Development and validation of q-absorbance ratio method for simultaneo

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 648 IJAMSCR |Volume 5 | Issue 2 | Apr - Jun - 2017 www.ijamscr.com Research article Medical research Development and validation of q-absorbance ratio method for simultaneous estimation of arterolane maleate and piperaquine phosphate in pharmaceutical dosage form Seju D Patel Dr Neha Tiwari Mrs Vanita Marvaniya Department of Quality Assurance A-one Pharmacy College Anasan Ahmedabad Gujarat382330 India Corresponding Author: Seju D Patel Email id: patelseju0007gmail.com ABSTRACT New Spectrophotometric Q-Absorbance Ratio method has been developed for the simultaneous estimation of Arterolane maleate and Piperaquine phosphate in tablet dosage form. UV spectrophotometric method methanol was used as a solvent. Objective To develop simple accurate linearity precision and reproducible UV Spectroscopic method for Arterolane maleate and Piperaquine phosphate in routine analysis. Method Aliquots of stock solution were further diluted with methanol to get working solution of 2.5-8.75μg/ml for Arterolane maleate and 12.5-43.5 μg/ml for Piperaquine phosphate working standards were scanned between 200 - 400 nm which shows the maximum absorbance at 276nm. Results The Iso -absorptive point was found to be 242 nm. Calibration curve were linear over a concentration range of 2.5- 8.75 μg/ml for Arterolane maleate and 12.5- 43.5 μg/ml for Piperaquine phosphate. Accuracy of method was determined through recovery studies which were found 99.36-101.22 for Arterolane maleate and 99.46- 100.80 for Piperaquine phosphate. Method was found to be reproducible with relative standard deviation for intra- day and inter-day precision to be 1.5. Conclusion This method was found to be simple Accurate precise and reproducible. The proposed UV method can be applicable for the simultaneous estimation of both the drugs in tablet dosage form. Keywords: Arterolane maleate Piperaquine phosphate Q-Absorbance Ratio method Analytical method validation Methanol. INTRODUCTION Arterolane maleate AM is chemically known as N-2-amino-2-methylpropyl-2-cis-dispiro admantane-2 3’-1 2 4 trioxolane-5 1”- cyclohexan-4”-yl acetamide: maleate. Arterolane maleate is synthetic peroxide which acts as anti- malarial agent by rapid acting as blood ISSN:2347-6567 International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 649 schizonticides against all blood stages of plasmodium falciparum without having effect on liver stages. Its molecular structure is uncommon for pharmacological compounds in that it has both an ozonide group and an adamantane substituent 1. Fig. 1: Chemical Structure of Arterolane maleate Piperaquine phosphate PQP is chemically known as 1 3-bis 4-7-chloroquinoline-4-yl piperazin-1-yl propane: Phosphoric acid. It is a bisquinoline of an antimalarial drug used as a prophylaxis and which shows good activity against chloroquine-resistant plasmodium strains 23 Fig. 2: Chemical Structure of Piperaquine phosphate Combination of AM and PQP is available in tablet dosage form in the ratio of 150:750 mg. AM is official in Indian Pharmacopoeia 2014 4. PQP is official in United State Pharmacopoeia 3. But combination of these drugs is not official in any pharmacopoeia. The combination of AM and PQP has been approved by Central Drug Standard Control Organization CDSCO on dated 19/10/2011 5. Very few methods like HPLC 6-8 Capillary zone electrophoresis9 LC-MS10-13 have been reported as a single or in combination with other drugs .Literature reveals that there is no single UV spectroscopic method for AM and PQP in pharmaceutical dosage form. So the present study aim at development of a simple accurate precise method for simultaneous estimation of AM and PQP in pharmaceutical dosage form by Q- Absorbance Ratio method. MATERIALS AND METHODS Apparatus and Instrument Double beam UV- visible spectrophotometer Shimadzu model 1800 Spharmaspec having two matched quarts cells with 1 cm light path Electronic analytical balance BL-220H pH meter LI- 610.All instruments and glass wares were calibrated. Reagents and Materials Arterolane maleate and Piperaquine phosphate gifted by Gitar Laboratories Ahmadabad India SYNRIAM Tablet procured from local market. Methanol AR Merck Pvt. Ltd India Preparation of standard stock solution 10mg of AM and 25 mg of PQP were placed in 100 ml volumetric flask and dissolved in 75 ml of Methanol and the volume was made up to the mark

