分析および生物分析技術ジャーナル

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Optimization and Validation of a Reverse-Phase High Performance Liquid Chromatography Assay with Ultra-Violet Detection for Measuring Total L-Ascorbic Acid in Food and Beverage Products

Olivia L Parbhunath, Fanie Rautenbach, Glenda Davison and Jeanine L Marnewick

In accordance with national and international regulatory standards, namely ISO/IEC 17025, the validation of chromatography methods is becoming necessary. This study provides an optimized and fully validated reversephase high performance liquid chromatography (RP-HPLC) assay with ultra-violet (UV) detection for the measurement of L-ascorbic acid (L-AA) in fruit, vegetable and food products.
Several commercial fruit juices and teas, fresh fruit and vegetables and food extract products were analyzed using a high performance liquid chromatographic system with UV detection. Chromatographic separation of L-AA was achieved on a reverse phase C₁₈ 150 mm×4.6 mm, 0.5 μm column with UV detection of 245 nm at room temperature. Distilled water/acetonitrile/formic acid (99: 0.9: 0.1, v/v/v) at a flow rate of 1 mLmin^-1 was used as the mobile phase, in isocratic mode. Samples were extracted in 4.5% metaphosphoric acid solution and filtered through a 0.45 μm membrane. The method was validated for accuracy, precision, linearity, range, limit of detection, limit of quantification, specificity, stability, robustness and system suitability in accordance with ISO 17025 validation requirements. Validation results demonstrated a linear response within a range of 5 to 125 μg/mL with a correlation coefficient of 0.999 was obtained. Mean recoveries ranged from 99 to 103% and 92 to 96% for L-AA standards and samples, respectively. The method was found to be precise (COV’s <5%) and specific with no interferences from coexisting peaks. The LOD and LOQ were 0.61 μg/mL and 1.84 μg/mL respectively.
The successful optimization and validation of the proposed method should make it easily applicable for routine laboratory analysis of L-AA measurement in various fruit and vegetable products.