Studies On Drug Quality Control And Metabolism In Vitro

Dipfluzine hydrochloride(Dip) is a novel piperazines calcium antagonist, which were firstly composed in School of Pharmacy, Hebei Medical University. Dip selectively dilated vertebral artery, basilar artery and coronary artery. And the effects of Dip were more potent than those of flunarizine. In addition, Dip increased cerebral blood stream, improved brain ischemic injury, attenuated the brain infarct size induced by caremral ischemia, and inhibited platelet aggregation and thrombus formation. Dip may be potentially useful to treat ischemic brain injury. Nowadays, the study of Dip is in pre-clinical research, which has no reasonable or efficient standard to control its quality. Our research aims to study the quality of Dip and its preparation in order to establish a base of drawing a quality standard. The metabolites of Dip were studied by microbial transformation method.Clavulanate potassium is aβ-lactamase inhibitor, which is structurally related to the penicillins. It is a fermentation product of Streptomyces clavuligerus. It inactivates a wide variety ofβ-lactamases by blocking the active sites of theses enzymes. Nowadays, the determination of clavulanate potassium is usually tested by HPLC. In the present study, the simple, fast and sensitive capillary zone electrophoresis and high performance liquid chromatography-mass spectrometric(HPLC-MS) methods have been developed for determination of clavulanate potassium and its preparation.Part one Studies on quality control of dipfluzine hydrochlorideObjective: To study physical and chemical properties of dipfluzine hydrochloride which relate to its quality in order to draw a quality standard.Methods: (1) Physical and chemical properties :①Solubility : According to ChP(2005), the solubility of dipfluzine hydrochloride was investigated in ordinary solvent.②Melting point : The melting point of dipfluzine hydrochloride was determinined by using the method from ChP(2005). (2) Identification :①Chemical method : Take some dipfluzine hydrochloride, heat it in water. Keep it to be cold, add some alkaloid precipitating agent, and observe the phenomenon. Chloride and fluoride were identified by using the methods from ChP(2005).②U V : Take the sample and reference substance of dipfluzine hydrochloride, and dissolve it with ethanol absolutely to a concentration of 18μg/ml. Scan the solutions from 200nm to 400nm wavelength.③HPLC : Take some dipfluzine hydrochloride and make a solution having a known concentration of 0.1mg/ml with mobile phase. The mobile phase was buffer solution-acetonitrile-methanol(43:10:47, adjusted pH to 3.5 with phosphoric acid)..The detection wavelength was set at 230nm. Record the chromatogram and compare it with that of the reference. (3) Test :①Ordinary impurity : According to the appendix of the ChP(2005), the fluoride, residue on ignition and heavy metal was checked.②Related substances : HPLC was adopted to test the related substances in dipfluzine hydrochloride. (4) Assay : Nonaqueous potentiometric titration, UV and HPLC were used to determine dipfluzine hydrochloride, and the results of three methods were compared.Results: (1) The physical and chemical properties :①Solubility : dipfluzine hydrochloride can be easily dissolved in trichlormethane or methanol, dissolved in ethanol or acetoacetate, slightly dissolved in acetone or 0.1mol/L HCl, and can not be dissolved in water, benzene, aether and 0.1mol/L NaOH.②Melting point : dipfluzine hydrochloride's melting point was the range from 212℃to 215℃. (2) Identification :①Chemical method : The sample showed white praecipitatum when added alkaloid precipitating agent. The identification of chloride and fluoride showed a positive reaction.②UV : The sample had maximum absorption at 228nm and 243nm, being the same as the reference.③HPLC : Retention time of dipfluzine hydrochloride in chromatogram was the same as the reference. (3) Test :①ordinary impurity : After the test, the residue of dipfluzine hydrochloride was not more than 0.20%. The assay of heavy metal was not more than 10ppm. The content of fluoride was not more than regulation.②The related substances : Chromatography condition of HPLC was determined : the system was carried out using Restek C18 column and the mobile phase of buffer solution-acetonitrile-methanol(43:10:47, adjusted pH to 3.5 with phosphoric acid) with flow rate of 1.0ml/min and the detection wavelength was set at 230nm. The LOD was 2.0ng. (4) Assay : The results which we got from three methods were consistent. The assay of the sample was not less than 98.5%. At last, nonaqueous potentiometric titration was ascertained as the method of assay.Conclusion: The methods of identification, test and assay for dipfluzine hydrochloride were established, which provided a guideline with quality control.Part two Studies on quality control of dipfluzine hydrochloride for injectionObjective: To study the method of identification, test and assay for dipfluzine hydrochloride for injection in order to establish a base of drawing a quality standard.Methods: (1) Identification :①Chemical method : Take some sample into water, add some alkaloid precipitating agent, and observe the phenomenon. Chloride was identified by using the method from ChP(2005).