Optimised reverse-phase chromatography for clinical detection of vitamins A and E in serum

Abstract

Vitamin A and E are indispensable fat-soluble vitamins that play pivotal roles in promoting human health and well-being. Accurate quantification of these vitamins in biological samples is critical. Thus, we aim to develop and optimise a sensitive method suitable for simultaneous analysis of vitamin A and E. We introduced a method for simultaneous detection of Vitamin A and E using reverse-phase high-performance liquid chromatography (RP-HPLC). Calibration curves and quality control samples were prepared using a 4% of Bovine Serum Albumin (BSA) matrix. The analytes and internal standard (ISTD), Dodecanophenone were effectively separated using a polar column and detected via UV detector at wavelengths of 255 nm, 298 nm, and 325 nm respectively, with a total run time of 30 min. Results: The method demonstrated excellent linearity with regression coefficients (r²) > 0.995. The optimisation process of the HPLC method affirmed its precision and reliability, meeting analytical chemistry standards. Subsequently, this validated method was applied to serum samples obtained from patients and a population cohort exhibiting distinct clinical pathologies, including individuals without colorectal cancer (CRC), those with symptomatic and asymptomatic CRC. Conclusions: In conclusion, this HPLC method offers a reliable means for routine quantification of vitamin A and E in human serum, holding promise for enhancing nutritional research and clinical diagnostics. Its novelty lies in the use of a surrogate BSA matrix with a polar RP-HPLC column and multi-wavelength detection, enabling robust quantification of endogenous analytes in clinically relevant cohorts.