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METHOD DEVELOPMENT AND VALIDATION OF QUETIAPINE FUMERATE BULK AND IN TABLET DOSAGE FORM BY UV AND HPLC

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METHOD DEVELOPMENT AND VALIDATION OF QUETIAPINE FUMERATE BULK AND IN TABLET DOSAGE FORM BY USING UV AND HPLC :

METHOD DEVELOPMENT AND VALIDATION OF QUETIAPINE FUMERATE BULK AND IN TABLET DOSAGE FORM BY USING UV AND HPLC Under the guidance of S.SHANMUGAPRIYA.M.Pharm . Submitted by (09404116) 1

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DRUG PROFILE QUETIAPINE FUMERATE Molecular structure Chemical name : 2-(2-(4-dibenzo [1, 4] thiazepine- 11-yl- 1-piperazinyl) ethoxy) ethanol Description : white crystalline powder Solubility : Soluble in water Molecular formula : C 21 H 25 N 3 O 2 S Melting point : 172 0 c – 173°c Molecular weight : 883.11g/mol Category : Anti-Psychotic 2

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LITERATURE REVIEW Radha Krishna S, et.al ., reported reverse phase liquid chromatographic method was developed or the of related substance and degradants of Quetiapine Fumarate bulk drug. Chromatographic separation between Quetiapine Fumarate its related substances. The separation was achieved using C 18 , (150X4.6 mm, 3.5 μm). Mobile phase 5mM ammonium Acetate as mobile phase A and Acetonitrile as Mobile phase B . flow rate at 1.0 ml/min. The column temperature was maintained at 40°C and the detection wavelength was 220 nm. The injection volume was 10 μL. 3

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Nikam D.S, et.al., reported derivative Spectrophotometric methods were developed for determination of Quetiapine fumarate in pharmaceutical formulation. Second order derivative ultraviolet Spectrophotometric methods were developed. Spectrophotometrically, Quetiapine fumarate was determined by measuring the 2 D -values at 254.76nm with 0.1 N HCl as background solvent. Dhaneshwar S.R. , et.al., reported a stability-indicating HPTLC method for quantitative analysis of quetiapine fumarate both as the bulk drug and in formulations has been established and validated. The stationary phase was silica gel. Mobile phase toluene–methanol 8:2 ( v / v ). R F : 0.37 ± 0.02. Densitometric analysis of quetiapine fumarate was performed in absorbance mode at 254 nm. 4

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Mandrioli R, et.al., developed High-performance liquid chromatographic (HPLC) method for the analysis of the novel antipsychotic drug quetiapine in plasma has been developed. The analysis was carried out on a C 8 (150×4.6 mm i.d., 5 μm). Mobile phase: a mixture of acetonitrile, methanol and pH 1.9 phosphate buffer. Triprolidine was used as the internal standard. Retention time 10min 5

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Soma Raju V , et.al., reported isocratic rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of quetiapine hemifumarate in bulk active pharmaceutical ingredient. chromatographic separation was achieved on a C 18 stationary phase with simple mobile phase combination delivered in a isocratic mode and quantification was by ultraviolet detection at 225 nm. flow rate of 1.0 mL min. injection volume 3 μL . 6

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La wrence Albers J, et.al., reported an original HPLC and UV method has been developed for the simultaneous determination of quetiapine and the geometric isomers of the second-generation antidepressant fluvoxamine. The analytes were separated on a reversed-phase C 8 column (150mm×4.6mm i.d., 5 μ m). Mobile phase composed of acetonitrile (30%) and a 10.5mM, pH 3.5 phosphate buffer containing 0.12% triethylamine (70%). The flow rate was 1.2ml/min. Detection wavelength was 245nm. 7

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The literature survey carried out and it revealed that several analytical methods have been reported for estimation of drug so the objective of work is to develop a new UV AND HPLC method for estimation of Quetiapine Fumerate in pure form. Hence present study have been planned to develop a specific precise, accurate, linear, simple and rapid UV AND HPLC method as per ICH guide lines for the method development of quetiapine fumerate in pure form. AIM AND OBJECTIVE OF WORK 8

