Optimization and Validation of a UV–HPLC Method for Vitamin C Determination in Strawberries (Fragaria ananassa Duch.), Using Experimental Designs

Abstract

Vitamin C or total ascorbic acid (TAA) in fruits can be assumed as ascorbic acid (AA) plus dehydroascorbic acid (DHA) content. The aim of this work was to optimize and validate, using experimental designs, a new high-performance liquid chromatographic method for vitamin C determination in strawberries. The mobile phase (MP) consisted of a 0.03 M sodium acetate/acetic acid buffer, 5% methanol. For optimization, a Box–Behnken design was used (three factors at three levels: (a) pH of MP, 3.8–5.8; (b) wavelength, 240–270 nm; and (c) flow rate, 0.5–1.2 ml min⁻¹). Responses were: AA and TAA areas, peak widths, and retention times. A global optimization was performed using the Derringer desirability function, and a value of 0.84 was reached for the combination of design factors: A = 5.8, B = 251 nm, and C = 1.15 ml min⁻¹. Method validation, using AA standard solutions, included: linearity study, limits of detection and quantification, and calibration and analytical sensitivity quantifications. Precision and accuracy were studied in strawberry extracts. The coefficients of variation (percent) were: AA, 1.5%; TAA, 1.8%, and DHA, 4.9%. Accuracy was evaluated with AA standard spiked in 30–150% range of the expected amount of analyte in real samples. The joint confidence elliptical region test and t test were employed for the study of the difference between recoveries (percent) and the ideal 100%. The robustness was analyzed using a fractional factorial design (3⁴⁻²), and an AA recovery study after slight changes in operative variables was performed. The results indicate that the optimized method was linear, sensible, precise, accurate, and robust.