ADVANCED BIOANALYTICAL TECHNIQUES.pptx

ADVANCED BIOANALYTICAL TECHNIQUES USE FOR VARIOUS SAMPLE DETECTION AND REASERCH PURPOSE

1. Presented by: Mr. Pratik S. Kapase M.Pharm 1 st Year (2 nd Sem) (Pharmaceutical Chemistry) ADVANCED BIOANALYTICAL TECHNIQUES PUNE DISTRICT EDUCATION ASSOCIATION’S Seth Govind Raghunath Sable Pharmacy College, Saswad Guided by: Dr. Smita J. Pawar H.O.D (Pharmaceutical Chemistry) 11-06-2023 1

2. CONTENTS 11-06-2023 2

3. LEARNING OBJECTIVE 11-06-2023 3

4. INTRODUCTION Bioanalysis is covering the identification and quantification of analytes in biological samples. Bioanalysis is not only measuring of small molecules such as drugs and metabolites but also to identify large molecules such as proteins and peptides. Bioanalysis has an important role to perform the toxicokinetic (TK), pharmacokinetic (PK) and pharmacodynamics (PD) studies of new drugs. Bioanalysis is also established in clinical, preclinical and forensic toxicology laboratories. It is growing rapidly and providing new challenges for the analytical chemists. These challenges have been resolved by the introduction of bioanalytical technique as a modern approach to disease diagnosis and therapy. 11-06-2023 4

5. BIOANLYTICAL METHOD DEVELOPMENT AND VALIDATION Today bioanalysis is an essential part in toxicological evaluation and in pharmacokinetic and pharmacodynamics studies during drug development. Additionally bioanalytical method validation is a crucial for the quantitative determination of various types of analytes in biological matrices. In pharmaceutical research companies the development of comprehensive bioanalytical methods is very important during the process of drug discovery and development The bioanalysis procedure includes 11-06-2023 5

6. CLASSIFICATION OF BIOANALYTICAL TECHNIQUES Spectrophotometry Microscopy Immunological & Radioisotopes techniques Hyphenated separation techniques Protein precipitation Ligand binding assay Chromatography Extraction techniques 11-06-2023 6

7. ADVANCED BIOANALYTICAL TECHNIQUES Spectrophotometry: Absorptions and Reflectance spectroscopy Near infrared techniques (UV-VIS-NIR techniques) Photoacoustic spectroscopy (PAS) Raman and infrared spectroscopy Imaging techniques: Computed Tomography (CT) Magnetic Resonance Imaging (MRI) Magnetic Resonance Spectroscopy (MRS) Positron-emission Tomography (PET) Single Photon Emission Computed Tomography (SPECT) 11-06-2023 7

8. ADVANCED BIOANALYTICAL TECHNIQUES 2) Microscopy: Confocal laser scanning microscopy (CLSM) Total internal reflection fluorescence (TIRF) microscopy 3) Immunological & Radioisotopes techniques: Radioimmunoassay (RIA) Enzyme immunoassay (EIA) Fluro immunoassay (FIA) Chemiluminescence immunoassay (CLIA) Liposome immunoassay (LIA) Cloned enzyme donor immunoassay (CEDIA) Flow injection immunoassay (FIIA) methods Capillary electrophoresis immunoassay (CEIA) 11-06-2023 8

9. ADVANCED BIOANALYTICAL TECHNIQUES 4) Hyphenated separation techniques: LC-MS Tanden MS-HPLC OR MS-UHPLC GC-MS CE-MS 5) Protein precipitation 6) Ligand binding assay 7) Chromatography: a) HPLC-UV/VIS b) Super critical fluid chromatography 8) Extraction Techniques Liquid-Liquid Extraction Solid-Phase Extraction 11-06-2023 9

10. LC-Tandem MS BIOANLYTICAL TECHNIQUIES LC-MS/MS, or Liquid Chromatography-Tandem Mass Spectrometry, is an analytical technique that combines the separation power of liquid chromatography (LC) with the sensitive and selective detection capabilities of mass spectrometry (MS). LC-MS/MS is commonly used in various fields, including pharmaceuticals, clinical diagnostics, environmental analysis, and forensic science, for the identification, quantification, and characterization of compounds in complex mixtures. Principle: The principles of LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) involve the combination of two powerful analytical techniques: liquid chromatography (LC) and tandem mass spectrometry (MS/MS). The LC-MS/MS system offers enhanced selectivity, sensitivity, and specificity for the analysis of complex samples. 11-06-2023 10

11. PRINCIPAL OF LC-MS/MS Liquid Chromatography (LC): Separation Mobile Phase Retention Time Mass Spectrometry (MS): Ionization Mass Analysis Detection Tandem Mass Spectrometry (MS/MS): Precursor Ion Selection Fragmentation Product Ion Analysis Multiple Reactions Monitoring (MRM) 11-06-2023 11

