JOURNAL CLUB PRESENTATION (20L81S0713-PA)
The document describes the development of a new magnetic solid phase extraction (MSPE) adsorbent called polyDOPA@Ag-MNPs for the analysis of trace beta-blockers in biological samples. PolyDOPA@Ag-MNPs were synthesized by reducing silver ions on the surface of magnetic nanoparticles coated with poly(3,4-dihydroxyphenylalanine). The adsorbent was able to isolate beta-blockers from sample matrices using a magnetic field. Optimization of the MSPE method identified pH 7, 2 minutes adsorption time, 4 mg polyDOPA@Ag-MNPs, methanol containing 1% acetic acid as the eluent, 2 minutes elution
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JOURNAL CLUB PRESENTATION (20L81S0713-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 Presented by Ms. M. Sudheeshna (20L81S0713) Department of Pharmaceutical Analysis Under the guidance of Mr. Kranthi Kumar, M.Pharm Assistant Professor Department of Pharmaceutical Analysis As a part of curricular requirement for M. Pharm 2nd year 1st semester Journal Club Presentation
- 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, Authors 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 Methods • Results and Discussion • 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 • β-blockers are widely used to treat various cardiovascular disorders such as hypertension, heart arrhythmias, and ischemic heart disease. • Apart from their clinical uses and therapeutic values, β-blockers are known to improve heart function by relaxing heart muscles and reducing heart rate. • They are often misused by athletes in professional competing sports, fatal adverse drug events following the use of β-blockers have been observed in hospitalized patients in internal medicine. • The inherent relevance of β-blockers in doping, forensic science, and toxicological contexts, as well as the need for optimum dosage adjustment in clinical settings has led researchers to develop analytical methods that can be used to accurately monitor these drugs. 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 • Analytical methods including HPLC, 2D HPLC and HPLC-MS have been developed for use in the analysis of β-blockers. • To overcome these limitations, various sample pretreatment methods such as protein precipitation (PP), matrix solid-phase dispersion (MSPD), liquid-liquid extraction (LLE), solid-phase microextraction (SPE) and magnetic solid-phase extraction (MSPE) have been applied. • Among these sample preparation methods, an obvious matrix background is still observed in the sample extract following the use of PP, and a large volume of organic solvents is used in LLE and SPE. • However, these drawbacks are avoided with MSPE, in which analytes are isolated from the sample matrix by a magnetic field, thus simplifying the sample pretreatment process
- 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 • Magnetic nanoparticles (MNPs) are a series of easily synthesized MSPE materials with large surface-to-volume ratios. • 3,4-dihydroxyphenylalanine (DOPA) is easily self-polymerized in weakly alkaline aqueous media, forming a poly(3,4dihydroxyphenylalanine) (polyDOPA) coating. • PolyDOPA shows excellent adhesive ability on various material surfaces through metal bidentate coordination and hydrogen bonding . • PolyDOPA-coated MNPs (polyDOPA-MNPs) have been widely applied as extraction adsorbents. • PolyDOPA-modified surfaces provide abundant active sites to immobilize molecules and grow shells through the Michael addition/Schiff base reactions, coordination, and redox, owing to the presence of many functional groups such as catechol, amine, carboxyl, and quinone moieties on the surface.
- 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 • The catechol group of DOPA can be used as a reducing agent to anchor the surface of metal nanoparticles. • A one-step in situ reduction process has been reported for the preparation of Ag@DOPA@Hg nanostructures to detect Hg2+ using a colorimetric method. • Au@DOPA nanoparticles have also been synthesized for catalytic and environmental applications. • Based on these reports, we developed novel nanosilver-functionalized MNPs with an interlayer of polyDOPA (polyDOPA@Ag-MNPs) and used them as MSPE adsorbents for the analysis of trace β-blockers in complex biological samples. • The adsorption performance of polyDOPA (polyDOPA@Ag-MNPs) towards β- blockers was investigated, and a method involving MSPE coupled with (FTICR- MS) for the analysis of the investigated β-blockers was developed.
