Study of Ethylenediaminetetraacetic Acid (EDTA)

Indian E-Journal of Pharmaceutical Sciences 01[01] 2015 www.asdpub.com/index.php/iejps ISSN: 2454-5244 (Online)

Original Article

Zero order and area under curve spectrophotometric methods for determination of Aspirin in pharmaceutical formulation

Mali Audumbar Digambar*, Hake Gorakhnath, Bathe Ritesh

Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India

*Corresponding Author Abstract Mali Audumbar Digambar Objective: A simple, accurate, precise and specific zero order and area under curve Department of Pharmaceutics, spectrophotometric methods has been developed for determination of Aspirin in its tablet Sahyadri College of Pharmacy, Methwade, dosage form by using methanol as a solvent. Sangola-413307, Solapur, Maharashtra, India Methods: (1) Derivative Spectrophotometric Methods: The amplitudes in the zero order E -mail: [email protected] derivative of the resultant spectra at 224 nm was selected to find out Aspirin in its tablet dosage form by using methanol as a solvent. (2) Area under curve (Area calculation): The proposed area under curve method involves Keywords: measurement of area at selected wavelength ranges. Two wavelength ranges were selected Aspirin, 218-227 nm for estimation of Aspirin. UV visible Result & Discussion: The linearity was found to be 5-25 μg/ml for Aspirin. The mean % Spectrophotometry, recoveries were found to be 99.47% and 100.43% of zero order derivative and area under AUC, curve method of Aspirin. For Repeatability, Intraday precision, Interday precision, % RSD were Method Validation, found to be 0.6416, 0.0046 and 0.9819, 0.8112 for zero order and 0.7257, 0.8731 and 0.7528, Analgesic, Accuracy. 1.7943 for area under curve method respectively. Limit of Detection and Quantitation were found to be 0.4823μg/ml and 1.5182μg/ml for zero order and 0.8272μg/ml and 2.5351μg/ml for area under curve method respectively. Assay results of market formulation were found to be 100.54% for zero order and 100.89% area under curve method respectively. The proposed method has been validated as per ICH guidelines and successfully applied to the estimation of Aspirin in its Tablet dosage form. Conclusion: The developed methods can be concluded as accurate, sensitive and precise and can be easily applied to the pharmaceutical formulation.

1. Introduction 2. Materials and Methods Aspirin is an Aspirin (ASP); 2-acetoxy benzoic acid [Figure 1A] 2.1 Apparatus and instrumentation is cyclo oxygenase inhibitor. It is used as an analgesic, antipyretic, anti- A shimadzu 1800 UV/VIS double beam spectrophotometer inflammatory and anti thrombic agent.[1,2] It is one of the most widely with 1cm matched quartz cells was used for all spectral measurements. used anionic drugs in the world.[3,4,5] Once ingested, aspirin is rapidly Single Pan Electronic balance (CONTECH, CA 223, India) was used for hydrolyzed in the body to produce salicylic acid, which is the compound weighing purpose. Sonication of the solutions was carried out using that is primarily responsible for the pharmacological activity of an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India). Calibrated aspirin.[6,7] It suppresses the production of prostaglandins and volumetric glassware (Borosil®) was used for the validation study. thromboxanes due to inactivation of the cyclooxygenase enzyme.[8,9,10] 2.2 Materials Literature survey revealed several analytical methods UV- Reference standard of Aspirin API was supplied as gift sample spectrophotometry[11,12] and HPLC[13,14] have been reported in bulk, Cipala Health Care (Pune, India). Tablet sample with label claim 500 mg pharmaceutical dosage form for determination of Aspirin. To our notice, per tablet were purchased from local market Solapur. so far no UV- spectrophotometric method using Zero order and Area 2.3 Method development under Curve (AUC) has been reported for the determination of Aspirin in 2.3.1 Preparation of Standard and Sample Solutions bulk and tablets. Hence an attempt has been made to develop new Zero Stock solution of 10μg/ml of Aspirin was prepared in Methanol order and Area under Curve Spectrophotometric methods method for for zero order and area under the curve spectrophotometric analysis. The estimation of Aspirin in bulk and pharmaceutical formulations with good standard solutions were prepared by dilution of the stock solution with accuracy simplicity, precision and economy. Methanol in a concentration range of 5,10,15,20 and 25 μg/ml with Molecular formula: C24H26N2O4 Methanol or zero order and area under the curve spectrophotometric Molecular weight: 406.47424 g/mol methods. Methanol was used as a blank solution. [10,11]

