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

Ferin, Rita; Pavão, Maria Leonor; Baptista, José

http://hdl.handle.net/10400.3/2747

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Authors

Ferin, Rita

Pavão, Maria Leonor

Baptista, José

Abstract(s)

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.

Description

Keywords

Ascorbic Acid Uric Acid Plasma Antioxidants HPLC-UV Ion-exclusion Atherosclerosis

URI

http://hdl.handle.net/10400.3/2747

Citation

FERIN, R., PAVÃO, M.L. e BAPTISTA, J. (2013). "Rapid, sensitive and simultaneous determination of ascorbic and uric acids in human plasma by ion-exclusion HPLC-UV", Clinical Biochemistry, 46(7-8): 665-669. http://dx.doi.org/10.1016/j.clinbiochem.2013.01.006.