The Quality Control Of Pharmaceutical Manufacturing Process Based On Liquid Chromatography-tandem Mass Spectrometry

Objective:This dissertation was to focus on the quality control of pharmaceutical manufacturing processusing the ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)and to provide the strategies for the improvement of manufacturing process of drugs via the research data obtained by UPLC-MS/MS.The impurities in cefepime hydrochloride for injection were to be analyzed by UPLC-Q-TOF-MS/MS.The traceful suspicious chemical components in an antimicrobial creamwere to be detectedby UPLC-QQQ-MS/MS,and the adulterated herbal in Xiaohuoluo Pill were to be identified and the contents of the poisonous and active components in Xiaohuoluo Pill were to be measured by UPLC-MS/MS.The marker components of sulfur-fumigated Chrysanthemi flos(Juhua,a commonly used traditional Chinese herb)were also to be found out to distinguish the sulfur-fumigated or the unsulfur-fumigated Chrysanthemi flos by using UPLC-Q-TOF-MS/MS.The quality control of raw materials,the cleaning validation and the concocted processing of raw herbal materialsshould be paid more attention during the drug manufacturing process to ensure the batch stability and quality reproducibility for drugs.Methods:The UPLC-Q-TOF-MS/MS technique was used to analyze the impurities in cefepime hydrochloridefor injection.Impurities A,C and E recorded in USP42 were identified by retention time andprimary mass spectra.Other 10 unknown impurities with high response of mass spectrum in cefepime hydrochloride for injection were detected and subjected for MS/MSanalysis.Also,some degradation components in stress testing were identified and characterized,which was used to infer the molecular structures of the unkown impurities in cefepime hydrochloride for injection.The trace of betamethasone and dexamethasone introduced intoan antimicrobial cream during the manufacturing process was detected by UPLC-MS/MS.An UPLC-Q-TOF-MS/MS approach was applied to analyze the characteristic components of Paeoniae Radix,which was added illegally in Xiaohuoluo pill,and a HPLC-PDA method was established to detect Paeoniflorin in Paeoniae Radix.At the same time,we also establish an UPLC-MS/MS method for confirming Paeoniflorin component existed in Xiaohuoluo pill.An UPLC-MS/MS method with the multiple reaction mornitoring mode(MRM)was set to determinate aconitine,mesaconitine,hypaconitine and benzoylaconitine,benzoylmesaconine,and benzoylhypacoitine in Xiaohuoluo pill from the adulteration of peony roots.The UPLC-Q-TOF-MS/MS method was applied to research the effection of sulfur fumigation procedure on Chrysanthemi flos.There was a comparison of the two total ion chromatograms between the sulfur-fumigated and the unprocessed Chrysanthemi flos.Results:Impurity A,C,E and other 10 unknown impurities of cefepime hydrochloride for injection with high response were detected by UPLC-Q-TOF-MS/MS method.There were three isomer impurities.They were inferred as?₃isomer and(6-H,7-H)isomers of Cefepime with the same molecular formula of C₁₉H₂₄N₆O₅S₂.The other three impurities are the decarboxylation products of Cefepime losing a carboxy group at C2 position.The molecular formula of these three impurities were inferred as C₁₈H₂₄N₆O₃S₂.Impurity of m/z 370.0641 with formula C₁₃H₁₅N₅O₄S₂ was also detected,and it was inferred as(2RS)-2[[(Z)-2-(2-amino-4-thiazolyl)2-(methoxyimino)a cetamido]-methyl]-1,2,5,7-tetrahydro-7-oxo-4H-furo[3,4-d][1,3]thiazine in the literature.These seven impurities were inferred as degradation products of Cefepime.While the impurity of[M+H]⁺m/z428.0681 was inferred as the process imputity as the substitution product of Cefepime with the N-methypyrrolidine group at C3 position substituted by-CH₂OCH₃.The molecular formula of this impurity was inferred as C₁₅H₁₇N₅O₆S₂.Impurity of[M+H]⁺m/z894.1786 was inferred as polymer impurity of Cefepime with the molecular formula of C₂₃H₅₇N₈O₁₀S₉.An oxidative degradation products from the excipient arginine was also detected.The