INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES

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1 International Journal of Universal Pharmacy and Bio Sciences 2(4): July-August 2013 ITERATIAL JURAL F UIVERSAL PHARMACY AD BI SCIECES IMPACT FACTR 1.89*** ICV 3.00*** Pharmaceutical Sciences Research Article!!! Received: ; Accepted: METHD DEVEPMET AD VALIDATI F PIPERAQUIE PHSPHATE AD ARTERLAE MALEATE I CMBIED DSAGE FRM BY RP-HPLC TECHIQUE Swathi Induru * 1, aira ayeem 2, Sandeep kolluru 1 1 Dept of Pharmaceutical Analysis, 2 Dept of Pharmaceutical Chemistry Krupanidhi College of Pharmacy, Bangalore KEYWRDS: Piperaquine Phosphate, Arterolane Maleate, RP- HPLC, Combined Dosage Form. For Correspondence: Swathi Induru * Address: Department of Pharmaceutical Analysis, krupanidhi college of Pharmacy, #12/1,Chikkabellandur village, Carmalaram post, Varthur, Hobli, Bangalore swathi.induru@gmail.co m ABSTRACT A reverse phase HPLC method has been developed for the simultaneous determination of Piperaquine phosphate (PQP) and Arterolane maleate (AM) in combined dosage form by using C 18 Thermo Hypersil BDS, (10 X 4.6 mm X 5.0µ) column and the mobile phase was prepared by mixing methanol: potassium dihydrogen phosphate buffer (ph- 2.3) in the ratio of (50:50). Retention times of PQP and AM were 1.13 min and 1.83 min respectively. This method shows to be linear (r 2 >0.99), precise (RSD<2%), accurate recovery of (98-102)% of PQP and (98-102)% of AM. specific and robust. 424

2 ITRDUCTI: Piperaquine phosphate is chemically known as 7-chloro-4-[4-[3-[4-(7-chloroquinolin-4- yl)piperazin-1-yl]propyl] piperazin-1-yl]quinoline; phosphoric acid [1]. Arterolane maleate is chemically known as [(-(2-amino-2-methylpropyl)-2-cis-dispiro(adamantane-2,3'- [1,2,4]trioxolane-5',1"- cyclohexan)-4"-yl]acetamide: maleate [2]. PQP and AM acts as Anti- Malarial drugs. PQP interferes with the degradation of haemoglobin by parasitic lysosomes, helps in damaging of plasmodial membranes. AM causes lipid peroxidation, damages endoplasmic reticulum, inhibits protein synthesis and ultimately results in lysis of the parasite [3]. Highly Sensitive, Selective, HPLC method will be very useful for the estimation of PQP and AM in combined dosage formulations. Literature survey reveals need for simultaneous estimation of drugs. Few methods were reported by HPLC [4-7], LC-MS [8-11], Capillary Zone electro phoresis [12]. The purpose of this study was to develop sensitive, simple, precise, accurate method for Simultaneous estimation of PQP and AM in bulk and combined dosage form. Fig o.1 Chemical Structure of Piperaquine phosphate and Arterolane maleate Cl CH 3 H 2 H 3 C H H P H H H H MATERIALS AD METHDS Apparatus: Cl Separation and estimation was carried out using HPLC (Waters-2469 with PDA detector), column used in experiment was C 18 Thermo Hypersil DS, 4.5µ (250 X 5.4 mm) analytical balance used was LABIDIA, Digital ph meter LABIDIA-PHA,. The mobile phase was prepared by mixing Methanol: potassium dihydrogen phosphate Buffer (ph-2.3) in the ratio of (50:50) was CH 4 filtered and degassed. Injection volume is 10µL and the detection was at 240nm. Reagents and solutions: Pure sample of piperaquine phosphate and arterolane maleate and other reagents such as methanol, double distilled water of HPLC grade and potassium dihydrogen phosphate. 425

3 Preparation of standard drug solutions Weighed accurately about 15.0 mg of the PQP and 75.0 mg AM pure drug sample and transferred into 100ml dry standard volumetric flask to this added 20ml of water and sonicated 10 minutes and make up the volume with water. From the above solution transferred 5 ml to 5o ml volumetric flask and diluted with water. Preparation of sample drug solution Randomly selected 10 tablets and determined the average weight of tablets and crushed into fine powder. Weigh and transferred tablet powder equivalent to 750 mg of PQP into a 1000 ml of volumetric flask, to this added 250ml of water and sonicated for 10 minutes and make up the volume with water. Further diluted 5ml of this solution to 50 ml with water and mix well. Filtered the solution. Marketed Formulation: Table o.1 Details of marketed Formulation Brand name Content Mfg. Company Synriam Preparation of mobile phase PQP & AM (750mg & 150mg Respectively) Ranbaxy The mobile phase was prepared by mixing methanol: potassium dihydrogen phosphate (ph-2.3) in the ratio of (50:50) and was filtered and degassed. Selection of mobile phase for method ptimization and experimental condition: Several trial had been taken for the proper optimization of RP HPLC method by changing different mobile phase with different ratio. And finally the mobile phase for optimised condition methanol: potassium dihydrogen phosphate (ph-2.3) in the ratio of (50:50)was selected and chromatogram was shown in Fig no.2. Fig : 2 Chromatogram of PQP and AM for ptimised method 426

