Determination of Cinchona alkaloids and Vitamin B6 by high-performance liquid chromatography with fluorescence detection

A simple and specific method has been developed for the simultaneous determination of the four major Cinchona alkaloids and their dihydroderivatives and pyridoxine hydrochloride (Vitamin B6) by high-performance liquid chromatography (HPLC) with fluorescence detection ((lambda)em=420 nm with (lambda)ex=330 nm). The chromatographic separation was performed on a Phenomenex Prodigy ODS column (5 (mu)m, 250 mm×3.2 mm i.d.), recommended for basic compounds, under isocratic reversed-phase conditions. The method allowed a good peak shape and an effective resolution of the tested compounds. The extraction of alkaloids from the Cinchona succirubra bark was carried out in mild and fast conditions (ambient temperature, 20 min) by ultrasonication. The procedure showed to be advantageous respect to a reference method, which involved Soxhlet extraction. The results were compared statistically by means of the Studentʹs t-test and the variance ratio F-test; no significant difference was found. The method was reproducible (relative standard deviations in the range of 1.0–5.0% for the different alkaloids) and gave quantitative recovery of alkaloids added to bark samples (97.8–105%). For additional informations a photoreactor was arranged between the analytical column and the detector and the online post-column photochemical conversion (irradiation=254 nm) was investigated. Vitamin B6 was shown to be highly photosensitive, giving significantly different fluorescence spectra with and without UV irradiation. The proposed method was successfully applied to the quality control of Cinchona bark, liquid extract and cosmetics.