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MATERIALS AND METHODS S.No Chemicals used Make 1 Potassium di hydrogen phosphate Analytical grade 2. Methanol Analytical grade 3 Ortho Phosphoric acid Analytical grade 4. Water HPLC and spectroscopy grade 5 Acetonitrile HPLC grade Drug sample Quetiapine Fumerate pure drug sample and was generously gifted by Local industry,The formulation Quetiapine tablets containing 200 mg of Quetiapine. Reagents and chemicals All the chemicals used were of analytical grade and procured from Qualigens India Ltd., Rankem Chemicals Ltd. The chemicals used for the study were, 9

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UV SPECTROSCOPIC METHOD Preparation of standard stock solution: Standard solution of quetiapine fumerate was prepared by dissolving 10mg of quetiapine fumerate in 10ml of water to get concentration 1000μg/ml. Different aliquots of above solution in the range 0.1 to 0.5ml were transferred into series of 10ml volumetric flask and volume made upto the mark with water to obtain the concentrations10 to50μg/ml. Scanning ranges was finalized for study and solutions were scanned on spectrophotometer in the uv range of 230-350nm. 10 Preparation of Sample Solution: Sample label claim 200 mg. The average weight was determined with 20 tablets, which were grounded in a mortar until fine powder. Accurately weighed amount of powder equivalent to 10mg of Quetiapine fumarate was quantitatively transferred to a 100 ml calibrated flask with the aid of Water. The volume was made up to mark, sonicate for 10 min. From above solution 0.2ml was transferred to 10ml calibrated flask and made up to mark with the aid of water to obtained the concentration 20 μg /ml and filtered through whatman Filterpaper no.1.Then the solution was scanned at 254nm.

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The method was validated with reference to linearity, accuracy, precision, and Limit of detection& Limit of quantification. Linearity Linearity was performed by taking from stock solution (1mg/ml) aliquots of 0.1 , 0.2, 0.3, 0.4 and 0.5 mL were taken in 10ml volumetric flasks and diluted upto the mark with water such that the final concentration of Quetiapine fumerate in the range of 10 to 50 μg/ml . Under the experimental conditions described the graphs obtained by plotting concentration Vs absorbance. Accuracy The accuracy of the proposed methods was assessed by recovery studies at three different levels i.e. 80%, 100%, 120%. The recovery studies were carried out by adding known amount of standard solution of three different levels. The resulting solutions were then re-analysed by proposed methods. Precision Precision of the methods was studied as intra-day, interday. Intra-day study was performed by analyzing, the three different concentration of drug for three times in the same day. Inter-day precision was performed by analyzing three different concentration of the drug for three days in a week. METHOD VALIDATION Limit of Detection and Limit of Quantitation: The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. 11

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HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY Mobile phase: A mixture of 50 volume of Buffer, 40 volume of Acetonitrile (HPLC grade) and 10 volumes of methanol was prepared. The mobile phase was sonicated for 10min to remove gases. Buffer preparation: 8g of potassium dihydrogen was weighed and dissolved in 100ml of water and volume was made up to 1000ml with water. Adjust the pH to 3.0  0.05 using dilute Ortho phosphoric acid. The buffer was filtered through 0.45μ filters to remove all fine particles and gases. Diluent Prepared a mixture of buffer: Acetonitrile: Methanol in the ratio of 50: 40: 10 which was used as diluent for dilution of standard stock solution. Standard solution of Quetiapine Fumerate: 10mg of Quetiapine fumerate was dissolved into 10ml of diluent to get 1mg/ml solution; sonic ate for 5 min and mix. 12

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Sample solution of Quetiapine Fumerate: Sample label claim is 200mg. The average weight was determined with 20 tablets, which were grounded until fine powder. Accurately weighed amount of powder equivalent to 10mg of Quetiapine fumerate was quantitatively transferred to 10 ml of calibrated flask with the aid of diluent. From the above solution 2ml was transferred to 10 ml calibrated flask and volume was made up to mark, sonicate for 10min and filtered. 13

