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

Abstract

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 phytometabolites, 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.

Characterization and quantification of chemical constituents in Fuzhuan brick tea using ultra‐high‐performance liquid chromatography–mass spectrometry

“…The fragmentation of phenolic acids in negative mode is usually characterized by the elimination of CO 2 and the hexosyl moiety from the parent ions. Peaks 3 and 5 exhibited parent ions at m/z 169.01 and 153.01 and fragments at m/z 108.02 and 125.02 were identified as gallic acid [15] and protocatechuic acid [19], respectively.…”

“…Peaks 4, 8, 9, 12, and 14 possessed a product ion at m/z 191.05, suggesting a possible substitution by quinic acid. Peak 4 with an [M − H] − ion at m/z 343.06 was characterized as galloylquinic acid [16,21], while peak 12, with m/z 353.08, was characterized as chlorogenic acid [19], according to the reference. Peak 8 showed an MS ion at m/z 179.03, implying a possible substitution of caffeic acid, which was identified as neochlorogenic acid [19,22,23].…”

“…Peak 4 with an [M − H] − ion at m/z 343.06 was characterized as galloylquinic acid [16,21], while peak 12, with m/z 353.08, was characterized as chlorogenic acid [19], according to the reference. Peak 8 showed an MS ion at m/z 179.03, implying a possible substitution of caffeic acid, which was identified as neochlorogenic acid [19,22,23]. The characteristic ion of peak 9 at m/z 163.04 implied the possible substitution of p-coumaric acid, as reported previously [15], and was confirmed to be coumaroylquinic acid.…”