stability indicating RP HPLC method for estimation of ziprasidone HCl

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Reverse phase HPLC, stability indicating method, ziprasidone, force degredation studies.

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PRESENTED BY Mr. Mahale Manoj V. (M. Pharm. IV SEMESTER QUALITY ASSURANCE) GUIDED BY Dr. C.S.Magdum. M.Pharm , PhD. Rajarambapu College of Pharmacy, Kasegaon. A Presentation On Stability Indicating RP-HPLC Method for Estimation of Ziprasidone hydrochloride in bulk and Marketed formulation. 12/06/2012 1

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CONTENTS INTRODUCTION OBJECTIVE OF WORK PLAN OF WORK DRUG PROFILE LITERATURE SURVEY MATERIALAND INSTRUMENTS EXPERIMENTAL RESULT AND DISCUSSION SUMMARY AND CONCLUSION REFERENCES 12/06/2012 2

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OBJECTIVE OF WORK To develop UV spectrophotometric method for determination of Ziprasidone hydrochloride in bulk and in capsule formulation. To develop HPLC method for estimation of Ziprasidone hydrochloride in bulk and marketed formulation. To validate the developed methods as per the ICH guidelines To study Stability Indicating method of drug. 12/06/2012 3

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Forced Degradation Studies Selection & Procurement of Drug Study of Physical Properties of Drug Literature Survey Optimization of Analytical Method U V. Spectra Analysis Validation of Developed Method PLAN OF WORK 12/06/2012 4 Result & Discussion Summary and Conclusion

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Chemical Name : 5‐{2‐[4‐(1,2‐benzisothiazol‐3‐yl)‐1‐piperazinyl] ethyl}‐ 6-chloro-1,3dihydroindol-2-one hydrochloride monohydrate. Molecular Weight : 449.40 Molecular Formula : C 21 H 22 Cl 2 N 4 OS.H 2 O Solubility : Very soluble in methanol, slightly soluble in isopropyl alcohol and in hot tetrahydrofuran and practically insoluble in water. Melting Range : Should between 220° and 224 o C. DRUG PROFILE 12/06/2012 5

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EXPERIMENTAL Method Development for Determination of Ziprasidone hydrochloride in bulk & Pharmaceutical Dosage Formulation PART-I A] UV Spectrophotometric Methods B] RP-HPLC Method PART-II Forced degradation Studies 12/06/2012 6

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Selection of Solvent System for Spectrophotometric methods Criteria for solvent selection No any interaction with analyte Minimum absorbance in UV range. Easily available. Low cost Solvent System Selected:- 1 M methanolic hydrochloride was selected as a solvent in developed method. UV Spectrophotometric Methods 12/06/2012 7

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Preparation of standard stock solution 10 mg of pure ZHM in 10 mL Methanol 100 μg / mL of ZHM 1ml in 10 ml solvent Selection of Analytical Wavelength UV Spectrophotometric Methods 12/06/2012 8

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Analysis of Marketed formulation Marketed Formulation Label Claim. (mg/capsule) Percentage Purity (%) S.D. * % R.S.D * Zipsydon 40 mg 97.54 0.75 0.7689 20 mg 98.85 0.605 0.61 Assay was performed by using commercial capsules Zipsydon (Sun Pharma) containing 20mg and 40 mg per capsule. The percentage purity of Ziprasidone Hydrochloride was calculated . *Results are average of three determinations. Assay limit for Ziprasidone HCl was found to be not less than 97.5 % w/w and not more than 102 % w/w as noted in certificate of analysis of the drug. UV Spectrophotometric Methods 12/06/2012 9

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Validation of U V Method 1 . Linearity and Range Parameters. Results. λ max (nm) 315nm Linearity Range, μg / mL 10-70 μg/mL Slope (m) 0.0140 Intercept (c) 0.01128 Correlation coefficient 0.9989 UV Spectrophotometric Methods 12/06/2012 10

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Precision The precision studies for the proposed method was carried out as per ICH guideline at the given wavelength (315nm).The intraday and interday precisions of the proposed methods were determined by estimating the corresponding responses 3 times on the same day and on 3 different days. Parameters SD* % RSD Morning 0.0026 0.4872 Afternoon 0.0005 0.1058 Evening 0.0017 0.3183 Intra-day precision studies Parameters SD* % RSD Day First 0.0005 0.1058 Day Second 0.0036 0.6281 Day Third 0.0005 0.1011 Inter-day precision studies . *Results are average of three determinations. UV Spectrophotometric Methods 12/06/2012 11

