JOURNAL CLUB PRESENTATION (20L81S0703-PA)
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The document presents a journal club presentation discussing the role of Traditional Chinese Medicine (TCM) in drug discovery and development, emphasizing the importance of quality control. Fingerprinting analysis, particularly using mass spectrometry, is highlighted as a robust method for assessing TCM quality, identifying constituents, and distinguishing between normal and abnormal batches. The findings suggest that MRM-based fingerprinting offers superior sensitivity and robustness compared to conventional methods.
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JOURNAL CLUB PRESENTATION (20L81S0703-PA)
- 1. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1 Journal Club Presentation As a part of curricular requirement for M. Pharm II Year I Semester Presented by G.HIMABINDU (Reg.no:20L81S0703) Department of Pharmaceutical Analysis Under the guidance/mentorship of Mr. S. Shakir Basha M. Pharm Assistant Professor Department of Pharmaceutical Analysis
- 2. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2 Title, Author’s and Affiliations
- 3. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3 Introduction Materials and reagents Results and Discussion Authors conclusion My conclusion References Contents
- 4. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4 • Now a days Traditional Chinese medicine is not only used for primary health care but also in drug discovery and drug development. • TCM holds a significant promise for the treatment of chronic and multi factorial diseases such as diabetes and cardiovascular diseases. • TCM shows batch to batch variations due to their different geographical origin, post harvest processing, agricultural practices and manufacturing process. Introduction
- 5. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5 • Quality control therefore plays a crucial role in assuring the safety and effectiveness and consistency of TCM. • Fingerprinting analysis has been increased as a powerful tool for assessing quality and chemical equivalence of TCM. • For assessing the quality of TCM, chromatographic fingerprints still have some limitations. • For eg., the congruence coefficient have been applied to quantitatively compare the similarity between the two fingerprints.
- 6. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6 • Featured with high sensitivity and selectivity, mass spectrometry has emerged as an essential tool in traditional medicine research. • Among various MS scan modes, multiple reaction monitoring (MRM) is considered as a reliable method for detecting specific constituents of interest. • MRM based fingerprint approach to quality assessment of TCM, in which ideally hundreds of constituents could be simultaneously monitored.
- 7. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7 • TCM samples were first subjected to liquid chromato analysis (Q- TOF-MS) analysis for chemical identification. • Robustness of fingerprints was systematically investigated by four indices, i.e., Clustering analysis by principal component analysis(PCA). Similarity analysis by congruence coefficient. The number of separated peaks. The peak area proportion of separated peaks.
- 8. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8 • Qishen YiQi pill is a TCM drug product widely prescribed for cardiac dysfunction, which is derived from four herbs. • Astragalus membranaceus • Salvia miltiorrhiza • Panax notoginseng and • Essential oil from dalbergia odorifera • By using MRM fingerprinting to this complex preparation could detect different sorts of constituents in a sensitive and robust manner.
- 9. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9 • HPLC grade acetonitrile and formic acid from merk and roe respectively. • Ultra pure water was prepared by a milli-Q plus water purification system. • Some of other reagents used were all of analytical grade. • Reference standards including Materials and Reagents
- 10. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10 S.no Reference Standards 1. Caffeic acid 2. Salvianolic acid 3. Rosmarinic acid 4. Ferulic acid 5. Astragaloside IV 6. Notoginsenoside 7. Calycosin
- 11. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11 • Accurately weighed QSYQ (1 mg) were dissolved in 1ml of 10% methanol (V/V). • After vortexed for 30 s, centrifuged at 10,000 rpm for 10 min • The supernatants were subjected to the fingerprinting analysis. • Solutions of reference standards were individually prepared in methanol to corroborate chemical identification results. Sample Preparation
- 12. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12 Parameters Measurement Instrument Acquity UPLC system Coupled with a Triple TOF 5600 plus. Ionisation method Electrospray Scan range 100-1500 Source voltage -4.5 kV Source temperature 550 °C Curtain gas pressure 30 psi Nitrogen gas pressure 50 psi Instrumentation
- 13. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13 Instrument Agilent 1100 coupled with variable wavelength detector VWD wavelength 203 nm Column Zorbax SB C18 column Mobile phase A- 0.1% formic acid- water B- Acetonitrile Flow rate 0.4 ml/ min Elution mode Linear gradient Injector volume 10 μL
- 14. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
- 15. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15 • Robustness of fingerprints was evaluated in terms of clustering analysis similarity analysis number of separated peaks and the peak area proportion of separated peaks. • The standard angle cosine measure and angle cosine measure after min-max normalisation were used to calculate the similarity index. • The number of peaks represents the chemical information captured by analytical method. Robustness Evaluation
- 16. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16 Sample Information
- 17. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17 • By applying these approaches, a total of 100 constituents were identified from QSYQ including: 45 saponins 19 phenolic acids 14 flavonoids 1 anthraquinone 16 unambiguously • The compounds are identified by comparison with the reference standards in terms of retention times and mass spectra.
