Identification, Validation and Standardization of Bioactive Molecules Using UPLC/MS-QToF, UHPLC and HPTLC in Divya-Denguenil-Vati: A Penta-Herbal Formulation for Dengue Fever

For the last fifty years, Dengue has been one of the most common mosquito-borne arboviral infections which has spread over the tropical and subtropical world. Divya-Denguenil-Vati (DNV) has been formulated by blending five specific herbs for effective resolution of Dengue fever. In the present study, we aimed to identify, develop, validate, and standardize methods for Divya-Denguenil-Vati (DNV), on UHPLC and HPTLC analytical platforms, with rapid, sensitive, accurate and rugged attributes. At first, 97 phyto-constituents were identified by UPLC/MS-QToF in Divya-Denguenil-Vati. UHPLC method was then developed and validated for simultaneous determination of gallic acid, 5-HMF, protocatechuic acid, magnoflorine, methyl gallate, berberine, rutin, ellagic acid, β-ecdysone and rosmarinic acid in DNV. Four selected markers, gallic acid, rosmarinic acid, magnoflorine and rutin were further developed and validated on HPTLC. Analytical processes were validated as per ICH Q2 (R1) guidelines; and were found linear (r(2) > 0.99), sensitive, precise (%RSD < 5%), and accurate, as indicated by high recovery values (88–105%). The limit of detection and quantification were also established for these phyto-metabolites, with their respective RSDs within 5% limits. Finally, these validated methods were employed to test twenty six different commercial batches of DNV. The quality, reproducibility and consistency of DNV have been well established using these developed and reliable analytical tools. These analytical strategies successfully set a path forward for robust quality evaluation and standardization of Divya-Denguenil-Vati, and other related herbal formulations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10337-022-04183-7.