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Parameter μg / mL LOD 0.941 LOQ 2.843 Limit of Detection (LOD) & Limit of Quantitation (LOQ). Accuracy (recovery study): The accuracy of the methods was determined by calculating the recoveries of Ziprasidone Hydrochloride by the standard addition method. Recovery data of proposed methods Drug Label claim Amount of drug taken (mg) Amount added (%) % mean recovery ±S.D (n=3) ZHM 40mg 40 50 99.78 ±0.82 40 100 98.7±0.92 40 150 99.55± 0.88 Formulation Label claim (mg) Amount found (mg) % label claim ±S.D. (n=3) Zipsydon 40 39.73 99.34±0.56 UV Spectrophotometric Methods 12/06/2012 12

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Optimization of Mobile Phase . 1.Methanol :Water 80:20 v/v, 1 ml/min, 219 nm. 2.Methanol: Phosphate buffer pH5.5, 70:30v/v, 1 ml/min,219 nm RP-HPLC Method 12/06/2012 13 3.Methanol: Phosphate buffer pH 4.0, 80:20v/v, 1 ml/min, 219 nm

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4.Methanol: Phosphate buffer pH 4.0, 60:40v/v, 1 ml/min, 219 nm RP-HPLC Method . Methanol: Phosphate buffer pH 3.2, 70:30v/v, 1 ml/min, 219 nm 12/06/2012 14 6.Methanol: Phosphate buffer pH 3.2, 60:40v/v, 1 ml/min, 219 nm

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Acetonitrile: Phosphate buffer25Mm. pH 4.0, 60:40v/v , 1 ml/min, 315 nm 8. Methanol: Acetonitrile , 80:20v/v, 1 ml/min, 315 nm RP-HPLC Method 12/06/2012 15

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Trail 10.Acetonitrile: Phosphate buffer25Mm. pH 3.2, 60:40v/v , 1 ml/min,252 nm. Trail -11.Acetonitrile: Phosphate buffer25Mm. pH 3.2, 40:60v/v , 1 ml/min, 219 nm. Retention Time Area Theoretical plates (USP) Asymmetry 2.833 27619612 5569 1.26460 Retention Time Area Theoretical plates (USP) Asymmetry 3.950 11055218 6569 1.29456 Finalization of Mobile Phase Trail 10 and 11 are within the level of acceptance. ZHM shows symmetrical peak in both the chromatograms without any interference. As the wavelength 219 shows more detection of same concentration of analyte and less amount of ACN required ,hence was selected for further analysis . RP-HPLC Method 12/06/2012 16

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Analysis of Marketed Formulation Marketed formulation Label Claim Drug Content (%) + SD %RSD Zipsydon 40mg 99.22% + 1.051 1.04 For the estimation of marketed formulation of ZHM, 20 µg/ml solution was injected .The peak area of standard and marketed was compared. The % drug content in marketed was found to be 99.22% w/v. RP-HPLC Method 12/06/2012 17

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HPLC Method Validation Linearity and Range Concentration (µg/ml) Mean Area ( n=3) SD %RSD 10 13048103 90295.36 0.692019 20 26676285 4005.873 0.015017 30 40201256 55736.77 0.138644 40 52233673 21027.85 0.040257 50 65340910 31019.27 0.047473 50814.29 Parameters Results Linearity Range 10-50 µg/ml Correlation coefficient. ( r 2 + SD) 0.9998 + 0.0001 Slope + SD 1307960 + 273.01 Intercept 217688 RP-HPLC Method 12/06/2012 18

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Precision The precision studies was carried out as per ICH guidelines by estimating the corresponding response three times on the same day and on three different days at concentration of 20 µg/ml (intra-day and inter-day precision). Intra-day precision Inter-day precision Mean Area ( n=6) SD %RSD Mean Area ( n=6) SD %RSD 26677364 4798.23 0.0170 26671851 260135.57 0.975 The Limit of detection and Limit of Quantitation LOD and LOQ was calculated by using formula LOD = 3.3  / S LOQ = 10  / S where, σ =Standard deviation of response. S = slope of calibration curve. The LOD and LOQ values found to be 0.1282 µg/ml and 0.3885 µg/ml for ZHM respectively. RP-HPLC Method 12/06/2012 19