4 Table: 2 Chromatographic conditions for the optimized method Parameters Description Column C 18 Thermo Hypersil BDS, (10 X 4.6 mm X 5.0µ) Mobile phase Methanol: Buffer (50:50) Injection volume 10 µl Flow rate 1.2 ml/min Detector Wavelength 240 nm Column Temperature 40 C Sampler Temperature 25 C Run Time 4 min Assay: Assay of marketed tablet formulation containing PQP (750mg) and AM (150mg) was performed by preparing the sample solutions as described earlier in the preparation of the sample. The assay of the commercial sample was calculated by comparing the areas of standard and sample peaks. The assay of marketed formulation synriam was found to be within the limit; the chromatogram is shown in Fig 3. Fig:3 Chromatogram for the Assay of marketed Formulation This optimized method was validated in terms of linearity, accuracy, precision, specificity, as per ICH guidelines [13]. Linearity: The linearity of calibration curves (peak area ratio V s concentration) in pure solution was checked over the concentration ranges of about % (Assay concentration in mcg/ml) for PQP and AM. The total eluting time was less than 4.0 min. The calibration curves were linear in the studied range. and equations of the regression analysis were obtained: y = 80651x, R 2 =0.999 for PQP and y = 14057x, R 2 =1.0 for AM. Accuracy: Accuracy of the method was determined by recovery experiments at spiked levels of 50%, 100%, 150%. The recovery studies were carried out three times, the percentage recovery and percentage relative standard deviation were calculated. 427

5 Precision: The precision of the analytical method was studied by analysis of multiple sampling of homogeneous sample. Specificity: A solution containing a mixture of tablet was prepared using sample preparation procedure and injected into the system, to evaluate possible interfering peaks. RESULTS AD DISCUSSI Validation: Method Precision Table 3. Method Precision Results for Piperaquine phosphate Test no. Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Avg. wt (mg) Wt. taken (mg) Area (Inj.1) Area (Inj. 2) Avg. Area % RSD Assay (mg/tab) Assay (%) Average Assay In mg = In % = Table 4. Method Precision Results for Arterolane maleate Test no. Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Avg. wt. taken (mg) Wt. taken (mg) Area (Inj.1) Area (Inj. 2) Avg. Area % RSD Assay (mg/tab) Assay (%) Average Assay In mg = In % = System suitability tests were carried out on freshly prepared standard solution and all the parameters are within limit. Results were shown in table o.5. TABLE : 5 System suitability Data Parameters PQP (± %RSD) AM (± %RSD) Retention Time 1.13± ±0.14 Theoretical plate 9348± ±0.34 Tailing Factor 1.14± ±0.23 ± %RSD = Percentage Relative Standard Deviation. 428

6 The method was linear in the concentration range of µg/ml for PQP and µg/ml for AM, with a correlation coefficient of for PQP and 1.0 for AM. Fig: 4 Linearity plot of PQP and AM The study of accuracy of the developed method has been done. The recovery was found in the range of 99.3%for PQP and 99.7%for AM shown in Table.6, indicating the accuracy of method and the % RSD of PQP and AM is 0.14 and 0.23 respectively. Concentration of Spiked level % TABLE: 6 ACCURACY DATA F THE AALYSIS F PQP AD AM Amount Std added µg/ml Total amount found µg/ml % Recovery µg/ml Mean PQP AM PQP AM PQP AM PQP AM

7 CCLUSI The Proposed RP-HPLC method is suitable for simultaneous determination of PQP and AM in Combined dosage form without any interferences form each other. The accuracy of the methods was assessed by recovery studies at three different levels. The method was found to be precise as indicated by the repeatability analysis, showing % RSD less than 2. All the parameters for both the drugs met the criteria of ICH guidelines for method validation. The developed method may be recommended for routine and QC analysis of the investigated drugs to provide simple, accurate and reproducible quantitative analysis for the determination of determination of PQP and AM in combined formulation. ACKWLEDGEMETS The authors are grateful to Management, Krupanidhi College of Pharmacy for their continuous support and encouragement and for providing the necessary facilities. REFERECES: 1. PubChem Compound [Cited 2013 july 31]; Available from: 2. PubChem Compound [Cited 2013 july 31]; Available from: 3. Tripathi KD Essentials of Medical Pharmacology. 6 th ed. Jaypee Brothers Medical Publishers ;2008.p Debrus B, Lebrun P, Kindenge JM, Lecomte F, Ceccato A, Caliaro G, MbayJMT, Boulanger B, Marini RD, Rozet E, Hubert Ph, (2011), Innovative high-performance liquid chromatography method development for the screening of 19 antimalarial drugs based on a generic approach, using design of experiments, independent component analysis and design space, J. of Chromatogr A, 1218, 31, Tarning J, Singtoroj T, Annerberg Ashton AM, Bergqvist Y, White J, Day PJ,. Lindegardh,(2006), Development and validation of an automated solid phase extraction and liquid chromatographic method for the determination of in urine, J of Pharm Biomed Ana, 41, 1(11), Te-Yu Hung, Davis TEM, Ilett KF,(2003), Measurement of piperaquine in plasma by liquid chromatography with ultraviolet absorbance detection, J. of Chromatogr B, 791, 1(2), Wahajuddin, Raju KSR, Taneja I, (2013), Bioanalysis of antimalarials using liquid chromatography, TrAC Trends in Ana Chem, 42,

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