Rapid, sensitive and simultaneous determination of ascorbic and uric acids in human plasma by ion-exclusion HPLC-UV

Abstract

OBJECTIVES: Ascorbic (AA) and uric (UA) acids act as antioxidants and are capable to react with biologically relevant oxidants. We aimed to developed a simple, rapid, sensitive, and accurate ion-exclusion HPLC-UV methodology for the simultaneously determination of AA and UA in human plasma. METHODS: Analytical pre-requisites, such as the use of heparin as an anticoagulant and meta-phosphoric acid as a stabilizer were added for accurate and reliable measurements. Chromatographic separation was achieved by an isocratic elution on a HEMA-BIO 1000 SB analytical column using a phosphate buffer, pH 2.4, as a mobile phase. RESULTS: Results indicated an excellent linearity with correlation coefficients (r²) ≥ 0.999. The LOD of AA and UA was 1.02 and 1.42 nmol/mL, respectively, while LOQ ranged from 0.306 to 0.426 nmol/mL. A great repeatability for both antioxidants was found, where the CV (%) values for intra-day were lower than 1.8% and under 6.5% for the inter-day assay. The recovery of AA ranged from 92% to 96% and from 99% to 100% for UA. CONCLUSION: This validated method allows the determination of both antioxidants within 10 min, and is well suited to routine measurements and/or high-throughput clinical analysis. The methodology was applied to assess the antioxidant status of a group of Azorean subjects.