- 18. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18 • Multiple MS data processing methods were employed complimentarily for chemical identification. • Identification of peaks was assigned based on literature, data base matching and then further confirmed via MS2 fragmentations. • Besides MS2 databases there are: Global Natural Products Social molecular network(GNPS) MassBank Human metabolome database(HMDB) MassGraphs, were also used for direct compound identification. Results and Discussion
- 19. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19 • The technique was used to see whether normal/abnormal batches of QSYQ could be precisely distinguished by the two fingerprinting methods. • Normal samples from S1-S5 which were produced by different batches of raw materials could be clustered in to one group in PCA scores plot. • S6-S10 each with an abnormal herbal material were well differentiated from S1. Clustering Analysis by PCA
- 20. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
- 21. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21 • The congruence coefficient is widely adopted to quantitatively characterize the similarity between the two fingerprints by calculating the cosine of the angle. • Generally an average or set of samples serves as the reference fingerprint to compare with. • All the tested samples including the abnormal ones showed high similarity to the reference under the test conditions. • Above samples indicating that this algorithm was not suitable for differentiating the normal and abnormal samples in test condition. Similarity Analysis By Congruence Coefficient
- 22. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22 • NS reflects the resolution of a chromatographic method and it is a direct metric for robustness evaluation. • Similarly, PS represents the information reliably acquired by a fingerprint. • Peaks with resolution not less than 1 were counted in analyst or chem station, and their proportions were added to obtain peak area proportions. • Many minor peaks were overlapped or merged by larger peaks in UV fingerprint when the conditions varied. Evaluation By The Number And Peak Area Proportion Of Separated Peaks
- 23. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23 • In MRM mode, peak overlap and merger can be largely avoided as analytes are monitored in separate channels by the paired mass filters. • These results highlighted the superiority of MRM based fingerprinting method in terms of robustness, sensitivity and coverage. • Notably, the similarity indices between the reference and S6, S7 and S8 were all below 0.85.
- 24. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
- 25. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25
- 26. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 26 • Through the MRM approach, MRM transitions of herbal constituents could be rapidly constructed independent of chemical standards. • The proposed method was successfully used to assess the quality and capable of differentiating normal/abnormal samples in reliable and accurate manner. • The results of MRM based fingerprint possessed much better coverage and robustness than the conventional UV fingerprint. Authors conclusion
- 27. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 27 • Finally I conclude that, although angle cosine measure after min-max normalisation was proposed for similarity analysis herein for similarity analysis of MRM fingerprints. • More appropriate algorithms or indices need to be developed for such large scale fingerprints in which hundreds of constituents are detected. My conclusion
- 28. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 28 1. Wang, Z. Li, X. Zhao, et al., A network study of Chinese medicine xuesaitonginjection to elucidate a complex mode of action with multicompound, multi-target, and multipathway, Evid-based Compl. Alt. 2013 (2013), 652373. 2. S.L. Lee, J.H. Dou, R. Agarwal, et al., Evolution of traditional medicine to botanical drugs, Science 347 (2015) S32eS34. 3. H. Ge, M. Turhong, M. Abudkrem, et al., Fingerprint analysis of Cirsium japonicum DC. Using high performance liquid chromatography, J. Pharm Anal.3 (2013) 278e284. References
- 29. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 29 4. H.S. Xiong, L.X. Yu, H.B. Qu, A weighting approach for chromatographic fingerprinting to ensure the quality consistency of botanical drug products, Anal. Methods 6 (2014) 476e481. 5. H.Y. Fu, J.W. Guo, Y.J. Yu, et al., A simple multi-scale Gaussian smoothing- based strategy for automatic chromatographic peak extraction, J. Chromatogr. A 1452 (2016) 1-9. 6. Y.H. Lai, H.A. Chen, Z.Y. Qu, et al., The robustness test for the HPLC conditions of the fingerprint of notoginsenosides, Chin. J. Nat. Med. 4 (2006) 107-110.
- 30. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 30