12. Quantification and Data Analysis: Calibration Curves: Known concentrations of standard compounds are used to construct calibration curves relating ion intensity or peak area to the concentration of the analytes. Quantification: The concentration of analytes in the sample is determined by comparing the ion intensities or peak areas of the sample against the calibration curves. Data Analysis: Specialized software is used for data processing, including peak integration, quantification, identification, and reporting of results. 11-06-2023 12

13. INSTRUMENTASION OF LC-MS/MS HPLC CONSIST LC PART : Solvent system (mobile phase) Pumps ( up to 6,000 psi) Mixer Injector Column (fast LC 15-50mm)(micro LC 20-150mm) Mass spectrometer Ion sources Electrospray ionization Atmospheric pressure chemical ionization Atmospheric pressure photoionization Mass Analyzer Quadrupole Time of flight Ion trap Fourier transform-ion cyclotron resonance 11-06-2023 13

14. INSTRUMENTASION OF LC-MS/MS 11-06-2023 14

15. LC-MS/MS is bioanalytical method used for the quantification of Encorafenib and Binimetinib as a first line treatment for advanced melanoma Optimization of Chromatographic Conditions and MS Detections The present study aimed to develop and validate a fast and sensitive method to quantitatively determine ENF and BNB in rat plasma. Presentative total ion chromatograms for blank rat plasma spiked with spebrutinib (IS) at a concentration of 100 ng/mL and overlays of the LC–MS/MS analysis of binimetinib (1.14 min), encorafenib (1.86 min) at concentrations of 0.5–3000 ng/mL and IS (0.73 min) at a concentration of 100 ng/mL. Multiple reaction monitoring (MRM) mass spectra and the expected fragmentation pathway of encorafenib, binimetinib, and spebrutinib (IS) Representative total ion chromatograms of rat plasma spiked with LLOQ (A), MQC (B), and HQC (C); for encorafenib (1.86 min), binimetinib (1.14 min) and IS (0.73 min). 11-06-2023 15

16. LC-MS/MS is bioanalytical method used for the quantification of Encorafenib and Binimetinib as a first-line treatment for advanced melanoma A newly developed and fully validated LC-MS/MS bioanalytical assay was used to analyze ENF and BNB in rat plasma. The developed wide range of calibration curves of the proposed assay allowed efficient quantitation of pharmacokinetic parameters after oral administration of binimetinib (3.8 mg/kg) and encorafenib (20 mg/kg). The present approach is distinguished by appropriate extraction recovery with the lack of matrix interference. Results also confirmed the high sensitivity of the developed method as low as 0.2 ng/mL with a total run time of 2 min, which rendered the developed assay applicable for effective routine assays in pharmacokinetic studies 11-06-2023 16

17. CONCLUSION By combining the separation power of LC and the specificity of MS/MS, LC-MS/MS provides a robust and sensitive analytical technique for the identification and quantification of compounds in various applications, such as pharmaceutical analysis, clinical diagnostics, environmental monitoring, and metabolomics. 11-06-2023 17

18. AKNOWLEDGMENT First of all, I would like to thank our Principal Dr. R. S. Chavan for giving me opportunity to present my seminar & giving me your valuable time. I would like to thank Dr. S. J. Pawar , Vice Principal & HOD, Pharmaceutical Chemistry for giving me opportunity to present my seminar and guiding me for selection and presentation of my seminar topic for his motivation to present my seminar. Also, I would like to thank Dr . A. P. Kale, Prof. J. R. Jagtap & Prof. G. B. Nigade for their motivation. Last but not the least, I would like to thank my seniors & my friends for contributing their time to listen me & supporting me. 11-06-2023 18

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20. REFERENCE Hussain, M.R.M.; Baig, M.; Mohamoud, H.S.A.; Ulhaq , Z.; Hoessli , D.C.; Khogeer , G.S.; Al-Sayed, R.R.; Al- Aama , J.Y. BRAF gene: From human cancers to developmental syndromes. Saudi J. Biol. Sci. 2015, 22, 359–373. [CrossRef] [PubMed] Long, G.V.; Eroglu, Z.; Infante, J.; Patel, S.; Daud, A.; Johnson, D.B.; Gonzalez, R.; Kefford, R.; Hamid, O.; Schuchter, L.; et al. Long-Term Outcomes in Patients With BRAF V600-Mutant Metastatic Melanoma Who Received Dabrafenib Combined with Trametinib. J. Clin. Oncol. 2018, 36, 667–673. [CrossRef] [PubMed] 11-06-2023 20

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