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 650 with Methanol to obtain the solution of 100 µg/ml and 250 µg/ml respectively. Preparation of working standard solution Suitable aliquots of above solution were diluted up to the mark with methanol to get the concentration range of 2.5-8.75 for AM and 12.5- 43.5 for PQP. Selection of Detection Wavelength AM 5 µg /ml and PQP 25µg/ml in Methanol both the solutions were scanned over range of 390- 190nm against Methanol as blank using medium scan speed. The sampling wavelength for analysis Overlay spectra shows Absorption maxima λmax of AM 276 nm Absorption maxima λmax of PQP 216 nm Isobestic point 242nm Fig. 3: overlay zero order absorption spectra of standard solutions of AM 2.5-8.75μg/ml and PQP 12.5- 43.75μg/ml in methanol. Calibration curve for AM and PQP To check linearity of the method working standard solution having concentration in range of 2.5-8.75 μg/ml for AM and 12.5-43.5 μg/ml for PQP were prepared from the standard stock solutions of both drugs. The absorbance was measured at 276 nm λmax of AM and at 242 nm iso-absorptive point. Calibration curves were constructed by plotting concentration vs absorbance. METHODOLOGY Absorbance ratio method uses the ratio of absorbance at two selected wavelengths one which is an iso-absorptive point and other being the λ- max of one of the two components. From the overlay spectra of two drugs it is evident that AM and PQP show an iso-absorptive point at 242 nm. The second wavelength was selected 276 nm which is the λ-max of AM. Working standard solutions having concentration 2.5 3.75 5 6.25 8.75 μg/ml for AM and 12.5 18.75 25 31.25 43.75 μg/ml PQP were prepared in methanol and the absorbance at 242 nm iso-absorptive point and 276 nm λ-max of AM were measured and absorptivity coefficients were calculated using calibration curve. The concentration of two drugs in the mixture can be calculated using following equations. CX QM – QY / QX -QY × A1/ax1........... 1 CY QM – QX / QY -QX × A1/ay1 ………… 2

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 651 Where A1 and A2 are absorbances of mixture at 276 nm and 216 nm ax1 and ay1 are absorptivities of AM and PQP at 276 nm ax2 and ay2 are absorptivities of AM and PQP respectively at 216 nm QM A2 / A1 QX ax2 / ax1 and QY ay2 / ay1. Quantitative estimation of AM and PQP in marketed Tablet Formulation Formulation Label claim for content drug is as follow Arterolane maleate: 150mg Piperaquine phosphate: 750mg Twenty tablets were finely powered. A quantity of powder equivalent was weighed and transferred to 100 ml volumetric flask. 60 ml methanol was added to the same flask and sonicated for 15 min. The volume was made up to 100 ml with methanol. The solution was first filtered using Whitman filter paper No.41 and than through 0.45 µ filters paper in order to remove the excipient. After filtration aliquots solutions were prepared by taking 5 ml sample stock solution. Volume was made up to 100 ml with methanol to produce of 5 µg/ml of AM and 25 µg/ml of PQP. VALIDATION OF DEVELOPED METHOD Linearity The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. Linear correlation was obtained between concentration vs absorbance of AM and PQP. The Linearity spectra and calibration curves of these two drugs at 276 nm and 216 nm are shown in Figure respectively. Calibration curve data of AM and PQP are shown in Table 1 and 2. Accuracy Accuracy is the closeness of the test results obtained by the method to the true value. To study the accuracy 20 tablets were taken and analysis of the same was carried out. Recovery studies were carried out by addition of standard drug to the sample at 3 different concentration levels 80 100 and 120 taking into consideration percentage purity of added bulk drug samples. Results are shown in table 3 and 4. Precision The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous sample. It provides an indication of random error in results and was expressed as RSD. Intermediate precision Reproducibility Variations of results within same day and amongst days are called as reproducibility. It includes following parameter Intra-day reproducibility A variation of results within same day is called intraday variation. It was determined by repeating calibration curve 3 times on same day. Results are shown in. Inter-day reproducibility Variation of results amongst day is called interday variation. It was determined by repeating calibration curve daily for 3 different days. Results are shown in. RESULTS AND DISCUSSION Linearity Table 1: linearity data for AM at 242 nm and 276 nm in methanol AM Concentration µg/ml Absorbance 242nm Concentration µg/ml Absorbance 276nm 2.5 0.262 2.5 0.181 3.75 0.385 3.75 0.265 5 0.532 5 0.375 6.25 0.685 6.25 0.458

