| ObjectiveBased on the innovation application of Watson LIMS management system and most advanced UPLC-MS/MS technology,a fast and sensitive method was to be developed for simultaneous determination of diphenoxylate and its active metabolite diphenoxylic acid in human plasma.Then the method should be validated and applied to a clinical pharmacokinetic bioequivalence study of compound diphenoxylate tablets in fasting conditions in healthy subjects.Methods1.Detection was performed by electrospray ion source in the positive mode?ESI+?,with the transitions of diphenoxylate?m/z 453.3?187.1?,diphenoxylic acid?m/z425.2?249.2?,diphenoxylate-d4?m/z 457.3?191.2?,diphenoxylic acid-d4?m/z429.3?253.2?,respectively.The analytes were isolated on a Thermo BDSHYPERSIL C18?50 mm×2.1 mm,2.4?m?,the mobile phases for eluting werethe mixture of water with 0.1%formic acid coupled with 4 mM ammoniumacetate?mobile phase A?and acetonitrile?mobile phase B?.2.The bioanalytical methods was Validated according to the CFDA,FDA,EMEAguide,domestic and foreign references.The validation is about the specificity,selectivity,accuracy,precision,extraction recovery of the method,and stability ofbiological samples under different storage conditions during the analysis wereinvestigated.3.The randomized,open,double-crossover,dual cycle,single oral dose clinical trialwas completed and Cooperated with the First Affiliated Hospital of HenanUniversity of Science and Technology.Plasma samples were collected at differenttime points from 0 to 72 hours before and after administration.The bioequivalenceof the compound diphenoxylate tablet after oral administration in the fasting stateof healthy subjects was assessed according to the pharmacokinetic parameterscalculated by industry standard software Phoenix WinNonlin.4.The Watson LIMS information management system was introduced into thisresearch,responsible for the process management and data management of theproject.participating in the establishment and verification of bioanalyticalmethods,the design of clinical trials,the management of biological samples,andthe integration of data results.Results1.The UPLC-MS/MS method established in this study is simple,rapid and sensitive,and the separation of diphenoxylate and diphenoxylic acid was achieved in 5 min.The lower limit of quantitation?LLOQ?of diphenoxylate and diphenoxylic acid is0.1 ng/mL and 0.5 ng/mL respectively.The retention time of diphenoxylate was2.25 min,which was linear in the concentration range of 0.1 ng/mL10 ng/mL,and the retention time of diphenoxylic acid was 1.95 min,the linearity is goodranging from 0.5 ng/mL to 50 ng/mL.And the correlation coefficients of the twoanalytes are both above 0.99.2.The method has good selectivity and specificity for diphenoxylate anddiphenoxylic acid,and there is no residual or matrix effect exist.The intra-assayaccuracy of diphenoxylate ranged from-3.31%to 9.04%,CV%<8.14%;Inter-assay accuracy was 3.95%to 7.64%,CV%<5.27%.The intra-assay accuracyof diphenoxylic acid is in the range of-6.42%8.46%,CV%<8.18%,theinter-assay accuracy is-1.24%2.20%,and the inter-assay CV%<7.08%,whichproves that the method has good repeatability.In addition,the extraction recoveryrate for the diphenoxylate and diphenoxylic acid and the corresponding internalstandard were both higher than 90%,which proved that the extraction efficiency ofthe analytes and the internal standard were great.At the same time,the stabilityresults proved that the diphenoxylate and diphenoxylic acid were stable undervarious storage conditions during the treatment.3.The pharmacokinetic parameters and equivalent analysis results of the 24 subjectsin the fasting study were as follows:the 90%confidence interval of diphenoxylatewas 80.65%91.56%,the 90%confidence interval of the AUC0-t-t was89.97%100.02%,the 90%confidence interval of the AUC?0-??was92.08%103.25%;As for the active metabolite diphenoxylic acid,the 90%confidence interval of Cmax was 82.33%101.16%,the 90%confidence interval ofAUC0-t ratio is 86.68%100.77%,and the 90%confidence interval of the AUC?0-??was 85.74%100.53%.The above results show that 90%confidence interval ofCmax,AUC0-t,and AUC?0-??</sub>the reference of diphenoxylate and its activemetabolites diphenoxylic acid are all within the range of 80%to 125%,whichconform the standards of CFDA,FDA,EMEA.Therefore it is considered to bebioequivalent between the test preparation and the reference preparation of thecompound diphenoxylate tablet.4.The introduction and application of Watson LIMS information managementsystem significantly improved the efficiency and quality of the method validation,biological sample analysis and data integration management.Watson LIMS madethe project more regulatory compliance and traceability,and ensured the analysisdata accurate and true.Conclusion:For the first time,the rapid and sensitive UPLC-MS/MS method was established to simultaneously determine diphenoxylate and diphenoxylin in plasma,which was successfully applied to the pharmacokinetic study of diphenoxylate in healthyvolunteers.At the same time,the Watson LIMS information management system was introduced innovatively,and the bioequivalence study of compound diphenoxylate tablets in the fasting state of healthy subjects was completed with high efficiency and high quality.This study provides a template and reference for technological andmanagement innovation and internationalization of standards for bioequivalence research in small and medium-sized laboratories in China. |
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