②UV : Take the sample and reference substance of dipfluzine hydrochloride, and dissolve it with ethanol absolutely to a concentration of 18μg/ml. Scan the solutions from 200nm to 400nm wavelength.③HPLC : Take some sample and make a solution having a known concentration of 0.1mg/ml with mobile phase. The mobile phase was buffer solution-acetonitrile-methanol(43:10:47, adjusted pH to 3.5 with phosphoric acid). The detection wavelength was set at 230nm. Record the chromatogram and compare it with that of the reference. (2) Test :①Related substances : HPLC was adopted to test the related substances in dipfluzine hydrochloride for injection.②Content uniformity : The content uniformity of sample was tested by using the method from ChP(2005). (3) Assay : UV, HPLC and capillary zone electrophoresis were used to determine dipfluzine hydrochloride for injection, and the results of three methods were compared.Results: (1) Identification :①Chemical method : The sample showed white praecipitatum when added alkaloid precipitating agent. The identification of chloride showed a positive reaction.②UV : The sample had maximum absorption at 228nm and 243nm, being the same as the reference.③HPLC : Retention time of dipfluzine hydrochloride for injection in chromatogram was the same as the reference. (2) Test :①The related substances : The limit of impurity was 2.0%. The LOD was 2.0ng.②Content uniformity : The content uniformity of sample was consistent with regulation. (3) Assay : The results which we got from three methods were consistent. Excipients had no interference with the results. HPLC was determined to be a method to test the assay for its preparations.Conclusion: The methods of identification, test and assay for dipfluzine hydrochloride for injection were established, which provided a guideline with quality control.Part three Studies on metabolism of dipfluzine hydrochloride in vitroObjective: To find dipfluzine hydrochloride metabolites by means of microbial transformation and anaerobic culture of intestinal tract bacteria, and identify the chemical structures of the metabolites.Methods: (1) Microbial transformation experiment : Firstly, we selected a robust strain for the transformation of dipfluzine hydrochloride by screening several kinds of strain, then we got a pure transformation product and confirmed its structure by its MS data. (2) Culture solution of intestinal bacteria were produced from rat feces and then incubated with dipfluzine hydrochloride to find whether there were metabolites by HPLC-PDA.Results: Microbial transformation experiment : after screening, Sporotrichum sp., AS 3.2882 was selected to transform dipfluzine hydrochloride for its potential metabolic capability and good reproducibility. We got a metabolite of dipfluzine hydrochloride from AS 3.2882, whose chemical name was 1-diphenylmethyl-4-(4-fluoro phenyl-4-hydroxyl) piperazine. We found no metabolites in the culture solution of intestinal bacteria of dipfluzine hydrochloride.Conclusion: We got a pure product by microbial transformation and confirmed its structure.Part four Determination of clavulanate potassium and its preparations by capillary zone electrophoresisObjective: To estebalish a simple and rapid capillary zone electrophoresis method for the analysis of clavulanate potassium and its preparations.Methods: The determination was performed on the following conditions : uncoated fused–silica capillary column (50cm of effective length), running buffer : 20mmol/ L phosphate electrolyte solution (pH 6.0), running voltage : 25kV, sample injection method (20kV×5s), column temperature : 20℃, detection wavelength : 214nm.Results: The linear range of clavulanate potassium was 0.05208～0.3125mg/ml(r=0.9996, n=6), the average recovery of amoxicillin-clavulanate potassium(7:1) dispersible tablet was 99.2% with RSD of 0.77%(n=9), that of amoxicillin-clavulanate potassium(4:1) suspension was 99.4% with RSD of 1.0%(n=9), the LOD was 1.953μg/ml.Conclusion: The method is simple, rapid and accurate, it provides a new reliable means for the quality control of clavulanate potassium and its preparations.Part five Determination of clavulanate potassium and its preparations by LC-MSObjective: To establish an LC-MS method for the determination of clavulanate potassium and its preparations.Methods: The Restek C18 column was used, and the mobile phase was 5mmol/L amine acetate buffer-acetonitrile (90:10) at a flow rate of 0.7ml/min. Column temperature was 25℃. The analysis was detected by a negative electrospray ionization(ESI) method under multiple reaction monitoring (MRM) mode. Results: The linear range of clavulanate potassium was 10.33～ 1033ng/ml(r=0.9989, n=6), the average recovery of amoxicillin-clavulanate potassium(7:1) dispersible tablet was 98.5% with RSD of 1.2% (n=9), that of amoxicillin-clavulanate potassium(4:1) suspension was 99.0% with RSD of 1.1% (n=9), the LOD was 1.033ng/ml.Conclusion: The method is rapid and accurate, it provides a new reliable means for the quality control of clavulanate potassium and its preparations.