A simple microHPLC-UV method for the simultaneous determination of retinol and α-tocopherol in human plasma. Application to intrahepatic cholestasis of pregnancy

摘要

(a) Objectives: the development of a simple, rapid, sensitive, and accurate HPLC-UV method for the simultaneous determination of retinol and ?±-tocopherol in human plasma, suitable for the evaluation of these vitamins in pregnant women suffering from intrahepatic cholestasis of pregnancy (ICP). (b) Design and methods: chromatographic separation of vitamins was achieved with a microcolumn BDS HYPERSIL C18 (100 mm ?— 2.1 mm, 2.4 ??m) using a C18 guard column. The developed and validated HPLC method proposed in this study was applied in the assessment of plasma concentrations of retinol and ?±-tocopherol in normal pregnancy (n = 37) and in pregnancy complicated with ICP (n = 19). (c) Results: the validation of the method showed excellent linearity for retinol and ?±-tocopherol (r2: 0.998 and 0.997), high sensitivity (LOD: 0.02 and 0.11 ??M, LOQ: 0.07 and 0.36 ??M), and high recovery (96.0a€“104.0% and 99.3a€“102.0%), respectively. The method requires a low sample volume and all procedures of sample preparation and analytical determination take less than 20 min to be completed. The evaluation of the results in ICP patients showed that ?±-tocopherol significantly diminished compared to normal pregnant women (13.1 ?± 2.9 vs. 22.4 ?± 2.2 ??M, p 0.05) whereas retinol remains unchanged (1.27 ?± 0.21 vs. 1.25 ?± 0.10 ??M). (d) Conclusions: a simple, accurate, reliable, selective, highly sensitive and cost effective method suitable for routine determination of retinol and ?±-tocopherol in biological samples is proposed. It was possible to determine the decrease of ?±-tocopherol in ICP patients by this method.