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

Abstract

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 reverse- phase high performance liquid chromatography (RP-HPLC) assay with ultra-violet (UV) detection for the measure- ment 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 us- ing a high performance liquid chromatographic system with UV detection. Chromatographic separation of L-AA was achieved on a reverse phase C 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 quantifica- tion, 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.