Aparajitha.K et al. / Journal of Pharmacy Research 2012,5(5),2814-2817 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Spectrophotometric and RP-HPLC methods for the estimation of troxipide. Aparajitha.K*, V.Shankarananth1, L.Nagamallika1, P.Jyosthna1, D.Narmada1 and M.Padmavathamma1. *-Sri Vishnu college of Pharmacy,Bhimavaram,AP,India. 1- Sri Padmavathi school of Pharmacy, Tiruchanoor, Tirupati-517503, AP, India. Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012

ABSTRACT The present work describes spectrophotometric and RP-HPLC methods for the estimation of Troxipide, an antiulcer drug. Troxipide obeyed Beer’s law in a concentration range of 2-10 µg/mL exhibiting maximum absorption at 259 nm. Spectrophotometric method is extended to tablet dosage form and there is no interference from any pharmaceutical additive and diluent. Chromatographic estimation (only bulk drug) was carried out on Phenomonex Gemini C-18 (250x4.6 mm,5µ) column using a mobile phase consisting of potassium dihydrogen phosphate buffer -methanol (70:30).The eluent was monitored at 260 nm. The results have been validated statistically.

Key words: Troxipide, Spectrophotometric and RP-HPLC estimation.

INTRODUCTION: Troxipide is used in the treatment of gastroesophageal reflux disease. Chemi- Choice of solvent: Different solvents like water, methanol, 0.1N HCl, 0.1N cally it is 3-(3, 4, 5-Trimethoxybenzamido) piperidine. It is a gastro protec- NaOH and Phosphate buffer pH 6.4 were employed for the optimisation of tive agent with antiulcer, anti-inflammatory and mucus secreting properties the method. Distilled water gave a single distinct peak with good absorbance (1). It neither inhibits acid secretion nor has acid neutralizing activity, but has for the Troxipide. So, it was employed as the solvent. been clinically proven to heal and gastric ulcers. It has been postu- lated that Troxipide’s mucosal protective effect in gastric ulcer and gastritis is Preparation of Stock solutions: exerted via the inhibition of inflammatory responses and -medi- Standard Troxipde 100 mg was weighed and dissolved in 100 mL of distilled ated mucosal injury. It promotes ulcer repair by increasing regenera- water in a 100 mL volumetric flask. The flask was shaken and volume was tion of the ulcer base and causes healing of peptic ulcer(2-4). Literature sur- made up to the mark with distilled water to give a solution containing 1000 µg vey reveals that very few reports are available for estimation of troxipide by / mL (stock solution I). From the stock solution I, pipetted out 1 mL and spectrophotometry and HPLC in bulk and dosage forms(5-8). The review of placed into 100 mL volumetric flask. The volume was made up to mark with literature prompted us to develop a simple and accurate spectrophotometric distilled water to give a stock solution containing 10 µg / mL (stock solution and RP-HPLC method for the estimation of Troxipide. II).

Determination of : Structure of Troxipide: lmax From the standard stock solution II of Troxipide, appropriate aliquots were made with distilled water to obtain working standard solutions of concentra- tions from 1 to 10 µg / mL. Absorbance for these solutions were measured in the range of 200-400nm and the spectra was recorded.

Procedures for Tablet Dosage Forms : Ten Tablets were taken, weighed and finely powdered. The powder equiva- lent to 100 mg of Troxipide was accurately weighed and transferred to volu- metric flask of 100 mL capacity containing 25 mL of the distilled water and sonicated for 15-20 min. The volume was made up to the mark with distilled water to give a solution of 1000 µg / mL (stock solution I). The above solution was filtered through Whatmann filter paper. From this solution, 1 mL was MATERIALS AND METHODS: taken and diluted to 100 mL with distilled water to give a solution of 10 µg / mL (stock solution II) and used for the estimation of Troxipide. The solu- Spectrophotometric Assay Method: tions were measured for their absorbance at 259nm, using distilled water as a Materials: Pure Troxipide bulk drug was obtained as a gift sample from blank. Chiral Bio Life Sciences, Hyderabad, distilled water was prepared at our laboratory, Troxip Tab (Zuventis) 100mg was obtained from local market. Instruments: UV-Spectrophotometer (SHIMADZU) 1800 series with two RP-HPLC Assay Method: matched quartz cells and 1cm path length, Digital balance (Essae) and Ultrasonicator (Optics Technologies) were employed in this method. Materials: Pure Troxipide bulk drug was obtained as a gift sample from Chiral Bio- *Corresponding author. sciences Limited, Hyderabad. Methanol and water used were of HPLC grade Aparajitha.K, (Merck). Potassium dihyrogen Phsophate and Orthophosphoric acid Ana- M.Pharm, lytical grade (Merck). Sri Vishnu college of Pharmacy, Bhimavaram,AP,India.

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2814-2817 Aparajitha.K et al. / Journal of Pharmacy Research 2012,5(5),2814-2817 Instruments: Overlay Spectra of Troxipide Bulk : Digital balance (Essae), pH meter (Elico), Ultrasonicator (Optics technolo- gies), Millipore solvent filtration unit (Bros Scientifics), UV detector, HPLC system( SHIMADZU- LC- 20AT Prominence solvent delivery system) supported by LC solutions software. Analytical column-Phenomonex-Gemini C-18 column (250 mm X 4.6mm, id 5µ) were used in this study.

Preparation of Standard stock solution:

Standard Preparation: Weighed accurately and transfered about 50.0mg of Troxipide into 100ml volumetric flask, dissolved and diluted to volume with mobile phase. Pippetted out 1ml of this solution into 100ml volumetric flask and diluted to volume with mobile phase.

