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

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Abstract
A simplified method of sample preparation and high-performance 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.