Rapid HPLC method for the determination of vitamin A and E and cotinine concentration in human serum in women with CIN and cervical cancer

Abstract

Abstract Objective: The aim of this study was to elaborate on the analytical method for quantitative determination of retinol and α-tocopherol in serum of women diagnosed with CIN and cervical cancer. The basic problem in the analysis of the vitamins content in biological material is their low physiological concentration level and instability. Liquid chromatography with diode array detector (DAD) was applied. Material and methods: The material consisted of serum and urine collected from 12 women diagnosed with cervical intraepithelial neoplasia (CIN) and 16 diagnosed with cervical cancer. The method was evaluated for the following parameters: linearity, recovery, sensitivity, precision, accuracy, selectivity, stability, limit of quantification (LOQ) and limit of detection (LOD). Results: Results showed good linearity (r2 ≥0,99) in the range 0,1μg/ml-10mg/ml for retinol and 0,25μg/ml-15μg/ml for α-tocopherol. The Lower Limit of Detection was 0,15μg/ml for vitamin E and 0,05μg/ml for vitamin A. The within-run R.S.Ds were below 5,2% at all concentration levels and the between-run R.S.Ds were below 10,0% at all concentration levels. Conclusions: The advantage of this method is that it measures both compounds in a more rapid, reproducible and accurate manner when compared to the previous HPLC studies. The compounds (vitamin A and E and internal standards) are measured in the same sample at the same time. Quantitative determination of cotinine may reveal active smokers and subjects exposed to environmental tobacco smoke, which is independent measurable carcinogenetic co-factor. The following study is a part of a project determining non-viral causative agents in cervical carcinogenesis.