Simultaneous Quantification of Betulinic Acid, Lupeol, and β-Sitosterol in Madhuca longifolia Methanolic Extract of Bark by Liquid Chromatography-Tandem Mass Spectrometric Method.

Abstract

Liquid chromatography with tandem mass spectrometry is used widely used for the quantitative analysis of phytoconstituents present in medicinal plants to assess the quality of extract used for different investigations. A sensitive, precise, and accurate liquid chromatographic method with tandem mass spectrometric detection was developed for simultaneous quantification of lupeol, betulinic acid, and β-sitosterol in the methanolic extract of Madhuca longifolia bark. The three compounds were eluted with a stationary phase Gemini C18 column (50 × 2.0 mm, 3 μm id) and the temperature of the column was maintained by a column oven at 40 ± 0.3°C; mobile phase A (water and 0.1% formic acid) and mobile phase B [acetonitrile-methanol (50+50, v/v) and 0.1% formic acid] were used in a gradient mode and the flow rate was 0.4 mL/min. With these conditions, the retention time for betulinic acid, lupeol, and β-sitosterol was found to be 1.25, 3.08, and 3.53 minutes, respectively. The total run time was 5.0 min. Detection and quantitation of all three phytoconstituents were carried out by the mass spectrometer, a triple quadrupole equipped with atmospheric pressure chemical ionization, and multiple reaction monitoring using the predominantly positive ion mode and obtained much higher and more stable response nebulizer gas flow at 3.0 L/min. Linear responses were exhibited for all three phytoconstituents with a dynamic linear range of 10-100 μg/mL with the values of the regression coefficient more than 0.995 for betulinic acid, lupeol, and β-sitosterol. The values of percentage RSD for intraday and interday precision were found to be within the accepted limits for analytical methods (<15%). Selectivity, linearity, LOD, LOQ, accuracy, and precision were evaluated for all three phytoconstituents as per International Conference on Harmonization guidelines. The proposed method is accurate and sensitive and can be used for the routine quantification of betulinic acid, lupeol, and β-sitosterol from the herbal extract and its poly-herbal formulations.