High pressure liquid chromatographic determination and biophamaceutical studies of 9-methoxycanthin-6-one from eurycoma longifolia jack

Abstract
A specific and sensitive reversed phase high performance liquid chromatography method using fluorescence detection was developed to determine quantitatively the plasma concentration of 9-methoxycanthin-6-one in biopharmaceutical studies. The HPLC method entailed direct injection of plasma sample onto the column after deproteinization using acetonitrile. The mobile phase comprised acetonitrile arid distilled water (55 : 45, v/v). Analysis was run at a flow rate of 1.0 ml/min with the detector operating at an excitation wavelength of 371 nm and emission wavelength of 504 nm. The linearity curve was in the range of 1.6 ng/ml to 1600 ng/ml with the detection limit of 0.6 ng/ml. The mean recovery was approximately 108 %. The accuracy and precision of between-day and within-day were all less than 12 %. The compound, 9-methoxycanthin-6-one was isolated from the roots of Eurycoma longifolia Jack by sequential extraction and acid-base extraction methods followed by various chromatographic techniques. The pure compound was characterised using ultraviolet, infrared and nuclear magnetic resonance spectroscopies. Biopharmaceutical studies were then conducted on the compound using rats. The oral absolute bioavailability of the 9-methoxycanthin-6-one determined using oral and intravenous administrations revealed that it was poorly absorbed, with bioavailability of less than 1 %. The mean T max and Cmax values obtained from the oral route were 0.50 ± 0.19 hr and 30.33 ± 10.61 ng/ml respectively while the mean volume of distribution estimated from intravenous data was 0.049 L/kg. The intravenous plasma profile of the compound exhibited a two-compartment phannacokinetic behaviour, with an initial fast distribution phase followed by a slower elimination phase. Finally, investigations were also carried out to determine various factors that could result in the poor oral bioavailability observed, which included studying the chemical stability of the compound and the influence ofP-gp and CYP3A4 in its absorption. The compound was found to be stable, with no appreciable degradation detected in pH 1, 4 and 7. From the in vivo study using ketoconazole as inhibitor of CYP3A4 and P-gp, the result revealed that 9-methoxycanthin-6-one might be a substrate of CYP3A4 and P-gp when administered orally but merits further studies using a larger number of rats.
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Keywords
Liquid chromatographic determination , 9-methoxycanthin-6-one
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