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 652 8.75 0.882 8.75 0.675 Correlation coefficient: 0.995 Correlation coefficient: 0.998 Intercept:0.010 Intercept: 0.043 Slope: 0.019 Slope: 0.052 Regression Equation: y 0.019x+0.010 Regression Equation: y0.052x+0.043 LOD: 0.432 LOD: 0.657 LOQ: 1.315 LOQ: 1.992 Fig. 4: calibration curve for AM at 242 nm Fig. 5: calibration curve for PQP at 276 nm Table 2: Linearity data for PQP at 242 nm and 276 nm in methanol PQP Concentration µg/ml Absorbance 242nm Concentration µg/ml Absorbance 276nm 12.5 0.132 12.5 0.06 18.75 0.235 18.75 0.142 25 0.331 25 0.258 31.25 0.395 31.25 0.425 43.75 0.585 43.75 0.574 Correlation coefficient: 0.998 Correlation coefficient: 0.990 Intercept: 0.106 Intercept: 0.117 y 0.019x - 0.010 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 a r e a Conc.ppm arterolane y 0.052x - 0.043 0 0.2 0.4 0.6 0.8 0 2 4 6 8 10 a r e a Conc.ppm arterolane

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 653 Slope: 0.009 Slope: 0.019 Regression Equation: y 0.009x+0.106 Regression Equation: y0.019x+0.117 LOD: 0.403 LOD: 1.111 LOQ: 1.222 LOQ: 3.333 DISCUSSION AM and PQP were given linear response from 2.5-8.75 µg/ml and 12.5-43.5 µg/ml in Q- Absorbance Ratio method. Fig. 6: Calibration curve for PQP at 242 nm Fig. 7: Calibration curve for PQP at 276 nm ACCURACY Table 3: Table recovery data of AM for developed method level of recovery Concentration of Sample Taken μg/ml Concentration of Pure API spiked μg/ml Mean Total Concentration Found μg/ml Recovery Mean RSD 80 5 4 3.97 99.42 1.57 100 5 5 5.05 101.22 0.87 120 5 6 5.94 99.36 0.62 denotes average of three determination y 0.009x - 0.106 0 0.2 0.4 0.6 0.8 0 10 20 30 40 50 a r e a Conc.ppm piperaquine y 0.019x + 0.117 0 0.2 0.4 0.6 0.8 0 10 20 30 40 50 a r e a Conc.ppm piperaquine

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 654 Table 4: recovery data of PQP for developed method level of recovery Concentration of Sample Taken μg/ml Concentration of Pure API spiked μg/ml Mean Total Concentration Found μg/ml Recovery Mean RSD 80 12.5 10 9.97 99.46 1.17 100 12.5 12.5 12.42 99.46 1.08 120 12.5 15 15.11 100.80 1.53 denotes average of three determination DISCUSSION Result reveals that recovery of AM and PQP was within acceptance criteria given in ICH i.e. 98-102 METHOD PRECISION Intermediate precision Reproducibility The intra-day and inter-day precisions of the developed method was determined by analyzing corresponding responses in triplicate on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of AM 2.5 5 and 8.75μg/ml and PQP 12.5 25 and 43.5 μg/ml. Results were reported in terms of RSD. Table 5: Intra-day precision data for AM of 242 nm and 276 nm Concentration µg/ml Absorbance at 242 nm Absorbance at 276 nm Mean ± SD RSD Mean ± SD RSD 2.5 0.184 ± 0.002 1.129 0.194± 0.002 1.03 5 0.366 ± 0.002 0.567 0.359± 0.003 0.83 8.75 0.519 ± 0.004 0.778 0.500± 0.004 0.80 denotes average of three determination Table 6: Intra-day precision data for PQP of 242 nm and 276 nm Concentration µg/ml Absorbance at 242 nm Absorbance at 276 nm Mean ± SD RSD Mean ± SD RSD 12.5 0.133± 0.001 1.145 0.230± 0.002 0.86 25 0.335 ± 0.004 0.190 0.440± 0.003 0.68 43.5 0.582 ± 0.003 0.524 0.726± 0.004 0.55 denotes average of three determination Table 7: Inter-day precision for AM of 242 nm and 276 nm Concentration µg/ml Absorbance at 242 nm Absorbance at 276 nm Mean ± SD RSD Mean ± SD RSD 2.5 0.186 ± 0.001 0.819 0.197± 0.001 0.507 5 0.375 ± 0.003 0.935 0.365± 0.002 0.547 8.75 0.574 ± 0.003 0.611 0.611± 0.003 0.490 denotes average of three determination

