Rapid, sensitive and simultaneous determination of ascorbic and uric acids in human plasma by ion-exclusion HPLC-UV.
OBJECTIVE
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 (r(2))≥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.


CONCLUSIONS
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.
Department of Technological Sciences and Development, University of the Azores, Rua da Mãe de Deus, 9501-801 Ponta Delgada, Azores, Portugal. rborges@uac.pt
Rita
R
Ferin
Maria Leonor
ML
Pavão
José
J
Baptista
D000328:Adult; D000975:Antioxidants / Q000032:analysis*; D001205:Ascorbic Acid / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D014466:Ultraviolet Rays; D014527:Uric Acid / Q000097:blood*
D000975:Antioxidants; D014527:Uric Acid; D001205:Ascorbic Acid