- 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 • Ferric chloride hexahydrate (FeCl3·6H2O), sodium acetate, diethylene glycol, ethylene glycol, and acetic acid - Tianjin Damao Chemical Reagent Factory- china. • Sodium acrylate - Beijing Universal Century Technology Company - Beijing, China. • DOPA - Alfa Aesar Chemical Company - Shanghai, China. • Bupivacaine hydrochloride, metoprolol tartrate salt, acebutolol hydrochloride, and propranolol hydrochloride - Shanghai Macklin Biochemical Co., Ltd. - Shanghai, China. • HPLC-grade acetonitrile and methanol - Burdick & Jackson - USA. Materials and Reagents
- 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 Preparation of PolyDOPA@Ag-MNPs & MSPE Procedure Fig. 1. Schematic illustration of the synthesis of polyDOPA@Ag-MNPs and their application as MSPE adsorbents for the analysis of trace β-blockers in biological samples using FTICR-MS.
- 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 • All mass spectrometric analyses were performed using a Sarix X 7T Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany) in positive ion detection mode. • Accurate mass measurement was acquired with a 4M recording mode, and mass resolutions of more than 100,000 for target analytes was obtained. Mass spectrometric detection
- 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 Preparation and characterization of polyDOPA@Ag-MNPs : • The synthesis of polyDOPA@Ag-MNPs involved the reduction of silver cations into neutral silver atoms using polyDOPA, and the subsequent production of reactive quinones for the deposition of nanocomposites to immobilize molecules and grow shells. • The catechol in polyDOPA films is easily oxidized to quinone, inducing a localized reduction of metal ions. • Simultaneously, a polyDOPA film is spontaneously deposited outside the structure, acting as a protective layer to prevent the oxidation of the generated Ag . • Therefore, DOPA was the reductant and stabilizer for the formation of AgNPs during the reaction process, and polyDOPA@Ag-MNPs were synthesized in situ. Results and Discussion
- 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 • The magnetic characterization of COOH-MNPs and polyDOPA@Ag-MNPs was performed using a vibrating sample magnetometer (VSM) with a LakeShore7404 instrument at an applied field of 15 and a temperature of 25 °C. • The saturation magnetization values of COOH-MNPs and polyDOPA@Ag-MNPs were 95.5 A·m2/kg and 52.2 A·m2/kg, respectively. • These results indicate that the particles exhibited an excellent magnetic response. • Compared with COOH-MNPs, the decrease in the saturation magnetization value of polyDOPA@Ag-MNPs demonstrated that polyDOPA@Ag was successfully modified on the surface of COOH-MNPs.
- 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 • The zeta potential and size distribution characteristics were determined using a Zetasizer nano ZS (Worcestershire, UK). • The zeta potentials of COOH-MNPs and polyDOPA@Ag-MNPs at pH values from 4–9 were compared. • With increasing pH, the zeta potential of COOH-MNPs and polyDOPA@Ag-MNPs both decreased gradually and finally plateaued out. • The zeta potential magnitude of polyDOPA@Ag-MNPs was lower than that of COOH-MNPs at each pH, indicating the successful immobilization of polyDOPA@Ag on COOH-MNPs. Zeta Potential and Size Distribution Characteristics
- 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 • The particle size and surface morphology of the COOH-MNPs and polyDOPA@Ag-MNPs were determined by transmission electron microscopy. • Both particles were spherical, with diameters of approximately 300 nm. • There seemed to be no significant difference between COOH-MNPs and polyDOPA@Ag-MNPs following visualization of their TEM images. • To further prove the successful preparation of polyDOPA@Ag-MNPs, the composite of the particles was verified by energy dispersive X-ray (EDX) spectroscopy. • The weight percentage of Ag in the polyDOPA@Ag-MNPs was 4.25%. Particle Size and Surface Morphology
- 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 Fig. 2. (A) VSM curves of COOH-MNPs and polyDOPA@Ag-MNPs; (B) zeta potential of COOH-MNPs and polyDOPA@Ag-MNPs at different pH values (n = 3); (C) size distribution of COOH-MNPs and polyDOPA@Ag-MNPs; and (D) energy dispersive X-ray (EDX) spectrum of polyDOPA@Ag-MNPs.
