Development and validation of an analytical method for simultaneous quantification of ascorbic acid and ascorbyl palmitate in liposomes

Abstract

Ascorbic acid (AA) and ascorbyl palmitate (AP) are attractive antioxidants, widely used in food industry. But they have a low stability because they can be degraded by different factors. There are several studies using nanotechnology through encapsulation to protect these compounds and increase their stability in different particles, such as liposomes. After encapsulation of AA and AP in liposomes, the actives need to be quantified. The aim of this study was to develop and validate a HPLC method for simultaneous quantification of AA and AP in liposomes. The method was validated according to the International Conference on Harmonization guidelines for validation of analytical procedures and the following parameters: specificity, linearity, detection and quantification limits, precision, accuracy, and robustness. The mobile phase was composed of acetonitrile:NaH2PO4 buffer 0.02 mol.L-1 pH 2.5:methanol (85:10:5, v/v) at a flow rate of 0.6 mL/min, isocratic elution and the column was a LiChroCART® 250-4. The chromatographic run was set to 13 minutes and AA and AP were detected at 243 nm. The method demonstrates specificity and has no interference from the excipients. The method showed linearity between 5 - 30 μg.mL-1, and the correlation coefficient represented by r2 = 0.9995 and r2 = 0.9996 for AA and AP, respectively. The analysis of precision and accuracy showed a low relative standard deviation (<1.55%) and a sufficient recovery percentage of AA (95.77%) and AP (101.24%). The procedure provided specificity, linearity, precision, accuracy and robustness, indicating that the method can be applied to the quantitation of AA and AP in liposomes.