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Stability indicating high-performance thin layer chromatography method for estimation of ascorbic acid in Hibiscus sabdariffa L. aqueous extract

Background: High performance thin layer chromatography (HPTLC) has become a routine analysis technique due to its advantages of low operating cost, high sample throughput and need minimum sample clean up. 
Aim: To develop and validate an accurate, specific, repeatable and stability-indicating HPTLC method for the determination of ascorbic acid in presence of their degradation products for assessment of stability of the ascorbic acid in Hibiscus sabdariffa L. preparations. 
Methods: Chromatographic sample separations employed thin layer chromatography plates (20 cm x10 cm) pre-coated with silica gel 60 F254 as a stationary phase. Solvent system consisted of acetone: toluene: water: glacial acetic acid (26:4:4:1 v/v) while scanning wavelength of 269 nm was used. Ascorbic acid was subjected to stress degradation conditions i.e. oxidation, dry heat treatment, photo degradation and hydrolysis under different PH. Method was validated according to international council for harmonisation guidelines. 
Results: Peaks of products formed were well resolved with different Rf values. The linear regression data for the calibration plots showed good linear relationship with r2 = 0.9912 in the concentration range 2-4 µg/ml. The mean value slope and intercept were -531.72 and 8040.22 respectively. 
Conclusion: A rapid, simple, accurate and specific HPTLC method for determination of ascorbic acid in Hibiscus sabdariffa L. preparations was developed and validated. In addition, this method separated all the degradants from stress conditions showing that it is a stability indicating method. The developed method could now be used in quality control of H. sabdariffa herb products.