mobile phase. Detection of both compounds was performed with diode-array detector, retinol was monitored at 325nm and alpha-tocopherol at 295nm. The linear dependence between peak area and concentration ranged from 0.25 to 10.00@mmoll^-^1 for retinol and 0.5-50.0@mmoll^-^1 for alpha-tocopherol. The limit of detection (LOD) for retinol was 0.02@mmoll^-^1 and limit of quantification (LOQ) was 0.07@mmoll^-^1. The limit of detection (LOD) for alpha-tocopherol was 0.1@mmoll^-^1 and limit of quantification (LOQ) was 0.3@mmoll^-^1. Retinol was eluted in 0.8min and alpha-tocopherol in 1.4min. The simultaneous analysis of vitamin A and E can be achieved in less than 2min. The implementation of monolithic column Chromolith Performance shortens the time of analysis of both vitamins four times in comparison with using traditional particulate column Pecosphere C18 (150mmx4.6mm), 5@mm. This fact may play an important role for routine clinical analysis of biological samples.