Optimization and validation of visible-spectrophotometry method for determination ascorbic acid in Jeruk Bali (Citrus maxima) fruit from Indonesia

The method validation is prelimnary step in a research when researcher has not familier yet with the instruments, procedures and different sample matrices. The aim is obtaining suitable analytical method. Spectrophotometry is an instrumental method which is simpler, faster and cheaper than other instrumental methods. This study optimized and validated Spectrophotometry method for the determination of ascorbic acid in Jeruk Bali Fruit (Citrus maxima). Ascorbic acid in acidic condition gives blue color when was reacted with ammonium molybdate, then the absorbance was observed in visible wavelength area. Optimization to determine ascorbic acid (250.0 μg) required the addition of 4.0 ml of 5% sulphuric acid, 1.5 ml of 3% metaphosphate acid, 1.5 ml of 5% ammonium molybdate and the resulting color was stabil within 20-30 minutes. Furthermore, optimized condition was applied to validate Spectrophotometry method for the determination of ascorbic acid in Jeruk Bali Fruit. Selectivity test obtained 701 nm as wavelength of choice, linierity test resulted Y=0.0019x+0.071 and r=0.9998 (p=0.000; p<0.01), Coeffisien of Variation (C.V)=0.3512%, recovery was (%) = 100.2128±1.7039. Using optimized and validated condition, the determination of ascorbic acid in Jeruk Bali Fruit from Indonesia =0.0208±0.0129 (%; w/w).