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Chromatographic Conditions: Instrument : Shimadzu pump LC – 2010CHT Detector : PDA detector Column : Phenomix Stainless steel Column C 18 (250 X 4.6 mm, 5µ) packed with ODS chemically bounded porous silica particles. Wavelength : 247nm Temperature : 40 o C Flow rate : 0.8ml/min Runtime : 7 min Sample size : 20  l Diluent : Buffer: Acetonitrile: Methanol (50: 40:10) Sample retention time : 4.69 ± 0.06 min 14

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HPLC Method validation Linearity study Linearity was performed by taking from stock solution aliquots of 0.0 1, 0.02, 0.03, 0.04 and 0.05 ml were taken in 10ml volumetric flasks and diluted upto the mark with mobile phase such that the final concentration of Quetiapine Fumarate in the range of 1 to 5 μg/ml . Volume of 20 μl of each sample was injected in five times for each concentration level and calibration curve was constructed by plotting the peak area versus the drug concentration. Accuracy as Recovery I t was done by recovery study. Sample solutions were prepared by spiking at about 50 %, 100% and 150 % of specification limit to Placebo and analyzed by the proposed HPLC method. Precision Precision of the methods was studied as intra-day, interday and repeatability. Intra-day study was performed by analyzing, the three different concentration of drug for three times in the same day. Inter-day precision was performed by analyzing three different concentration of the drug for three days in a week. Limit of Detection and Limit of Quantitation: The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. Specificity Parameters The specificity of the method was predicted by preparing diluent, sample, and excipients as placebo sample and injected into the HPLC system 15

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. RESULTS AND DISCUSSION UV-Spectroscopic Method S.No. Solvent Solubility status 1. Distilled water (HPLC and spectroscopic grade) Freely soluble 2. 0.1N Hydrochloric Acid Freely soluble 3 Methanol soluble 4. Acetonitrile soluble 5. Iso propyl alcohol Slightly soluble Concentration ( μg/ml) Absorbance 10 0.082 20 0.166 30 0.251 40 0.338 50 0.422 TABLE - 2 Linearity data TABLE-1 Solubility of Quetiapine 16

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Parameters Quetiapine Fumerate Nm 254 Beer’s law limits 10-50 μg/ ml Correlation coefficient 0.999 Regression equation Y=mx+c Y= 0.00852-0.0038 Intercept(c) -0.0038 Slope 0.00852 Std error 0.00894 Molar absorptivity 4021.16 Sandell sensitivity 0.117371 Drug Label claim mg/tab Amount found mg/tab Label claim (%) S.D.* S.E* Quetiapine fumerate 200 197.9 98.9 0.0052 0.0026 TABLE-3 Optical characteristic and linearity data TABLE-4 Assay 17

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%conc Amount added Amount found % of Recovery %RSD 80 16 15.82 98.9 2.04 100 20 18.70 99.3 1.193 120 24 23.89 99.6 1.154 Mean 99.2 S.NO Concentration µg/ml INTER DAY INTRADAY S.D %RSD S.D %RSD 1 10 0.001 1.23 0.0015 1.87 2 30 0.0015 0.611 0.0015 0.60 3 50 0.0025 0.595 0.00152 0.361 LOD in μg/ ml 0.075 LOQ in μg /ml 0.227 TABLE-5 Accuracy and Recovery TABLE-7 LOD and LOQ TABLE-6 Precision 18

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HPLC Formulation Labeled amount (mg) Amount found *(mg) %Amount found %RSD Quetipin 200 198.92 99.46 0.285 Component Observation Diluent No interference at RT of analyte peak. Placebo No interference at RT of analyte peak. TABLE-8 Assay TABLE-10 Specificity TABLE-9 Linearity 19 Concentration (μg/ml) Peak Area 1 2 3 4 5 272206 540163 806202 1062866 1329391

