Untargeted identification and targeted quantification of the marker compound in Gmelina asiatica L. using UHPLC-Q Exactive Orbitrap MS and validated UHPLC-TQ-MS/MS methods.

Gmelina asiatica L. plays a significant role in traditional medicine, with diverse applications in the Ayurvedic and Siddha systems and multiple pharmacological activities. However, no validated analytical method has been reported for its standardization and quality control through the quantification of its major phytoconstituent, despite its importance for pharmaceutical applications. Therefore, a sensitive and validated method for its standardization is required. In this context, liquid chromatography-mass spectrometry is one of the most suitable analytical techniques for this purpose. In this study, ultra-performance liquid chromatography-Q-Exactive-Orbitrap-mass spectrometry (UHPLC-Q-Exactive-Orbitrap-MS) and ultra-performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-TQ-MS/MS) were employed to identify the marker compound of Gmelina asiatica and subsequently quantify it using a validated method. UHPLC-Q-Exactive-Orbitrap-MS revealed 699 metabolites after applying filtration criteria. The marker compound was identified as verbascoside/isoverbascoside (m/z 623.165 [M - H]-) and subsequently distinguished by UHPLC-TQ-MS/MS using selected reaction monitoring (SRM) transitions and retention time in comparison with an authentic reference standard, confirming its identity as verbascoside. The developed UHPLC-TQ-MS/MS method was validated and demonstrated excellent selectivity, precision, accuracy, and linearity (R2 ≥ 0.9989) over the range of 0.5-100 µg L-1, with limits of detection and quantification of 0.005 and 0.016 µg L-1, respectively. The validated method was successfully applied to quantify verbascoside in Gmelina asiatica extract, yielding 48.18 ± 3.67 mg g-1 extract (4.82%, w/w). The relatively high abundance of verbascoside demonstrates its suitability as a marker compound for the standardization and quality control of Gmelina asiatica and supports its reported pharmacological activities. This study establishes a reliable and validated UHPLC-TQ-MS/MS method for verbascoside-based standardization of Gmelina asiatica.