A rapid analytical method for characterization and simultaneous quantitative determination of phytoconstituents in...

A rapid ultra performance liquid chromatography electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for identification and characterization of phytoconstituents with simultaneous quantitative determination of three major bioactive phenolics namely allylpyrocatechol-3,4-diacetate, eugenyl acetate and eugenol from thirteen landraces of Piper betle leaf extracts. Among the 29 phytoconstituents detected, 19 were identified and characterized based on their fragmentation pattern obtained via MS/MS by means of collision-induced-dissociation (CID) and by comparison of their retention time with authentic standards or by previously reported literature. The proposed method was fully validated in terms of linearity, LOD, LOQ, precision, stability and recovery. All calibration curves showed a good linear relationship (r² ≥ 0.9981). The precision evaluated by an intra- and inter-day study showed RSD ≤ 1.0% and good accuracy with overall recovery in the range from 96.14-98.46% (RSD ≤ 1.6%) for all analytes. The quantitative results showed that there were remarkable differences in the contents of the major bioactive phenolics in all the thirteen landraces of P. betle. The developed UPLC-ESI-MS/MS method was found to be simple, precise, sensitive and accurate. Hence, it can be reliably utilized as a quality control method for P. betle or derived herbal formulations. In vitro antimicrobial activity of the crude extract of P. betle landraces was also evaluated, and all extracts tested exhibited antifungal activity but none of the extracts were found to be active against bacteria even at 500 μg mL^-1 concentration.