Quality Analysis Of Bendamustine Hydrochloride

Quality investigation and specification establishment are one of the key points for R&Dof chemical drugs. Quality investigation plays a very important role for the references andfoundations in quality specification establishment of chemical drugs. Firstly, qualityspecification is a series of test method and index that described for the quality guarantee ofchemical drugs. Secondly, it is a basic requirement for stabilization of realizing curativeeffect of chemical drugs, and meanwhile it is a basis of quality control of chemical drugs.Quality specification of chemical drugs is a technique method and index for investigatingquality control and we use these method and index to give a test for the purpose to controlquality of chemical drugs. Stability testing can not only provide important reference datafor the term of validity of chemical drugs but also for drug production, transportation,storage and clinic use. From two aspects of the quality investigation and stability testing ofactive pharmaceutical ingredient （API） of bendamustine hydrochloride that we studied forquality control and developing draft of quality specification in this text. It is a basic datafor the quality specification establishment of bendamustine hydrochloride.We studied quality investigation of bendamustine hydrochloride that includeddescription, identification, inspection and assay, respectively.Description: Test results showed that bendamustine hydrochloride is like white or whitepowder of osteoporosis with odorless and tasted bitter easy to absorbmoist. It can be easyto dissolve in methanol, DMSO, DMF and glacial acetic acid and also can dissolve inwater and ethanol. However, it displayed slightly soluble in acetonitrile while insoluble indiethyl ether, ethyl acetate and methylbenzene. The scale of melting point of bendamustinehydrochloride is148℃～151℃.Identification: In this text, we showed three kinds of method to indentify bendamustinehydrochloride that included chemical method, infrared spectrophotometry （IR） and highperformance liquid chromatography （HPLC）, respectively. Results showed thatbendamustine hydrochloride displayed characteristic phenomenon in identification ofchloride ion. API of bendamustine hydrochloride was accordance with reference substancein IR graph and the retention time between peak of test article and reference substance. It’sselective, sensitivity, repeatability and convenience of these methods for identification ofbendamustine hydrochloride. Inspection: Studied the items of solution clarity and color, acidity, absorbance, drugsubstances, residual organic solvent, loss on drying, water content and residue on ignition.The results indicated that the aqueous solution of bendamustine hydrochloride was clarifyand colorless and the pH of its aqueous solution was3.20³.30. Its maximum absorptionwavelengths were235nm and330nm. Each single impurity was lower than0.05%, theresult was qualified. In residual organic testing, we assayed the contents of tetrahydrofuran,ethyl acetate, methanol, dichloromethane and methylbenzene, respectively. What’s more,the methodology was evaluated. The results turned out that the organic solvents mentionedabove were all within the scale of limitation and analytical procedure was valid. The resultof water content test was0.60%¹.50%. Loss on drying was tested at a constanttemperature of105℃and the loss weight of bendamustine hydrochloride was2.80%⁴.85%. The result of residue on ignition showed that the residue of bendamustinehydrochloride after burning of high temperature was0.15%⁰.20%.Assay: Two different kinds of method we used in this study for content assay wereHPLC and non-aqueous titration. The result of HPLC method for content was99.77%¹00.76%that without water content while the content was99.75%¹00.51%innon-aqueous titration method, respectively. Compared to HPLC for content assaybendamustine hydrochloride, non-aqueous displayed almost the same result. It can beconclude that both of two methods could be used to assay bendamustine hydrochloride.Information on stability of the bendamustine hydrochloride is an integral part of thesystematic approach to stability evaluation. Stability testing of bendamustine hydrochlorideincluded stress testing, acceleration testing and long term testing.Stress testing: Stress testing helps to determine the intrinsic stability of the molecule byestablishing degradation pathways in order to indentify the likely degradation products andto validate the stability indicating powder of the analytical procedures used. API powder ofbendamustine hydrochloride was placed under conditions of high temperature at60℃,high humidity at25℃/RH90%±5%and strong light at4500lx±500lx, respectively.And then we took the samples placed under the condition mentioned above at the5thdayand10th day to test the stability, respectively. The contents that we tested includedhygroscopicity, water content, inorganic impurities and content assay. From the data ofstress testing, we concluded that the solid powder of bendamustine hydrochloride wasstable under the conditions described above.Accelerated testing: API solid powder of bendamustine hydrochloride tested under conditions at40℃±2℃/RH75%±5%during six months storage. Accelerated testinghelps to determine the intrinsic stability of the molecule by accelerating chemical orphysical changes of drugs in order to provide necessary references for design, package,delivery and storage for preparation. We conducted for items of water content, contentassay and drug substances of bendamustine hydrochloride during the storage of the1thmonth,2thmonth,3thmonth and6thmonth, respectively. It turned out that API solidpowder of bendamustine hydrochloride was stable under the conditions mentioned aboveduring six months storage.Long term testing: The long term testing will be continued for a sufficient period oftime beyond9months to cover all appropriate re-test periods, under the furtheraccumulated data can be submitted to the Authorities during the assessment period of theRegistration Application. The solid powder of drugs storage under conditions at25±2℃/RH60%±10%beyond9months. The contents that conducted were the same asaccelerated testing during the storage of the0thmonth,3thmonth,6thmonth and9thmonth.API solid powder of bendamustine hydrochloride was stable under the conditionsmentioned above beyond9months storage.Electro-spray ion trap mass spectrometry: Bendamustine hydrochloride was directlyinjected into the instrument and the fragments of the samples were yielded by usingmulti-stage ion trap mass spectrometry. It mainly gave characteristic fragment ions by theelimination of H₂O、CO from the carboxyl and HCl, CH₃Cl, CH₂=CHCl from the nitrogen（N） of5-carbon at the positive ion mode for bendamustine hydrochloride. Compared tonegative ion model, it was easy to crack to a series of ion fragments at the positive modefor bendamustine hydrochloride. These results provide very important information for thestructural modification and the drug metabolic research of bendamustine hydrochloride.We studied degradation compounds from bendamustine hydrochloride in the process ofclinical administration and confirmed their structures. Analyze the degradation ofbendamustine hydrochloride aqueous solution of sodium chloride （0.5mg/mL） with theincrease of time under the condition of room temperature. The information of molecularweight of degradation compounds was obtained by the system of Thermo Scientific LCQFleet LC/MSⁿ. The elemental composition of the degradation compounds was acquired byThermo Scientific Q Exactive LC/MS². Degradation compounds of HP1and HP2increased by the time that bendamustine hydrochloride aqueous solution of sodiumchloride placed under the condition of room temperature. The molecular weights of HP1 and HP2were339and321that obtained by LCQ Fleet LC/MSⁿ, respectively. Elementalcomposition of HP1and HP2were C₁₆H₂₃O₃N₃Cl and C₁₆H₂₄O₄N₃that acquired by theequipment of Q Exactive LC/MS²that it is high solution and sensitivity. And the structuresof the degradation compounds were confirmed at last. Bendamustine hydrochloride proneto degradation compounds in the process of clinical administration and producedmonohydroxy compound of HP1and dihydroxy compound of HP2which are hydrolysisproducts. We suggest that bendamustine hydrochloride injection should be ready preparedand injected within the shortest time for the purpose of safe and effective of clinicalmedications.We draw draft quality standard of bendamustine hydrochloride based on the qualityinvestigation. The draft quality standard of bendamustine hydrochloride was stated in theappendix.