Figure 1:- The structural formula of Aspirin. © ASD Publisher All rights reserved. 40 Mali Audumbar Digambar et al/ Zero order and area under curve spectrophotometric methods for determination of Aspirin in pharmaceutical formulation 2.3.2 Area under curve (Area calculation) Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2 representing start and end point of curve region. The area under curve between λ1 and λ2 was calculated using UV probe software. In this study area was integrated between wavelength ranges from 218-227 nm. λ1 Area calculation: (α+β) = λ2 퐴푑λ Where, α is area of portion bounded by curve data and a straight line connecting the start and end point, β is the area of portion bounded by a straight line connecting the start and end point on curve data and horizontal axis λ1 and λ2 are wavelength range start and end point of curve region[15]. Fig. 2 Zero order derivative spectrum of Aspirin in Methanol 2.3.3 Assay Procedure (20µg/ml). Twenty tablets each containing 500 mg of Aspirin were weighed crushed to powder and average weight was calculated. Powder equivalent to 500 mg of Aspirin was transferred in 100 ml of volumetric flask. A 50 ml of Methanol was added and sonicated for 15 minutes. Then solution was further diluted up to the mark with Methanol. The solution was filtered using Whatmann filter paper no. 41; first 5 ml of filtrate was discarded. This solution was further diluted to obtain 15µg/ml solution with water subjected for UV analysis using Methanol as blank. Appropriate dilutions were made with Methanol from stock solution for both zero order and area under the curve spectrophotometric methods. [14,15]

Fig. 3 UV AUC spectrum of Aspirin in Methanol (20µg/ml)

Table 1: Assay of tablet dosage form Sr. No. Sample Solution Amount found (%)* Amount found Mean % Found Mean % %RSD zero % RSD Concentration Zero order derivative (%)* Auc zero order Found Auc order derivative Auc (µg/ml) derivative 1 15 99.32 102.48 2 15 100.58 100.21 99.47 100.43 0.7513 0.8258 3 15 98.52 98.61 *n=3, % RSD = % Relative Standard Deviation.

Fig. 4 Zero order derivative spectrum of Aspirin in Methanol (25µg/ml).

3. Results and Discussion relationship. Regression analysis was made for the slope, intercept and The zero order and area under the curve spectra for Aspirin correlation coefficient values. The regression equations of calibration were recorded at the wavelength of 224 nm and 218-227 nm curves were y=0.021x-0.017 (r2=0.995) at 224nm for zero order respectively. derivative spectrophotometry and y=0.023x-0.028 (r2=0.988) at 218-227 3.1 Linearity and Range nm for area under the curve spectrophotometry. The range was found to Under the experimental conditions described, the graph be 5-25 μg/ml for both zero order and area under the curve obtained for zero order and area under the curve spectra showed linear spectrophotometric methods.

y = 0.021x - 0.017 0.6 R² = 0.995 0.5 0.4 0.3 Ab. 0.2 ab 0.1 Linear (ab) 0 -0.1 0 5 10 15 20 25 30

Conc.

Fig.5 Linearity of Aspirin by Absorbance

0.7 0.6 y = 0.023x - 0.028 0.5 R² = 0.988 0.4 ab 0.3 AUC 0.2 0.1 0 -0.1 0 5 10 15 20 25 30

Conc.

Fig.6 Linearity of Aspirin by AUC

Fig. 7 Zero order derivative overlay of Aspirin at diff. Concentration.

Table 2: Stastical data for the calibration graphs for determination of Aspirin by Proposed methods.

Parameters Zero order derivative Area Under the Curve Linearity range (µg/ml)* 5-25 5-25 r2 0.995 0.988

