| Diabetes is a series of heterogeneous metabolic diseases with the feature of chronic high blood sugar, which is caused by absolute or relative lack of insulin for some reason and characterized by chronic high blood sugar with carbohydrates, and fat and protein metabolism disorder. With people’s living standards improving year by year, the number of patients is also increasing. Currently, there are four categories of oral diabetes drugs in the research phase, and they are oral insulin preparations, insulin secretion, insulin sensitizers and a-glucosidase inhibitors.Voglibose is a new type of a-glucosidase inhibitors, which is developed and listed in1994by Japan Takeda Pharmaceutical Industries, Ltd., and named Basen. Compared with other a-glucosidase inhibitors, voglibose has high activities, small doses and few side effects in the gastrointestinal tract. Due to no conjugate structure and chromophore group in the molecular, the voglibose could not be detected by analytical methods of HPLC/UV and HPLC/FLD. Domestic and foreign researchers have used the analysis methods of HPLC/UV, HPLC/ELSD, HPLC/RID, pre-column derivatization-GC, pre-column derivatization-HPLC and HPLC post-column derived fluorescence. Compared with other analytical methods, HPLC post-column derived fluorescence analysis is of better selectivity and higher sensitivity, which makes it suitable for the quality control of voglibose and other preparations.In this study, a HPLC post-column derivatization fluorescence method is established for the quality control of voglibose, which may have impact on the testing of post-column derivatization factors for the peak area of voglibose.This study is divided into two parts:the first part is mainly focused on the establishment and optimization of the HPLC post-column derived fluorescence analysis method; the second part is on the study of factors derived in the HPLC column. The main contents of these two parts are as follows:1Establishment and optimization of the HPLC post-column derivatization fluorescence methodZorbax SB-aq4.6*250mm5μm column is applied, with phosphate buffer containing different concentrations of sodium octanesulfonate as the mobile phase, fluorescence detector(excitation wavelength of350nm and emission wavelength of430nm, the PMT is set to16), taurine and sodium periodate as derivatization reagent, reaction bath temperature of100℃, the derivative reaction tube length of20m (inner diameter,0.5mm), cooling temperature of15℃and the cooling pipe length of2m (internal diameter,0.3mm).2Study of factors derived in the HPLC columnThis section uses the single-factor experimental design to examine the T-shaped three-way connection, the reaction temperature, the cooling temperature, derivatization reagent concentration, flow rate of voglibose and the mobile phase, and other post-column derived factors on the voglibose peak area. The conclusions are as follows:The T-shaped three connection has few effect on the voglibose peak area, but certain impact on the theoretical plates.Within a certain range, the voglibose peak area increases with the temperature of the reaction bath.Within a certain range, the cooling temperature has no effect on the voglibose peak area.The voglibose peak area is getting larger first and then smaller in accordance with the increasing derivatization reagent concentration, and larger with the concentration of voglibose increasing. The flow rate of mobile phase and the derivatization reagent have significant influences on the voglibose peak area. |
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