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 655 Table 8: Inter-day precision for PQP of 242 nm and 276 nm Concentration µg/ml Absorbance at 242 nm Absorbance at 276 nm Mean ± SD RSD Mean ± SD RSD 12.5 0.136± 0.002 1.841 0.170± 0.002 1.170 25 0.335 ± 0.004 1.240 0.436± 0.003 0.688 43.5 0.587 ± 0.002 0.354 0.754± 0.001 0.132 denotes average of three determination DISCUSSION Result reveals that SD and RSD of AM and PQP was within acceptance criteria given in ICH i.e. less than 1 and less than 2 respectively. So the proposed method for estimation of AM and PQP in précised in nature. Quantitation estimation of AM and PQP marketed formulation The proposed method was evaluate in the assay of table formulation containing AM and PQP. Three replicate determinations were carried out on tablets. assay found was for AM and that for PQP was . Result is shown in table 9. Table 9: Quantitative estimation of AM and PQP in marketed formulation Parameters SYNARIAM TABLET AM PQP Actual Concentration μg/ml Concentration Obtained μg/ml Assay RSD Limit 150 750 146.60 739 97.74 98.53 1.690 0.488 90-110 90-110 denotes average of three determination DISCUSSION assay of AM and PQP was found in an acceptance limit so this method could be used for analysis of this combination. CONCLUSION The described method enables the quantification of AM and PQP in combined tablet dosage form. The validation data demonstrates good precision and accuracy which prove the reliability of proposed method. This method was based on the determination of graphical absorbance at two wavelengths one being Iso-absorptive point for the two drugs 242 nm and the other being the wavelength of AM 276nm. Hence this Q- Absorbance Ratio method can be used routinely for quantitative estimation of both components in solid dosage form. ACKNOWLEGEMENTS The authors are grateful thank to Gitar Laboratories Ahmadabad India for providing a gift sample of standard Arterolane maleate and Piperaquine Phosphate. REFERENCES 1. Yuxiang Dong Wittlin Sergio: The Structure- Activity Relationship of the Antimalarial Ozonide Arterolane OZ277. J Med Chem 53 2010 481-491. 2. Valecha N: Arterolane maleate plus Piperaquine phosphate for treatment of uncomplicated Plasmodium falciparum malaria: A comparative multicenter randomized clinical trial. Clin Infect Dis 55 2012 663-671

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Seju D P et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-52 2017 648-656 656 3. US Pharmacopoeia 34 NF 29. The United State Pharmacopoeia Convention Rockville sep2014 https://mc.usp.org/monograph/piperaquine-phosphate 4. Indian Pharmacopoeia 6 th Edn: ministry of health and Family welfare Indian pharmacopeia commission Ghaziabad. India 2014 1084. 5. Central Drugs Standard Control Organization. List of Drug Approved For Marketing in India 2011. http://www.cdsco.nic.in/NEW20DRUG20UPDATE.DOC 6. Tarning J Singtoroj T Anneberg Ashton AM and White NJ: Development and validation of an automated solid phase extraction and liquid chromatographic method for the determination of in urine. Journal of Pharmacy Biomed Analysis 111 2006 213-218. 7. Te-Yu Hung Devis TEM and Ilett KF: Measurement of piperaquine in plasma by liquid chromatography with ultraviolet absorbance detection 12 2003 93-101. 8. Wahajuddin Raju KS and Teneja I: Bioanalysis of antimalarials using liquid chromatography. TrAC Trends in Analysis Chemistry 42 2013 186-204. 9. Amin NC Blanchin MD Ake M and Fabre H: Capillary electrophorosis methods for the analysis of antimalarials. Journal of chromatoghrphy Analysis 1276 2013 1-11. 10. Lindegardh N Anneberg A White NJ and Day NJ: Development and validation of a liquid chromatographic- tandem mass spectrometric method for determination of Piperaquine in plasma: Stable isotope labeled internal standard does not always compensate for matrix effects. Journal of chromatography B 12 2008 227-236. 11. Hodem EM Zanolari B Mercier T Biollaz J Keiser J and Decosterd LA: A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma. Journal of chromatography B 101 2009 867-886. 12. Lindegardh N Tarning J Toi PVHie TT Farrar J Singhasivanon P White NJ Aston M and Day NP: Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry. Journal of Pharm biomed analysis 493 2009 768-773. 13. Kirchhorfer C Keiser J and Huwyler J: Development and validation of a liquid chromatography/ mass spectroscopy method for pharmacokinetic studies of OZ78 a fascocidal drug candidate. Journal of Chromatography B 87828 2010 2770-2774. How to cite this article: Seju D Patel Dr Neha Tiwari Mrs Vanita Marvaniya. Development and validation of q-absorbance ratio method for simultaneous estimation of arterolane maleate and piperaquine phosphate in pharmaceutical dosage form. Int J of Allied Med Sci and Clin Res 2017 52: 648-656. Source of Support: Nil. Conflict of Interest: None declared.

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