Sample Preparation: Weighed accurately and transfered about 50.0mg of sample into volumetric flask, dissolved and diluted to volume with mobile phase. Pippetted out 1ml of this solution into 100ml volumetric flask and diluted to volume with mobile phase.

Solution injected No.of Injections Calibration of troxipide bulk: Blank (MP) 1 Standard preparation 6 S.No. Concentration Absorbance Sample 1 (µg / mL) at 259 nm

1 2 0.179 Chromatographic conditions: 2 4 0.354 · Column: 250 mm X 4.6mm, 5µ, Phenomonex-Gemini C-18 col- 3 6 0.566 umn. 4 8 0.735 · Flow rate: 1.2ml /min. 5 10 0.908 · Detector: UV. Linearity curve for Troxipde Bulk: · Wavelength: 260nm. · Injection Volume: 20µl. · Column oven temperature: 300C. · Run time: 60min. Time Mobile Phase Mobile Phase (minutes) A % B %

0.01 70 30 15.0 70 30 30.0 55 45 45.0 55 45 50.0 70 30 60.0 70 30

RESULTS AND DISCUSSION: The Beer’s law limit, Molar extinction coefficient, Regression equation and Percent relative standard deviation were calculated and shown in table. The absorbance was measured at 259 nm and the quantity of Troxipide was determined from the standard curve graph. The recovery studies were carried out at the different concentrations by spiking a known concentration of Parameter Result standard drug to the pre analysed sample and contents were reanalysed by proposed methods. The results of marketed formulation analysis and recov- lmax of the Drug 259nm ery studies were given in table. Beer’s Limit 2-10(µg / mL) Aliquot of standard Troxipide stock solution was taken in a different 10 ml Linearity: volumetric flask and diluted up to the mark with the mobile phase such that Regression Equation : Y = mX + C y = 10.894x + 0.0214 Slope (m) 10.894 the final concentration of Troxipide was in the range of 10-120 µg/ml. Each of the drug solution (20 µL) was injected three times into the column, and the Intercept (C) 0.0214 2 peak area and retention time were recorded. Evaluation was performed with Correlation Coefficient (r ) 0.9991 UV detector at 260 nm and a calibration graph was obtained by plotting peak Molar absorptivity 0.0895 X 104 area versus concentration of Troxipide. The plot of peak areas of sample against respective concentration of Troxipide was found to be linear in the

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2814-2817 Aparajitha.K et al. / Journal of Pharmacy Research 2012,5(5),2814-2817

Concentration of Absorbance Drug in mcg/ml at 259nm Peak Ret. Height Area Area % Theoretical Tailing time Plate Factor 2 0.182 4 0.361 1 4.386 1406 9158 0.023 9046.956 1.155 6 0.575 2 5.370 1053 10477 0.026 6541.758 1.278 8 0.741 3 5.951 4767 41672 0.103 9932.730 1.073 10 0.917 4 6.503 3220087 40057383 98.945 5923.640 1.832 5 7.553 7773 119445 0.295 6930.348 0.000 Quantification Results in Troxip tablet dosage form 6 10.313 7338 100782 0.249 9334.159 0.000 7 10.483 3760 45306 0.112 0.000 0.000 S.No. Sample Label Claim Amount % Label 8 27.824 1020 37881 0.094 12955.257 1.085 (mg) Found(mg) Claim found 9 32.210 1013 62434 0.154 6338.128 0.998 Total 3248216 40484538 100.000 1 Troxip tab 100mg 101.46 101.46

range of 10-120 µg/ml with correlation coefficient of 1.000. Linear regression least square fit data obtained from the measurements are given. The respec- tive linear regression equation being Y (-105593.8824) = 65647.112x + 45974.1647 for Troxipide. The regression characteristics, such as slope, intercept, and %RSD was calculated for this method. This method was validated by following standard validation protocol and all the parameters were found to be within acceptable limits (9-11).

CONCLUSION: From the above experimental data and validation results, both UV and HPLC assay methods for the estimation of Troxipide in bulk and pharmaceutical formulation (only UV method) is said to be rapid, simple, sensitive, precise, accurate and reliable for routine analysis in research institutions and quality control department in pharmaceutical industries.

ACKNOWLEDGEMENTS: The authors are thankful to Chiral Bio Life Sciences, Hyderabad for provid- ing gift sample of Troxipide. The authors are also thankful to Smt.P.Sulochana, M.A..,B.Ed.,L.L.B., Chairperson, Dr.D.Ranganayakulu, M.Pharm,Ph.D, Principal, Sri Padmavathi school of pharmacy, Tiruchanoor,Tirupathi for Chromatogram of Troxipide: providing facilities to carry out this work. We extend our thanks to Dr.K.K.Rajasekhar, A.Sreenivasa Charan, P.KeerthiSikha and Rajath.U for their help during this work.

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Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2814-2817 Aparajitha.K et al. / Journal of Pharmacy Research 2012,5(5),2814-2817 8. Ravipratap Pulla,B.S.Sastry,Y.Rajendraprasad and N.Appalaraju, 10. ICH,Good manufacturing practice for active pharmaceutical Estimation of Troxipide in tablet dosageform by RP- ingredients. International Conference of Harmonization. HPLC;International Journal of Pharmacy and Pharmaceutical IFPMA,Geneva,2000. Sciences.Vol 3, 4, 316-318,2011. 11. United States Pharmacopoeia.29th Revision, Asian edition, United 9. Singh.S., The ICH process its benefits, implication. RIPS,2000,1,2- states Pharmacopoeial convention, Inc. Rockvilla MD,2000,2129. 7.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2814-2817