A High performance thin layer chromatography (HPTLC) method was developed and optimized for
determination of colchatetralene, a new bioactive alkaloid isolated from the chloroform extract of an endophytic fungus,
isolated from surface sterilized seeds of Gloriosa superba Linn. The development of method involved evaluation and
optimization of the various stages of sample preparation, chromatographic separation, detection and quantification. The
method employed aluminum backed pre coated TLC plates (silica gel 60F254) as the stationary phase. The mobile phase
system consisted of ethyl acetate- methanol-water (10:1.5:1, v/v/v) at room temperature (25 ± 2°C). Densitometric
analysis was carried out in the absorbance/reflectance mode at the wavelength of 236nm. The system was found to give
compact spots for colchatetralene (Rf value of 0.36 ± 0.03). The linear regression analysis data for the calibration plots
showed good linearity (r2 = 0.99601 ± 0.002) in the concentration range 200–1200 ng spot-1. The method was validated
according to the International Conference on Harmonization (ICH) guidelines for precision, accuracy, recovery and
robustness. The limits of detection and quantification were 20 and 60 ng per spot, respectively.
Keywords: HPTLC, colchatetralene, endophyte, gloriosa superba, validation.
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