Validation of an UPLC-DAD Method for the Quantification of Phenolic Acids, Verbascoside and 6-epi-aucubin in Crescentia cujete Fruit

Background: The fruit pulp decoction of Crescentia cujete, commonly known as calabash, is traditionally used for the treatment of several respiratory diseases and is available as syrup formulations. Unfortunately, there is no detailed investigation on analytical methods for warranting the quality of these products. Aim and objective: To develop and validate an appropriate analytical method for the simultaneous quantification of transcinnamic acid, 4-hydroxybenzoic acid, verbascoside and 6-epi-aucubin in the decoction and commercial cough syrups of Crescentia cujete fruit. Materials and methods: A reversed-phase ultra-high-performance liquid chromatographic method coupled to a diode array detector (UPLC-DAD) was validated following the ICH guidelines. The chromatographic analysis was performed using a C18 column, the mobile phase system consisted of water and acetonitrile containing 0.1% formic acid, and UV chromatograms were recorded from 200 to 400 nm. Results: A new UPLC-DAD method was validated for the simultaneous quantification of trans-cinnamic acid, 4- hydroxybenzoic acid, verbascoside and 6-epi-aucubin in calabash-derived products. After successful validation, this method was applied for the quantification of the selected chemical markers in an in-house decoction and three commercial cough syrups. Among the selected chemical markers, 6-epi-aucubin was the main compound in the calabash decoction, while trans-cinnamic acid and 4-hydroxybenzoic acid were the major compounds in the commercial products. Verbascoside and 6-epi-aucubin were below the limit of quantification in all syrup samples. Conclusion: The proposed method was successfully applied for the analysis of three commercial syrup formulations and can be useful for standardization and quality control of raw and pharmaceutical calabash preparations.