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Abstract
A simple and rapid reversed - phase high performance liquid chromatographic (HPLC) method using an ultraviolet detector for the analysis of tropane alkaloids in feedstuffs and biological samples is presented.
Hyoscyamine, scopolamine and the internal standard bamifylline are adsorbed on a C18 cartridge employing the solid - phase liquid extraction technique for sample clean up and subsequent separation of those compounds and internal standard from endogenous interfering compounds and preconcentration.
Chromatographic analysis is achieved on a Lichrosorb RP-18 10μm reversed phase column and the mobile phase is an isocratic mixture of acetonitrile, methanol and 0.05 M ammonium acetate (20.9 : 27.9 : 51.2) at a flow rate of 1.30 ml/min. The edited alkaloids are detected at 210 nm. The retention time is 3.990 min for scopolamine, 6.934 min for hyoscyamine and 8.750 min for the internal standard bamifylline. The correlation of the integrated peak area ratios of scopolamine and hyoscyamine to internal standard with the concentrations of those compounds showed a linear relationship between 1.20 to 60.0 ppm for scopolamine and 1.25 to 40.0 ppm for hyoscyamine. The linearity, employing the clean up technique, of scopolamine and hyoscyamine is 1.20 to 15.0 ppm and 1.25 to 20.0 ppm respectively per 20 μl injection. The absolute detection limits are 12.05 ng and 13.25 ng for scopolamine and hyoscyamine respectively.
The method outlined in this paper is applied to the simultaneous determination of scopolamine and hyoscyamine in feedstuffs (soya and corn) and biological samples (eggs, blood serum and urine).
