Development and Validation of HPTLC-Densitometric Method Compared to Titrimetric Method for Determination of L-ascorbic Acid in Citrus Fruits

Abstract

Aims: The aim of this study is to develop and validate an efficient method for the specific determination of L-ascorbic acid in citrus fruits and to compare it with a usual method, method using 2,6-dichlorophenolindophenol Methodology: The research for a specific method for determination of L-ascorbic acid has led to development and validation of High Performance Thin Layer Chromatography – Densitometric (HPTLC-D) method. The validation criteria evaluated are response function, determination reading wavelength, limit of detection, precision of the technique, limit of quantification and recovery rate. The validated method was applied to citrus juice samples for quantification of L-ascorbic acid. Then a 2,6-dichlorophenolindophenol titrimetric assay of L-ascorbic acid in the same sample was performed for comparison of method results. Results: HPTLC-D method showed improved sensitivity on 360 ​​nm scanner and a good linearity relationship between standard concentrations and absorbance responses. The regression coefficient obtained is R2 = 0.99. The limit of detection (LOD) and the limit of quantification (LOQ) are respectively 3 ng and 9.5 ng per deposit. The method also exhibited good repeatability and precision, with intra-day (n = 3) and inter-day (n = 4) coefficients of variation (CV) of less than 6%. Application of the HPTLC-Dmethod to citrus juices yielded a recovery rate ranging from 97% to 99%. Conclusion: Comparison of results of the two methods shows that the contents obtained by titrimetry are greater than those obtained by HPTLC-D by 42.25%. This is explained by the presence of other redox compounds which are dosed at the same time as L-ascorbic acid. HPTLC-D makes it possible to specifically dose L-ascorbic acid.