AJPTI 111 10 2014 RPHPLC METHODS OF LEVOSULPHIRIDE PELLETS sandeep ra

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Sandeep Rajan Kolli et al. Asian Journal of Pharmaceutical Technology Innovation 02 08 2014 111-120 Asian Journal of Pharmaceutical Technology Innovation ISSN: 2347-8810 www.asianpharmtech.com 111 Research Article Received on: 01-10-2014 Accepted on: 09-10-2014 Published on: 15-10-2014 Validation and Development of New RpHPLC Methods of Levosulphiride Pellets Corresponding Author: Sandeep Rajan Kolli K.Mohini Kalyani P.Uday Bhaskar K.Vineela Sandeep Rajan Kolli C.R.R. College of Pharmacy Eluru- 534007 West Godavari DT Andhra Pradesh ABSTRACT A simple economic selective precise Reverse phase High Performance Liquid Chromatography method for analysis of levosulphride pellets 40 was developed and validated according to ICH guidelines. The quantification of the drug was carried out using grace smart 250mm × 4.6mm × 5μm or its equivalent in isocratic mode with mobile phase compressing of Buffer : Acetonitrile 70:30 The flow rate was 0.8ml/min and the detection was carried out by PDA detector i.e. 237 nm. The retention time for levosulphride pellets was found to be 2.3 min. The percent assay was found to be 98.98. The method of levosulphride pellets validated for precision accuracy linearity range specificity and robustness. Email Id- sndiprkgmail.com Key-words: levosulphride pellets Reverse phase High Performance Liquid Chromatography Validation. Cite this article as: Sandeep Rajan Kolli K.Mohini Kalyani P.Uday Bhaskar K.Vineela Validation and Development of New RpHPLC Methods of Levosulphiride Pellets Asian Journal of Pharmaceutical Technology Innovation 02 08 2014. C.R.R. College of Pharmacy Eluru-534007 West Godavari DT Andhra Pradesh

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Sandeep Rajan Kolli et al. Asian Journal of Pharmaceutical Technology Innovation 02 08 2014 111-120 www.asianpharmtech.com 112 Introduction: Chemically Levosulpiride is S---5-aminosulfonyl-N-1-ethyl-2-pyrrolinyl methyl-2 methoxyIbenzamide CAS No.23672- 07-3. a new antipsychotic agent belonging to the substituted benzamide group. Levosulpiride is only a weak D2 dopamine receptor antagonist. Furthermore in the D2 receptor family which includes D2 D3 and D4 receptors the affinity of levosulpiride for the D2 receptor is only 2-3 times greater than that for the D3 receptor this contrasts with typical antipsychotics which are 10-20 times more potent at D2 than at D3 1. At low doses 50-200 mg/day levosulpiride preferentially blocks dopamine auto receptors which are located on presynaptic neurons. At these doses levosulpiride is therapeutic for negative and cognitive symptoms of schizophrenia and for depressive and somatoform disorders. At high doses 400-800 mg/day levosulpiride blocks both dopamine presynaptic and postsynaptic D2 receptors and may therefore be effective for the positive symptoms of schizophrenia. Its low incidence of extrapyramidal side effects EPS is characteristic of a typical antipsychotic 23 . Material and Methods Chemicals and Reagents levosulphride pellets was obtained from hetero pharmaceuticals hyderabad. Acetonitrile HPLC grade Methanol HPLC grade Ammonium hydroxide AR grade ammonium acetate AR grade tetrabutyl ammonium hydroxide sulphateAR grade were of reagent grade. Instrumentation A HPLC LC-2010 SHIMADZU with Waters UV/VIS Detector/PDA detector grace smart 250mm × 4.6mm × 5μm was used. A Rheodyne injector with a 10 μl loop was used for the injection of sample. The HPLC system was equipped with Empower2 software for data processing. Chromatographic Condition The mobile phase containing Buffer: Acetonitrile 70:30 was found to resolve of levosulphride pellets. 1N ammonium Hydroxide solution was used for pH adjustment of buffer. The mobile phase was filtered on a 0.45 nylon membrane filter and then ultrasonicated for 30 min. The flow rate was set to 0.8ml/min. The drug shows good absorbance at 237 nm which was selected as wavelength for further analysis. All determinations were performed at constant column temperature 45 0 C. Preparation of buffer Buffer Preparation: Accurately weighed and transfer 3.85gm of ammonium acetate and1.1gm tetrabutyl ammonium hydrogen sulphate in 1000ml of water into a beaker and mix. Adjust the pH to 7.9 ±0.05 with 1N ammonium Hydroxide solution and dilute to 1:1 with water and mix. Filter the solution through 0.45μm nylon filter paper. Preparation of Mobile Phase Preparedly filtered and degassed mixture of buffer and Acetonitrile in the ratio of 70:30 v/v . Diluent solution Prepared a degassed mixture of water and Acetonitrile in the ratio 50: 50 v/v.

