Studies On Applying Mass Snectrometry And Microfluidic Chip To The Quality Assessment Of Traditional Chinese Medicine

Quality control plays a critical role in modernization and internationalization of traditional Chinese medicines(TCMs).and is critical for ensuring the safety,efficacy and consistency of TCMs.With years of development,great progress has been made in this realm,where chemical testing is currently the prevailing approach used for quality assessment of TCMs.However,due to their inherent complexity,quality assessment of TCMs still confronts with significant challenges,including assay performance and cost,and clinical relevance.which hampers the improvement of TCM quality standards.Generally,TCMs contain a myriad of diverse phytochemicals,thus quantification of one or several specific constituents of interest cannot fully account for the efficacy and quality.Therefore,chemical testing alone may not be sufficient to ensure quality and thus therapeutic consistency of TCMs.As a result,there has been increasing interest in developing biological assays that correlate with the drug's intended mechanism of action or clinical effect to complement the chemical tests.However,due to the uncertainty in the active constituents and mechanism of action,both chemical and biological tests confront with challenges,including assay performance and indicator selection.Moreover,due to the inherent distinction in analytes and protocols,it is challenging to conduct chemical and biological assays in parallel simultaneously.Therefore,how to integrate chemical and biological tests and thereby developing clinically relevant assays for quality assessment of TCMs,has become a critical technology issue in TCM studies.Since its emergence,biomarker is increasingly perceived to be an essential tool in drug development,regulation and clinical research.This trend has also led to the emergence of many research strategies and analytical techniques,which show great potential in bioassay studies of TCMs.Thus,employing techniques in biomarker researches would be a feasible and convenient way to develop testing methods for TCM quality assessment.In this work,we proposed a microfluidic chip-based approach and derivative multiple reaction monitoring(MRM)fingerprinting to assess the biological activity and chemical consistency of TCMs,respectively.Furthermore,we successfully integrated chemical and biological assays on direct analysis in real time-mass spectrometry(DART-MS)by using novel mass spectrometry probes,which facilitated rapid,sensitive,accurate and multiplexed analysis for biological activity and chemical consistency of TCMs.The main contents of this paper are summarized as follows:1.We developed a multiple biomarker assay to assess the biological activity of TCMs using microfluidics,where enzyme inhibition was employed to indicate the drug's activity and thereby evaluate drug quality.This approach was exemplified on QiShenYiQi Pills using thrombin and angiotensin converting enzyme as "quality biomarkers".Our results demonstrated that there existed variations in activity across different batches of the intermediates and preparations.Compared with chromatographic fingerprinting,the bioassay provided better discrimination ability for some abnormal samples.Moreover,the chip could function as "affinity chromatography" to identify bioactive phytochemicals bound to the enzymes.2.We proposed a derivative multiple reaction monitoring(MRM)-based fingerprinting method for consistency assessment of TCMs.MS2 of QTOF-MS produced major MS parameters for the derivative MRM on QqQ-MS,which offered rapid and direct transition design independent of chemical standards.The method exhibited high robustness and sensitivity in the case study of QiShenYiQi Pills,and was applicable to quality assessment of TCMs3.To address critical issues in biological tests of TCMs,which includes multiplexed detection and integration with the chemical tests,we proposed a DART-MS based method to assess the quality of TCMs,where chemical consistency and biological activity were simultaneously analyzed by MS fingerprinting and a multiple-enzyme assay,respectively.Moreover,novel enzymatic probes labeled with a 1-(2-pyrimidyl)piperazine moiety were designed for quantitative enzyme activity measurements,which not only displayed dramatic enhancements in MS sensitivity with up to 125-fold increase compared to unlabeled ones,but also facilitated the multiplexed enzyme assay.The method exhibited high efficiency and reliability in the case study of Danshen Inj ection with a total analysis time of just minutes per sample.