The Study On Quality Control And Stability Of The Innovative Drug Leonurine

The research on quality control and stability of drugs is an important part of drug pre-clinical studies. During the study on drug quality standards, it needs to do a check of drug quality to guarantee the safety and reliability of drugs, and guarantee the smooth progress of pre-clinical research, as well as the safety of post-marketing drugs. Drug storage conditions and validity of drugs are determined by the stability.According on the study on drug quality standards and stability, the contents and methods of the quality standards and stability of leonurine and the draft quality standards of leonurine were established. By elemental analysis, UV, IR, NMR (13C, 1H) and mass spectrometry for structural confirmation of leonurine, reliable raw material for the study on quality standards and stability was provided. The work was divided into the following two parts:the first part is as the main part titled the study on quality control and stability of the innovative drug leonurine, the second part as the deputy papers titled the pre-column derivatization technique applied in biological samples.The first partThe study on quality control and stability of the innovative drug leonurine 1、The quality control of leonurineThe quality standard of leonurine was investigated according to Chinese Pharmacopoeia and technical specifications of the drug quality standards. According to the structure and property of the leonurine, we selected the appropriate method for its quality control. The items of solubility, melting point, rotation, identification, examination of inorganic impurities, moisture, weight of moisture absorption, residue on ignition et al were studied to determine the appropriate test methods and results.The methods of residual organic solvents inspection, the related substances inspection and assay were established for the first time. Verification methodology was conducted, and was used for the determination of relevant contents of leonurine.The results indicated that organic solvent residues of leonurine met Chinese Pharmacopoeia requirements. The results of related substances inspection were between 0.89-0.98% and the main structure of the related substances was speculated. The leonurine content determined by gravimetric method, nonaqueous UV and high performance liquid chromatography were greater than 99%. 2、The stability study of leonurineStability of leonurine was conducted according to the guiding principles of drug stability test. Impact factors test, accelerated test and long-term test were carried out respectively. The sample characteristics, the main component content and related substances content of stability samples were analyzed.It’s found that leonurine is insensitive to light, temperature and humidity, but for leonurine has little moisture absorption, it can’t be stored in humidity. After the accelerated tests at 40℃, humidity of 75%, and long-term test at 25℃, humidity of 60%, the related substances content increased to 0.94% and 1.17% and main component content were decreased to 99.04% and 99.08%.For statistical analysis on the validity, if main component content of leonurine becomes 98.5%, the retention time is 12.3 months. So the leonurine is valid for 12.3 months.Leonurine should be stored under the condition of room temperature, dark and dry.3、The draft quality standards of leonurine and drafting instructionsThe draft quality standards of leonurine and drafting instructions were formulated and drafted according to the first two chapters of the research content. This chapter provides norms and basis for quality control of leonurine.The second part the pre-column derivatization technique applied in biological samples.In this study, we investigated a simple, sensitive and reliable liquid chromatography-fluorescence detection method for memantine hydrochloride in rat plasma which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-C1). For the first time, FMOC-C1 was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (IS, internal standard) were trapped with FMOC-C1 to form memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-C1 compositions, which can be very compatible for LC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C18 column (DIAMONSIL 150X4.6 mm, id 5μm) with a mobile phase consisting of acetonitrile and water at a flow-rate of 1.0 mL/min. The retention times of memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in rat plasma, the linear range was 0.025-5.00 mg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 4.5-10.5% and 7.5-14.3%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples. The pharmacokinetics of memantine hydrochloride was studied in six male Wistar rats following intragastric administration, and pharmacokinetic parameters were carefully estimated.