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Sandeep Rajan Kolli et al. Asian Journal of Pharmaceutical Technology Innovation 02 08 2014 111-120 www.asianpharmtech.com 113 Preparation of Standard solution Weigh down 75mg’s of Levosulphiride pellets in 100ml volumetric flask and add about 70ml diluent and sonicate to dissolve. Make upto the mark with diluent and mix. Take 5ml of above solution in 50ml volumetric flask and and make upto the mark with diluents. Preparation of Sample solution 188mg levosulphiride pellets sample equlivalent to 75mg levosulphiride into a 100ml volumetric flask. Add 70ml diluents and sonicated for 20 minutes with intermittent shaking and the solution was made up to volume with diluent and filtered through 0.45µ membrane. Placebo Preparation: 188mg placebo pellets equivalent to 75mg of levosulphride pellets into a 100 ml vf. Add 70 ml diluent and sonicate for 20 minswith intermitent shaking. Dilute to volume with diluent and mix.filter the solution through 0.45µ membrane filter. Method Validation 4 1 Calibration Curve of levosulphride pellets The linearity of levosulphride pellets responses at concentrations in the range of 20 to 80 ppm 56 was determined by preparing and injecting standard solutions 40μg/ml. 2 Specificity To demonstrate that diluents and placebo are not interfering with analytic peak. Solutions of levosulphride pellets standard sample and placebo were prepared individually at 0.040mg/ml concentration. The peak purity of analyte peak should be not less than 0.999 789. 3 System suitability: A Standard solution was prepared by using Levosulphride working standards as per test method and was injected Five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms by calculating the RSD from five replicate injections for levosulphride pellets retention times and peak areas 1011. 4 Precision Precision was measured in terms of repeatability of application and measurement. Repeatability of standard application was carried out using six replicates of the same standard concentration 40μg/mL for standard application 12-14. 5 Accuracy Recovery Accuracy Recovery of the method was tested by spiking 50 100 and 150 of levosulphride pellets working standard. The accuracy of the analytical method was established in triplicate across its range according to the assay procedure 15-19. 6 Robustness The robustness of the proposed method was determined by analysis of aliquots from homogenous lots by differing physical parameters like flow rate column temperature which may differ but the responses were still within the specified limits 20-25. 7 Limit of Detection and Limit of Quantification LOD and LOQ were determined from standard deviation and slope method as per ICH guidelines 25-27

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Sandeep Rajan Kolli et al. Asian Journal of Pharmaceutical Technology Innovation 02 08 2014 111-120 www.asianpharmtech.com 114 8 Sieve analysis 28-31 : Weigh about 50gm of test sample. Place the required sieves1420on top plate of instrument. Place previously weighed sample of pellet on top mos sieve. Now lock the both sieve clamp knob by turning it in clockwise direction. Set the desired power level and time for the test by increment and decrement key at the front panel for the power and time respectively. Press start key from the front panel the display shows the elapsed time and set value of the power. After completion of the passed through corresponding sieves and calculate the percentage 32-39. 9Bulk Density : Weight accurately 25gm of test sample and transfer onto 50ml graduated cylinder . carefully level the sample without compacting and read the unsetteled apparent volume vo to the graduated unit calculate the bulk density in g/ml by formula m/v0 40-42. 10 Water Analysis 42-44 : Fill the burette with karl fisher reagent with the help of rubber bellow. Fill the titration vesselwith 40ml of methonol. Swith on instrument to half opened position and press the start button 45-47. stop the ehrn the siren is heard. note down the initial burette readingI take about 3.0gm of pellets crushing weigh accurately and transfer about 0.2 gm of test sample w into the titration vessel. Switcj on the start button note down the final burette readingf 4849. when sound of siren is heard. Calculation the water content of test sample. w/w RESULTS AND DISCUSSION 1 Linearity: A linear relationship of levosulphride pellets across the range 20-80 ppm of the analytical procedure in triplicate. The range of concentrations was selected based on 20-80 ppm of the test concentration for assay. Peak area and concentrations were subjected to least square regression analysis to calculate regression equation. The correlation coefficient r2 was found to be 0.999 and shows good linearity. The data of the calibration curve was given in Table 1. Concentration ppm Average Area Statistical Analysis 0 0 Slope 58.76 20 1212 y-Intercept 27.40 30 1840 Correlation Coefficient 0.999 40 2360 50 2975 60 3535 70 4139 80 4727

