Determination of ascorbic acid in blood plasma or serum and in seminal plasma using a simplified sample preparation and High-Performance liquid chromatography coupled with UV detection

Abstract

A simplified method of sample preparation and highperformance liquid chromatography procedure using UV detection is described for the determination of ascorbic acid (AA) in blood plasma or serum and seminal plasma. Within two hours from collection samples are treated with dithioerythritol (DTE) and then stored under Argon at -80 °C. Prior to analysis, protein precipitation is initiated with the addition of cold methanol. AA elution is carried out on a C18 reverse phase column using dodecyltrimethylammonium bromide as an ion-pairing agent. The detection is accomplished by measuring ultraviolet absorption at 265 nm. The analysis time for sample is 10.5 min, the retention time of AA being 9.7 min. Within- and between-day coefficients of variation are 2.9% and 4.9% for blood serum, 1.0 % and 2.3 % for seminal plasma. Mean analytical recovery of 102.5 ± 3.7% was found analyzing a serum pool after addition of a standard amount of AA. AA levels are stable for at least 43 days under the described storage conditions. Detection limit (3x sd) was of 1.8 ng/injection. Mean concentration of blood serum AA levels, measured in 165 male subjects, aged 56–76 years, was 8.9 ± 4.0 mg/L (50.5 ± 22.7 µmol/1). Seminal plasma AA levels observed in 14 adult subjects, aged 23–35 years, ranged from 25.8 to 232.4 mg/1 (146.5 to 1319.6 µmol/l), with a mean concentration of 114.4 ± 69.0 mg/1 (649.6 ± 391.8 µmol/1). © 1991, Taylor & Francis Group, LLC. All rights reserved.