Determination of synephrine and octopamine in bitter orange peel by HPTLC with densitometry

This paper presents the development and validation of an improved method for the simultaneous analysis of synephrine and octopamine using high-performance thin-layer chromatography with densitometric detection. Separation was performed on silica gel 60F254 plates. The mobile phase is comprised of methanol, ethylacetate, methylene chloride and concentrated ammonia (2:2:1:0.05, v:v:v:v). The Rf values were 0.292 ± 0.0083 and 0.413 ± 0.0089 for synephrine and octopamine, respectively (n = 9). Ultraviolet absorbance detection at 277 nm was used for the alkaloids detection. Specificity, accuracy (recovery rates were between 96 and 99%) and precision (in both cases intra-day precision and inter-day precision were ≤ 2.0%) of the method were determined. Their amounts were calculated using the regression equations of the calibration curves which were linear in the range 0.2-1.2 µg/spot. The amounts of alkaloids in basic methanolic extracts of bitter orange peel measured by the method were 0.253 and 0.142% for synephrine and octopamine, respectively. Most of the factors evaluated in the robustness test were found to have an insignificant effect on the selected responses at 95% confidence level. The method was validated giving rise to a dependable and high-throughput procedure well suited to routine application.