Enhancement of Selectivity and Sensitivity for Berberine Determination by Extraction-Fluorophotometry

Abstract

Berberine has been determined by extraction based on the formation of ion-association complexes with anionic dyes such as tetrabromophenolphthalein ethyl ester', sodium picrate2, and Bromophenol Blue.3 Procedures in ion-associate extractions with dyes often become complicated. These methods are poor in selectivity because of strong interferences caused by extractable amines(HR3N+) and quaternary ammonium compounds(R4N+). Recently, flow injection methods4'5 using thermo-spectrophotometry for Berberine and acetylcholine (R4N+) determination have been developed to eliminate interferences from amines, but it is difficult to eliminate completely interferences from some amines and other quaternary ammonium compounds. On the other hand, a simple ion-pair high-performance liquid chromatography using ODS-silica gel has been reported for Berberine determination in crude drugs and oriental pharmaceutical preparations .6 However, the method with UV detector was insensitive and time-consuming. A simple, selective and sensitive method needs to be devised for the determination of berberine in complicated samples.We found that berberine is almost completely extracted into 1,2-dichloroethane (DCE) in the presence of sodium perchlorate. Its fluorescence intensity in DCE phase is enhanced remarkably compared with that in aqueous phase. In addition, since the emission spectrum of berberine does not overlap with those of other extractable compounds, berberine can be determined simply, sensitively and selectively. ExperimentalReagents and apparatus A standard berberine solution (1 X 10-3 M=mot dm 3) was prepared by dissolving 0.0407 g of berberine chloride dihydrate (Nacalai Tesque, Inc.) in 100 ml of distilled water. Working solutions were prepared by accurate t Present address: Department of Chemistry , Chungbuk National University, Cheongju 360-763, Republic of Korea.dilution. A perchlorate solution (1X10-3 M) was prepared by dissolving 0.070 g of sodium perchlorate monohydrate in 500 ml. A borate-phosphate buffer solution was made from equal volumes of 0.3 M potassium dihydrogenphosphate and 0.1 M sodium borate and the pHs were adjusted with 1 M sodium hydroxide and 0.5 M sulfuric acid. 1,2-Dichloroethane (DCE) was used without further purification. A Shimadzu Model RF-5000 fluorometric spectrometer with a quartz cell (polytetrafluoroethylene stopper attached) of 10-mm path length was used for fluorescence measurements.Hitachi-Horiba Model F-711 pH meter, Iwaki Model KM shaker and Kubota Model KS-4000 centrifuge were used. General procedurePut 0.5 -25.0 ml of berberine standard solution (5.0X10-' M), 10 ml of buffer solution (pH 5.5) and 5 ml of 1.0X10-3 M perchlorate solution into a 50-ml volumetric flask. Dilute the mixture with water to 50 ml. Transfer the mixture into a 100 ml-separatory funnel and shake with 10 ml of DCE for 5 min. After separation, the emission intensity of the organic phase is measured at excitation (Ex.) and emission (Em.) wavelengths, 355.2 and 516.8 nm. Results and DiscussionThe fluorescence spectra of ...