- 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 • To achieve a good yield of β-blockers during the extraction process, factors affecting MSPE, including sample pH, adsorption time, amount of polyDOPA@Ag-MNPs, type of eluent, desorption time, and volume of eluent were optimized using acebutolol, metoprolol, and propranolol as target analytes for examination. pH: • The sample pH, which determines the charge of the analyte and adsorbent, was investigated first. • The highest extraction efficiency was achieved when the pH of the sample solution was 7. • The dissociation constant (pKa) values of acebutolol, metoprolol, and propranolol are 9.20, 9.68, and 9.45, respectively Optimization of MSPE conditions:
- 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 • Therefore, these β-blockers are primarily cationic below their pKa values. • The electrostatic attraction between the analytes and the adsorbents should play a role in the adsorption process. • If the electrostatic interaction plays a key role in the adsorption, theoretically, the extraction efficiencies should be higher at a pH lower than 7.0. • pH range of 4.0–6.0, the extraction efficiencies were lower than those at pH 7.0, suggesting that non-specific interactions between the analytes and adsorbents, such as π-π stacking and hydrogen bonding forces, were also at play in the adsorption process. • Therefore, a sample pH of 7.0 was used in this study.
- 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 Adsorption Time: • Next, the adsorption time was investigated, and 2 min was found to be the optimal adsorption time • The extraction efficiency of polyDOPA@Ag-MNPs for each β-blocker increased with increasing amounts of adsorbent, with a plateau being attained at 4 mg. • Therefore, 4 mg polyDOPA@Ag-MNPs was used for further experiments. • The conditions of desorption, including the type of eluent, desorption time, and volume of eluent, were investigated.
- 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 Type of Eluent: • MeOH containing 1% HAc exhibited the best extraction performance. • The extraction efficiencies increased with increasing eluent time until a plateau of 2.0 min. • Subsequently, the eluent volume associated with the optimal extraction efficiency was recorded. • The extraction efficiency reached a maximum with an eluent volume of 1 mL, and a further increase in the eluent volume was unfavorable
- 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 Fig. 3. Effect of (A) pH, (B) adsorption time, (C) amount of polyDOPA@Ag-MNPs, (D) type of eluent, (E) desorption time, and (F) volume of eluent on the extraction efficiencies of polyDOPA@Ag-MNPs for β-blockers.
- 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 • Propranolol was selected as the target analyte to evaluate the adsorption capacity of polyDOPA@Ag-MNPs, and the adsorbed amount of the β-blocker was calculated according to Eq. (1). qe = (c0 − ce) V / m where, qe = the amount of propranolol adsorbed at equilibrium (mg/g) c0 and ce = initial and equilibrium concentrations of propranolol (μg/mL) V = volume of solution (mL) m = mass of polyDOPA@Ag-MNPs (g) Adsorption capacity of PolyDOPA@Ag-MNPs
- 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 • The Langmuir model was used to analyze the adsorption process of propranolol on polyDOPA@Ag-MNPs, and the model is expressed using Eq. (2). ce / qe = ce / qm + 1 / Kd qm where Kd = the energy of adsorption qm = is the maximum adsorption amount of propranolol (mg/g) • The relationship between ce/qe and ce was expressed as an equation, ce/qe = 2.57ce + 0.0043, with a determination coefficient (R2) of 0.9973. • The maximum adsorption capacity of polyDOPA@Ag-MNPs for propranolol was calculated as 0.39 mg/g after applying the Langmuir model.
- 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 Fig. 4. Correlation of initial propranolol concentration and amount of propranolol adsorbed by polyDOPA@Ag-MNPs.
- 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 • Acebutolol, metoprolol, and propranolol were added to phosphate solution (10 mM, pH 7.0) to form a series of matrix-spiked samples (with concentrations of 0.02, 0.05, 0.1, 0.5, 1, 5, 10, and 20 ng/mL) for construction of calibration curves. • Bupivacaine was used as the IS and was added into the eluent with a concentration of 10 ng/mL. • Good linearities were obtained in the concentration range of 0.02–20 ng/mL, with (r2) ˃ 0.9901 for analyses of the three target β-blockers. • The limits of detection (LOD) and quantification (LOQ) were from 3.5–6.8 pg/mL and 11.7–22.8 pg/mL, respectively, indicating desirable sensitivity of the developed MSPE-FTICR-MS method. Method validation
- 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 a) LOD and LOQ were determined as the concentrations producing signal-to-noise ratios of 3 and 10, respectively. b) Analysis of samples spiked with 1.0 ng/mL.
