Development and Validation of Derivatization Method for Simultaneous Quantitation of Asteracantha longifolia (L.) Nees Phytoconstituents by Liquid Chromatography–Electrospray Ionization–Mass Spectrometry/Mass Spectrometry

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ABSTRACT Background: Betulin, lupeol, stigmasterol, and β-sitosterol are the active phytoconstituents accountable for the practical application of Asteracantha longifolia (L.) Nees in ayurvedic treatments as antitumor, hypoglycemic, aphrodisiac, antibacterial, free radical scavenging, lipid peroxidation, and hepatoprotective agent. As an essential medicinal plant in modern and traditional medicine, regularly checking raw material’s quality attributes and quantifying the phytoconstituents is indispensable. Aim: The aim of this study was to optimize derivatization conditions of these four essential phytoconstituents with p-toluenesulfonyl isocyanate (PTSI) reagent and the development of a liquid chromatography–electrospray ionization (ESI)–mass spectrometry (MS)/MS method that aids in the simultaneous detection of these four phytoconstituents. Objective: Thus, derivatization enhances sensitivity for detection in the negative mode with an electron spray ionization source and tandem mass spectroscopy. Further, the sensitive and rapid analytical method for simultaneous quantitation of betulin, lupeol, stigmasterol, and β-sitosterol is subsequently validated in line with the guidelines by ICH and used to quantify the phytoconstituents in two over-the-counter herbal formulations and one plant extract isolated at laboratory scale under batch analysis. Materials and Methods: PTSI reagent of purity <98% procured from TCI and other chromatography reagents of MS grade were utilized in the optimization of an analytical method. Results: The developed LC-ESI-MS/MS-based method demonstrated specificity for the targeted phytoconstituents, with LOD and LOQ values of < 0.13 ng/mL and 0.4 ng/mL, respectively. Linearity was established with a correlation coefficient ≥ 0.998 over a concentration range of 0.2 ng/mL to 6 ng/mL. Precision, expressed as %RSD for peak response, was ≤ 5%. The method showed satisfactory recovery for betulin (98.5%-108.3%), lupeol (95.0%-109.3%), stigmasterol (101.9%-117.0%), and β-sitosterol (99.1%-116.2%). Conclusion: The reagent PTSI reacts rapidly and forms stable derivatives. The derivatization of active phytoconstituents betulin, lupeol, stigmasterol, and β-sitosterol has enhanced the detection sensitivity of the analytical method.