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Factor Level Retention Time (t R ) Flow Rate (ml/min). 0.9 - 1 2.99 1.0 0 2.87 1.1 +1 2.65 Mean + S.D (n=3) 2.83 + 0.172433562 Percentage of Acetonitrile in Mobile Phase(v/v). 38 -2 3.22 40 0 2.87 42 +2 2.62 Mean + S.D (n=3) 2.90 + 0.3013 pH of mobile phase. 3.1 - 1 2.90 3.2 0 2.87 3.3 +1 2.77 Mean + S.D (n=3) 2.85 + 0.0680 λ max 218 - 1 2.84 219 0 2.87 220 +1 2.92 Mean + S.D (n=3) 2.87 + 0.0404 Robustness RP-HPLC Method 12/06/2012 20

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Recovery Studies (Accuracy). Label Claim Amount Added % Total amount µg/ml Amount Recovered (µg/ml) % Recovery %RSD * 40mg 80 72 70.36 97.73 0.7660 100 80 79.60 99.49 0.1971 120 88 86.29 98.08 0.9845 Summary of Validation parameters of developed method- Parameters (units) Results Linearity Range (µg/ml) 10-50 µg/ml Correlation Coefficient + SD 0.9998 + 0.0001 Limit of Detection (µg/ml) 0.1282 µg/ml Limit of Quantitation (µg/ml) 0.3885 µg/ml Precision (%RSD) (n=3) Intra-day precision 0.0170 Inter-day precision 0.975 Recovery(%) (n=3) 80% 100% 120% 97.73 + 0.7660 99.49 + 0.1971 98.08 + 0.9845 Robustness Robust. RP-HPLC Method 12/06/2012 21

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Forced Degradation Studies Acid Induced Degradation For acid hydrolysis the solution was treated with 0.1 N HCl (Refluxed at 70-80 0 C for 8 hrs) no sufficient degradation observed hence further tried for 1N, 2N, for 12hrs, 24hrs, respectively at 70 0 -80 0 C, No sufficient degradation observed in this treatment hence concluded that ZHM is practically stable for acid. Stress Degradation Studies 12/06/2012 22

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Base Induced Degradation For base induced degradation the stock solution was treated with 0.1 N NaOH (Refluxed at 70-80 0 C for 8 hrs). Sufficient amount of degradation observed in 0.1N NaOH hence the ZHM said to be labile to base hence no further concentration of NaOH tried (i.e. 1N, 2N). 0.01 N NaOH also not tried because of no complete degradation was observed. The percentage degradation of drug was found to be 35.47 %. Stress Degradation Studies 12/06/2012 23

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Hydrogen Peroxide Induced Degradation For oxidative reaction the ZHM was treated with 3% v/v of H 2 O 2 (Kept at room temperature for 6 hrs). Sufficient amount of degradation observed at 3% v/v. The lower concentration of H 2 O 2 was also tried for the analysis but no sufficient degradation was observed hence as per classification system of degradation pathways the compound ZHM was said to be labile to H 2 O 2 . The percentage degradation of drug was found to be 46.98 %. Stress Degradation Studies 12/06/2012 24

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Thermal Degradation For thermal degradation the ZHM was subjected to the treatment to dry heat at 100 0 C for 5 hrs. The ZHM did not show any additional peak. The standard peak area and the thermal induced peak area of drug was compared, sufficient amount of degradation was observed hence the compound said as non stable for heat. Stress Degradation Studies 12/06/2012 25

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Photolytic Degradation For photochemical degradation no addition peaks were seen. The standard peak area and the resulting peak area of drug were compared. No sufficient amount of degradation was observed hence the compound said as stable for light. The resulting peak observed at t R 2.810. Stress Degradation Studies 12/06/2012 26

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Summary for stress testing of ZHM. Stress Condition Time % Recovery % Degradation Retention Time of Degradants Acid 2N HCl , Reflux 24 hrs 98.78 ------- ----- Base 0.1 N NaOH, Reflux 8 hrs 64.53 35.47 5.653 H 2 O 2 3% v/v at RT 6 hrs 53.02 46.98 1.660, 3.460 5.563 Photo-degradation (Sunlight) 5 days 96.87 ------- ------ Dry Heat 100 0 C 5 hrs 76.23 23.77 ------ Stress Degradation Studies 12/06/2012 27

