The objective of this study was to develop and to validate reversed phase high performance liquid chromatography (RP-HPLC) method for the determination of acid orange 7 (AO7) and Sudan II (SII) in blusher product. Separation of AO7 and SII was performed using C18 column (Thermo Synergy Gold 250 mm x 4.6 mm i.d., 5 µm) with mobile phase consisting of acetonitrile-water as solvent (1:1 v/v) and delivered in gradient manner. The mobile phase was delivered at flow rate of 0.9 mL/min, using column temperature of 30oC. RP-HPLC was then validated by assessing several performance characteristics which included selectivity, linearity and range, sensitivity, precision, accuracy, and robustness. The developed RP-HPLC was selective for separation of AO7 and SII. Over concentration of 10.326-41.304 µg/mL (AO7) and 9.967-39.869 (SII), the method was linear with coefficient of correlation (r) of > 0.999. The method was sensitive as indicated by low limit of detection and limit of quantification. The relative standard deviation (RSD) values during intra-assay and inter-assay were lower than those required by RSD Horwitz. The percentage of recovery were within acceptable ranges as required by AOAC method. The validated RP-HPLC method has been successfully applied for analysis of AO7 and SII in commercial blusher products, and no samples were detectable to contain both AO7 and SII.
Key words: Acid orange 7, sudan II, RP-HPLC, blusher products, validation.
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