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Label claim sample conc (µg) Amount added in µg Amount Recovered* in µg % Recovery* Average recovery (%) %RSD 200 mg 10 2 4 6 2.05 4.03 5.92 102.5 100.75 98.66 100.64 1.91 S.NO Concentration µg/ml Intraday Inter day S.D %RSD S.D %RSD 1 1 1734.81 0.63 1245.94 0.459 2 3 5666.9 0.70 1694.06 0.21 3 5 3064.8 0.23 2430.10 0.18 LOD in μg/ ml 0.0167 LOQ in μg /ml 0.0506 TABLE-11Accuracy TABLE-13 LOD and LOQ TABLE-12 precision 20

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S.No Method Label claim (mg/tab) Average % obtained S.D %RSD 1 Quantification For UV 200 98.9 0.336 0.33 2 Quantification For HPLC 200 99.46 0.284 0.285 TABLE-14 Quantification for comparative table of UV and RP-HPLC 21

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UV Spectrum of Standard UV spectrum of sample 22

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UV Linearity 23

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Chromatogram of standard 24

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Chromatogram of sample 25

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HPLC Linearity 26

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Quetiapine marketed by AstraZeneca as Seroquel and by Orion Pharma as Ketipinor , both as an quetiapine fumarate salt of the drug, is an atypical antipsychotic used in the treatment of schizophrenia , bipolar I mania, bipolar II depression, bipolar I depression, and used off-label for a variety of other purposes, including insomnia and anxiety disorders . The proposed analytical methods are simple, reliable, rapid, sensitive, reproducible and accurate for the estimation of Quetiapine The method adopted for our studies are Simple UV-Spectroscopic method RP-HPLC method The drug samples were analyzed by UV spectroscopy using distilled water as solvent and the average content of drug present in the formulation was found to be 197.9 mg (98.93%). A new RP-HPLC method was developed for both bulk drug and formulation. These proposed methods give reliable assay results with short analysis time (<5.0 mins) using the mobile phase of phosphate buffer: acetonitrile: methanol (50:40:10). The content of drug present in the formulation was found to be 198.92 mg (99.46%). All the above methods do not suffer from any interference due to common excipients. Therefore it was shown that the proposed methods could be successfully applied to estimate commercial pharmaceutical products containing quetiapine. Thus the above studies and findings will enable the quantification of the drug for future investigation in the field of analytical chemistry. Among the established analytical methods, RP-HPLC method was found to be more precise and accurate. The % RSD calculated for RP-HPLC method was very less when compared to UV method. Hence RP-HPLC method can be applied for regular analysis of Quetiapine from bulk drug and its dosage forms. SUMMARY AND CONCLUSION 27

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www.wikipedia.com 2. Radha Krishna S, Rao B.M. and Someswara Rao N. A simple and accurate reverse phase liquid chromatographic method was developed for the of related substance and degradants of Quetiapine Fumarate bulk drug. Rasayan J. Chem Vol.1, No.3 (2008), 466-474. 3. Nikam D.S, Bagade S.B Simple, fast and reliable derivative Spectrophotometric methods were developed for determination of Quetiapine fumarate in pharmaceutical formulation. Second order derivative ultraviolet Spectrophotometric methods were developed. International Journal of ChemTech Research, vol.1, no.4, pp 898-904, Oct-Dec2009. 4.Dhaneshwar S.R , Patre N.G, A sensitive, selective, precise, and stability-indicating HPTLC method for quantitative analysis of quetiapine fumarate both as the bulk drug and in formulations has been established and validated. Bio medical chromatography, vol22, pp1043-1055, Oct2008. REFERENCES 28

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5. Mandrioli R, Fanali S, Ferranti A and Raggi MA, A precise and feasible high-performance liquid chromatographic (HPLC) method for the analysis of the novel antipsychotic drug quetiapine in plasma has been developed. journal of pharmaceutical and biomedical analysis 30 (2002)969-97 6.Soma Raju V , a simple, sensitive isocratic rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of quetiapine hemifumarate in bulk active pharmaceutical ingredient. chromatographia, volume 70 no3-4 7. Lawrence J. Albers, Roberto Merli An original HPLCandUV method has been developed for the simultaneous determination of the atypical antipsychotic quetiapine and the geometric isomers of the second-generation antidepressant fluvoxamine. Journal of chromategrphy B, March2006,p 227-233. 29

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30 THANK U