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SUMMARY AND CONCLUSION 28 1. Simple UV spectrophotometric method was developed for estimation of ZHM in bulk and marketed formulation. 2.HPLC method developed for Estimation of ZHM Validated as per ICH guideline The LOD and LOQ of the methods are satisfactory The proposed method is found to simple be accurate, precise, specific, selective and reproducible intra and inter day variation were well within limit with RSD<2%. Found to be specific for degradation studies. 12/06/2012

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Ziprasidone HCl shows degradation for base (0.1N NaOH ) and Hydrogen peroxide (3% v/v). So it can be concluded that Ziprasidone HCl is labile to base and oxidative condition. Found to be stable for acid and photolytic degradation. Degradation was observed in dry heat treatment, hence it was found unstable to heat. 3.Forced Degradation Studies. 12/06/2012 29

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The developed HPLC method provide simple, accurate, reproducible and stability indicating for quantitative analysis for determination of Ziprasidone Hydrochloride in pharmaceutical dosage form, without any interference from the excipients and in the presence of its acidic, alkaline, oxidative, dry and photolytic degradation products. The chromatographic method was validated as per ICH guidelines. Statistical tests indicate that the proposed HPLC method reduce the duration of analysis and appear to be equally suitable for routine determination of Ziprasidone Hydrochloride monohydrate in pharmaceutical dosage form in quality control laboratories, where economy and time are essential. conclude that all these methods can apply for routine analysis of formulation and in bulk drug. SUMMARY AND CONCLUSION 12/06/2012 30

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REFERENCES Douglas A. Skoog , F. James Holler, Timothy A. Nieman , “Principles of Instrumental Analysis”, 5 th Edition, 2005, Saunders College Publishing. Harcourt Brace College Publishers, p;725-750 A. H. Backett , J. B. Stanlake ,” Practical Pharmaceutical Chemistry “, 4th edition, part II, CBS Publisher and Distributors, Delhi, 2001, p;255-280. Loyd R. Snyder et.al, “Practical HPLC Method Development “, 2nd edition, John Willey and sons, p;1-21. Willard, “Instrumental Methods of Analysis “7th edition, CBS Publisher and Distributors, Delhi. P;514 Chatwal GR, Sham KA, High performance liquid chromatography. In: Instrumental methods of chemical analysis. 5 th ed. Mumbai: Himalaya Publishing house: 2002. p: 2.626- 2.636. Frank Settle. Handbook of Instrumental techniques for analytical chemistry. NJ: Prentice Hall PTR: 1997. p:17, 19, 56, 57. ISO/IEC 17025. International standard: General requirements for the competence of testing and calibration laboratories. Geneva, Switzerland (1999). 12/06/2012 31

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ICH ; Validation of analytical procedures Text and methodology. Q2(R1).Fed Regist 2005; ICH, Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of analytical procedures: Methodology, ICH‐Q2B, 1997. ICH ; Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of analytical procedures, ICH‐Q2A, 1995. Jens T. Carstensen , C.T.Rhodes ; Drug Stability Principles and Practice Marcel Dekker series,3rd edition; p 331. ICH Harmonised Tripartite Guideline Stability Testing Of New Drug Substances And Products Q1A(R2).2003. Sumie Yoshioka , Valentina.J.Stella ;Stability of Drugs and Dosage Form, Kluwer Academic Publisher.p;4-33,139 George Ngwa ; Forced degradation studies, Drug Delivery Technology; vol 10 ;June 2010 Hildegard Brummer ., How To Approach A Forced Degradation Study Life Science Technical Bulletin, Issue 31; Jan 2011, p;1-4 Saranjit Singh, Monika Bakshi ; Guidance on Conduct of Stress Tests to Determine Inherent Stability of Drugs, April 2000, Pharmaceutical Technology Online, April 2000, p;1-14. Ana Paola Cione , Edivan Tonhi Stability Indicating Methods Bioagri Laboratorios p;25-36. Saranjit Singh, Monika Bakshi ; Development of Validated Stability Indicating Assay Methods – critical review, 28 ,2002, p;1011-40 12/06/2012 32

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