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Sandeep Rajan Kolli et al. Asian Journal of Pharmaceutical Technology Innovation 02 08 2014 111-120 www.asianpharmtech.com 115 Fig1: Linearity Plot Concentration Vs Response of levosulphride 2 Specificity :The specificity of the method was established by the peak purity of levosulphride pellets were assessed by comparing the retention time TR of standard levosulphride pellets .Good correlation was obtained between the retention time of standard and sample of levosulphride pellets. Fig2: Got a peak for std at an RT of 2.3 Fig3: Got a peak for sample at RT of 2.3 3 Precision : Precision studies were carried out in terms of repeatability. Six determinations of 100 concentration at 24 μg/mL level and the data given in a Table. The RSD was found to be below 2 and fulfilled the ICH guidelines criteria. Table 2. TABLE 2: Data of Precision Concentration 40ppm Injection Peak Areas of levosulphride Assay 1 2350.3960 98.81 2 2394.8147 100.68 3 2369.9285 99.62 4 2372.5720 99.64 5 2366.0735 99.67

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Sandeep Rajan Kolli et al. Asian Journal of Pharmaceutical Technology Innovation 02 08 2014 111-120 www.asianpharmtech.com 116 Statistical Analysis Mean 2370.757 98.68 SD 15.96292 0.663 RSD 0.67 0.67 4 Accuracy: The recovery of the added standard to the drug product sample was calculated and it was found to be 97.70-101.5 whichindicates a good accuracy of the method to that of the labeled claim. The obtained recovery results were given in Table 3. TABLE 3: Data of Accuracy Concentration of spiked level Amount added ppm Amount found ppm Recovery Statistical Analysis of Recovery 50 injection 1 20 20.15 100.79 MEAN 100.79 50 injection 2 20 20.17 100.88 50 injection 3 20 20.14 100.71 RSD 0.07 100 injection 1 40 39.53 98.81 MEAN 99.68 100 injection 2 40 40.28 100.68 100 injection 3 40 39.82 99.55 RSD 0.94 150 injection 1 60 60.34 100.76 MEAN 99.44 150 injection 2 60 59.38 98.966 150 injection 3 60 59.28 98.70 RSD 0.97 5 Lod And Loq : LOD and LOQ were calculated form the average slope and standard deviation form the calibration curve. LOD and LOQ were found to be 0.1920 g/mL and 0.5813 g/mL respectively indicating high sensitivity of the method 6 Bulk density : Bulk density Weight taken 25gm Volume unsettled 30 ml

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Sandeep Rajan Kolli et al. Asian Journal of Pharmaceutical Technology Innovation 02 08 2014 111-120 www.asianpharmtech.com 117 Bulk density 25/30 0.83 gm/ml 7 Water Analysis: w/w 28.8-27.42100/0.21000 1.4 8 Sieve Analysis: passed through 14 100- 2gm/50 gm 0.04 Retained on 20 100- 1gm/50 gm 0.02 9 LIMIT OF DETECTION AND LIMIT OF QUANTITATION LOD and LOQ: From the linearity plot the LOD and LOQ are calculated: LOD 3.3 σ S 1.47 LOQ 10 σ S 4.45 REFERENCES 1. B. K. Sharma Instrumental Methods of Chemical Analysis 23 rd ed. Goel Publishing House. Meerut 2004 C10 C11 C68. 2. Hobart H. Willard N. Howell Furman and Egbert. K. Bacon A Short Course in Quantitative Analysis 2 nd ed. D. Van Nostrand Company 1968 4-5. 3. Hobart H. Willard Lynne. L. Merritt J. J. A. Dean and A. S. Frank Instrumental Method of Analysis 5 th ed. CBS Publishers and Distributors New Delhi 1986 3. 4. D. A. Skoog F. James Holler and T. A. Nieman Principles of Instrumental Analysis 5 th ed. Thomson Brooks /Cole Publishers 2005 674. 5. Hobart H. Willard Lynne. L. Merrit Jr. John. A. Dean and Frank. A. Settle Instrumental Methods of Analysis 7 th ed. CBS Publishers and Distributors 1986 514. 6. Satinder Ahuja and Stephen. Scypinski Handbook of Modern Pharmaceutical Analysis Harcourt Science and Technology Company 2001 423. 7. P. D. Sethi HPLC Quantitative Analysis of Pharmaceutical Formulations 1 st ed. CBS Publishers 2001 69-70. 8. John. H. Kennedy Analytical Chemistry Principles 2 nd ed. Saunders College Publishing New York 756. 9. Frank. A. Settle Handbook of Instrumental Techniques for Analytical Chemistry Pearson Education Inc. 2004 151.

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