UPLC-MS/MS method used for the determination of betamethasone and dexamethasone in an antimicrobial cream showed a good linearity in the range of 1-41ng·m L^-1.Limits of quantitation(LOQ)and detection(LOD)of this method were12.5?g·kg^-1and 5?g·kg^-1,respectively.The method recoveries were from 100 to 108%.Four batches ofantimicrobial creams were detected to contain betamethasone ranging from84-1165?g·kg^-1,and dexamethasone was detected under LOQ in 2 batches of the creams.The microscopic characteristics of massive calcium oxalate crystals in paeonia lactiflora Pall that shouldn't be found in Xiaohuoluo pills were observed among six of sixteen batches of Xiaohuoluo Pills.Paeonia lactiflora is suspected to have been added into Xiaohuoluo pills.For studing this problem,the UPLC-Q-TOF-MS/MS method were applied to annlyze the characteristic components of Paeoniae Radix including paeoniflorin,albiflorin,4-epi-albiflorin,and Galloylpaeoniflorin.The four components of Paeoniae Radix were detected in the six batches of Xiaohuoluo pills withcalcium oxalate crystals,while not detected in Xiaohuoluo pills without calcium oxalate crystals.So the conclusion was obtained that roots of Paeonia lactiflora existed in those Xiaohuoluo pills.And Radix Paeoniae Alba(Baishao)was also found to be added into several products.The amounts of paeoniflorin in the six batches of Xiaohuoluo Pills ranged from 39.57?g·g^-1 to642.77?g·g^-1.For the adulteration of Paeony roots,UPLC-QQQ-MS/MS(MRM)method was applied to measure the contents of six kinds of aconitum alkaloids in Xiaohuoluo pills.Among sixteen batches of Xiaohuoluo Pills,the total amounts of aconitine,hypaconitine and mesaconitine ranged from 0.005?g·g^-1to 27.28?g·g^-1,and the total amounts of benzoylaconitine,benzoylhypacoitine,and benzoylmesaconine ranged from 45.57?g·g^-1to318.47?g·g^-1.There were significantly variations of the total content of Aconitum alkaloids among the researched Xiaohuoluo Pills,which indicated the quality of Xiaohuoluo Pillof different brands was not at the same level.The main components chlorogenic acid and isochlorogenic acid of Chrysanthemi flos were both partial sulfited.There were 11 new substances emerged in the sulfur-fumigated Chrysanthemi flos.The characteristic fragments of m/z 80 or m/z 81were produced by sulphurous acidpresented among the daughter scan spectrum of these 11 new substances.This phenomenon indicated the flowers were sulfur fumigated obviously.Conclusions:Eight unknown impurities in cefepime hydrochloride for injectionnot reported in literature were detectedby the UPLC-Q-TOF-MS/MS method,their molecular structures were derived from the obtained the information of mass spectra.The formation of these unkown impurities were preliminarily discussed,and the provided research data reminded that the quality of the excipient arginine should be controlled.The procedure causing to produce isomer impurities and the polymer impurities should be improved.And the procedure impurities also should be controlled strictly and marked in the specifications if necessary.The method established for quantification of trace dexamethasone and betamethasone in an antimicrobial creamwas sensitive and selective.It can be applied as the monitoring method for the cleaning procedure in the manufacture of this product and the supervision of the adulteration of glucocorticoids in creams of antimicrobial agent.The established HPLC-PDA method to inspect paeoniflorin emerged illegally in Xiaohuoluo Pills and the comfirmation mass sepetrometry as well as the quantitation method of six kinds of Aconitum alkaloids can be used for the quality control of Xiaohuoluo Pills.Identification of Sulfite compoundsin and the fingerprints of Chrysanthemi flos obtained by UPLC-Q-TOF mass sepetrometer can be used as a valid method to distinguish the sulfur fumigated flower from unfumigated one.It could also be used for the quality control of Chrysanthemi flos as raw materials in the production of Chinese materia medica preparation containing Chrysanthemi flos.