- 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 • The repeatability, experiment was performed by conducting six consecutive analyses of phosphate sample solutions (10 mM, pH 7.0) spiked with 1.0 ng/mL of each β-blocker in a single day. • For reproducibility, the experiment was performed by analyzing six phosphate solution (10 mM, pH 7.0)-spiked samples (1.0 ng/mL of each β-blocker) on six different days. • The obtained relative standard deviation (RSD) values of the ion intensity ratio of analyte to IS (Ianalyte/IIS) for each β-blocker. • The results indicated that our developed MSPE-FTICR-MS method had good repeatability, with RSD values ranging from 4.1%–5.6% for six replicate analyses. • In addition, our method showed desirable reproducibility, with RSD values ranging from 9.8%–10.6% for six parallel experiments conducted on different days.
- 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 • The recovery (%) of the analyte was calculated from the measured versus added amounts according to Eq. (3). % Recovery = cfound V1 / cspiked V0 × 100% cfound and cspiked = concentrations of analyte in the eluate and spiked standard solution V1 and V0 = volumes of the eluate and spiked standard solution, • To determine the recoveries of the three investigated β-blockers in blood, three blank human plasma samples were spiked with 1 and 10 ng/mL of the mixed standard. • Using the above equation, the recoveries were calculated to be in the range of 80.9%–91.0%. Real Sample Analysis
- 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
- 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 • The analytical performances of the spiked blood samples (10 ng/mL) with and without undergoing extraction using polyDOPA@Ag-MNPs were compared. • When the spiked blood sample was directly detected by the ESI-FTICR-MS method, the signals of the three investigated β-blockers were very weak (Fig. 5A). • However, the signals of the investigated β-blockers in the blood sample were clearly observed using the MSPE-FTICR-MS method.
- 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 Fig. 5. Mass spectra of (A) spiked blood (10 ng/mL of each β-blocker) without extraction (B) spiked bovine blood after extraction with polyDOP@Ag-MNPs.
- 31. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 31 Authors conclusion: • In conclusion, a polyDOPA@Ag-MNP-based MSPE coupled with FTICR-MS method was developed for the rapid analysis of trace β-blockers in biological samples. • The redox-active polyDOPA facilitates in situ formation of polyDOPA@Ag-MNPs, which not only effectively removes matrix interferences from biological samples, but also enhances detection sensitivity through the enrichment of targets. • By using our developed method, rapid and sensitive analysis of trace β-blockers in blood samples was successfully achieved, with an overall detection process of less than 10 min as well as limits of detection and quantification in the ranges of 3.5– 6.8 pg/mL and 11.7–22.8 pg/mL, respectively. Conclusion
- 32. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 32 My conclusion: • Matrix components in biological samples often affect the sample preparation step and influence the ionization efficiency of MS. • Overall, the results indicate that this method provides a novel technique for the sensitive detection of trace β-blockers in human blood samples.
- 33. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 33 1. M. Caban, P. Stepnowski, M. Kwiatkowski, et al.Determination of β-blockers and β-agonists using gas chromatography and gas chromatography–mass spectrometry – a comparative study of the derivatization step J. Chromatogr. A, 1218 (2011), pp. 8110-8122. 2. A. Mahmoudi, M. RajabiSelective determination of some beta-blockers in urine and plasma samples using continuous flow membrane microextraction coupled with high performance liquid chromatography J. Chromatogr. B, 1128 (2019), p. 121768. 3. V.M.F. Gonçalves, P. Rodrigues, C. Ribeiro, et al.Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid chromatography (2D-LC) J. Pharmaceut. Biomed. Anal., 141 (2017), pp. 1-8. References
- 34. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 34 4. J.Q. Cheng, T. Liu, X.M. Nie, et al. Analysis of 27 beta-blockers and metabolites in milk powder by high performance liquid chromatography coupled to quadrupole orbitrap high-resolution mass spectrometry Molecules, 24 (2019), p. 820. 5. M.A. Rosa, H.D. De Faria, D.T. Carvalho, et al.Biological sample preparation by using restricted-access nanoparticles prepared from bovine serum albumin: application to liquid chromatographic determination of beta-blockers Mikrochim. Acta, 186 (2019), p. 647.
- 35. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 35