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

• Published in: Clinical biochemistry Vol. 46; no. 7-8; pp. 665 - 669
• Main Authors: Ferin, Rita, Pavão, Maria Leonor, Baptista, José
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2013
• Subjects: Adult; Antioxidants - analysis; Ascorbic acid; Ascorbic Acid - blood; Atherosclerosis; Chromatography, High Pressure Liquid - methods; Female; HPLC-UV; Humans; Ion-exclusion; Male; Middle Aged; Plasma antioxidants; Reproducibility of Results; Ultraviolet Rays; Uric acid; Uric Acid - blood; Ion-exclusion; Ascorbic acid; Uric acid; Plasma antioxidants; HPLC-UV; Atherosclerosis
• ISSN: 0009-9120; 1873-2933; 1873-2933
• DOI: 10.1016/j.clinbiochem.2013.01.006

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. 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 indicated an excellent linearity with correlation coefficients (r2)≥0.999. The LOD of AA and UA was 1.02 and 1.42nmol/mL, respectively, while LOQ ranged from 0.306 to 0.426nmol/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. This validated method allows the determination of both antioxidants within 10min, 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. [Display omitted] ► Rapid and simultaneous determination of ascorbic and uric acids levels in plasma. ► The use of heparin as anticoagulant and MPA as stabilizer is required. ► Ion-exclusion HPLC-UV isocratic elution within 10min. ► Method offers an excellent precision and recovery for ascorbic and uric acids. ► Methodology suitable for high-throughput clinical analysis.