Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid
and uric acid play important role in protection of spermatozoa against free radicals. A method for the
simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV
detection and investigation their clinical significance as antioxidants protecting male germ cells against
oxidative damage are described. Semensamples were obtained fromconsecutivemalepartners of couples
presenting for a fertility evaluation.After liquefaction,the samples were centrifuged and the supernatants
were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the
separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 m, was used.
The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as
a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid:
the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from
spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both
ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 mol/L
and 323.7 ± 99.5 mol/L vs. 444.8 ± 245.5 mol/L and 316.6 ± 108.9 mol/L, p > 0.05). This assay is a simple
and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in
human seminal plasma.