© ASD Publisher All rights reserved. 42 Mali Audumbar Digambar et al/ Zero order and area under curve spectrophotometric methods for determination of Aspirin in pharmaceutical formulation 3.2 Accuracy 120% levels of 15µg/ml standard solution. For Area under curve (AUC) To study the accuracy of the proposed methods and to check was measured in wavelength range 218-227 nm and for zero order the interference from excipients used in the dosage forms, recovery derivative at 224 nm and results were obtained in terms of percent experiments were carried out by the standard addition method. The recovery. Three determinations at each level were performed and % RSD accuracy for the analytical method was evaluated at 80%, 100% and was calculated for each level. Table 3: Accuracy results for Aspirin. Sample Total amnt. Accuracy Std. % Recovery % Recovery Mean of Zero Mean of Auc % RSD Zero % RSD conc Added level conc zero derivatie Auc* derivative* derivative* derivative Auc (µg/) (µg/m) 80 15 12 27 102.32 100.38 100 15 15 30 101.07 102.36 100.54 100.89 0.538 1.705 120 15 18 33 98.24 99.94 *n=3, % RSD = % Relative Standard Deviation. 3.3 Precision To determine the precision of the method, Aspirin solutions at Acknowledgement a concentration of 20 μg/ml were analysed each three times for both zero Authors were thankful to by Cipla Health Care (Pune, India) for order and area under the curve spectrophotometric methods. Solutions providing gift sample of Aspirin. We would also like to thanks Principal for the standard curves were prepared fresh every day. and Management of Sahyadri College of Pharmacy, Methwade, Sangola, Table 4: Results of Intra and Inter Day Precision Solapur, Maharashtra, India for providing all the facilities to complete Parameters Intra Day Inter Day this work successfully. Precision Precision S.D* % RSD* S.D* % RSD* References Zero derivative 0.6416 0.9819 0.0046 0.8112 [1] Vora D. N., Kadav A. A., “Validated Ultra HPLC Method for the Area under the 0.7257 0.7528 0.8731 1.7943 Simultaneous Determination of Atorvastatin, Aspirin and their curve Degradation Products in Capsules,” Journal of Liquid 3.4 Sensitivity Chromatography & Related Technologies.2008; 18(31): 2821‐2837. The limit of detection (LOD) and limit of quantification (LOQ) [2] Na Wang, Fengguo Xu, Zunjian Zhang, Cheng Yang, Xiuhong Sun, were calculated by using the equations LOD = 3xσ/ S and LOQ = 10xσ/S, Jinheng Li. “Simultaneous determination of dipyridamole and where σ is the standard deviation of intercept, S is the slope. The LOD and salicylic acid in human plasma by high performance liquid LOQ were found to be 0.4823μg/ml and 1.5182/ml respectively for zero chromatography‐mass spectrometry,”. Journal of Biomedical order derivative and The LOD and LOQ were found to be 0.8275µg/ml Chromatography.2007; 22(2): 149 –156. &2.5351 µg/ml for area under the curve methods respectively. [3] Weale A. R., Cooper D. G., Hentschel, D. M., Dixon, B. S.,Feldman, H. I. 3.5 Analysis of the Marketed Formulation “Effect of Dipyridamole plus Aspirin on Hemodialysis Graft There was no interference from the excipients commonly Patency.” The New England Journal of Medicine. 2009; 360(21): present in the tablets. The drug content was found to be 99.34% and 720‐721. 101.18% zero order and area under the curve spectrophotometric [4] Ting Qin, Feng Qin, Ning Li, Shan Lu, Wei Liu, Famei Li.“Quantitative methods respectively. It may therefore be inferred that degradation of determination of dipyridamole in human plasma by Aspirin had not occurred in the marketed formulations that were high‐performance liquid chromatography‐tandem mass analysed by this method. The low % R.S.D. value indicated the suitability spectrometry and its application to a pharmacokinetic study”. of this method for routine analysis of Ranitidine in pharmaceutical Journal of Biomedical Chromatography.2009; 5(2): 1283. dosage form. [5] Maria J. Nozal, Jose L. Bernal, Laura Toribio, Juan J. Jimenez, Maria T. Table 5: Summary of validation parameters Martin,“ Validation of the removal of acetylsalicylic acid. Recovery Parameter Zero derivative AUC and determination of residues on various surfaces by high λ range 200-400 nm 218-227nm performance liquid chromatographic”. Journal of Chromatography A. Regression Equation Y=0.021x-0.017 Y=0.023x-0.028 (2000); 870(2): 69 ‐ 75. (y=mx+c) [6] M. Gandhimathi, T.K. Ravi, Annie Abraham, Renu Thomas, Measured wavelength 224nm 224nm Linearity range 5-25µg/ml 5-25µg/ml “Simultaneous determination of aspirin and isosorbide 5‐ Slope 0.021 0.023 mononitrate in formulation by reversed phase high pressure liquid Intercept 0.017 0.028 chromatography. Journal of Pharmaceutical and Biomedical Analysis. Correlation coefficient (R2) 0.995 0.988 (2003); 32(1): 1145 ‐1148. Limit of Detection (LOD) 0.4823 0.8272 [7] Patel RB, Shankar MB, Patel MR, Bhatt K.K. Simultaneous estimation µg/ml of acetylsalicylic acid and clopidogrel bisulfate in pure powder and Limit of Quantitation (LOQ) 1.5182 2.5351 tablet formulations by high-performance column liquid µg/ml chromatography and high-performance thin-layer chromatography. Accuracy (Mean % Recovery) 100.54 100.89 J AOAC Int. 2008; 91(4): 750-755. Precission (%RSD) 0.538 1.705 [8] Anandakumar T, Ayyappan V, Raghu Raman, Vetrichelvan T, Sankar

ASK, Nagavalli D. RP-HPLC analysis of aspirin and clopidogrel 4. Conclusion bisulphate in combination. Indian J Pharm Sci. 2007; 69(4): 597- No UV or Area under Curve spectrophotometric methods have 599. been described for the determination of Aspirin. Therefore simple, fast [9] Londhe SV, Mulgund SV, Deshmukh RS, Jain K S. Simultaneous and reliable derivative spectrophotometric methods were developed for HPTLC analysis of aspirin, atorvastatin calcium and clopidogrel the routine determination of Aspirin. The developed methods can be bisulphate in the bulk drug and in capsules. Acta Chromatogr. 2010; concluded as accurate, sensitive and precise and can be easily applied to 22(2): 297-305. the pharmaceutical formulation. © ASD Publisher All rights reserved. 43 Mali Audumbar Digambar et al/ Zero order and area under curve spectrophotometric methods for determination of Aspirin in pharmaceutical formulation [10] Fayed AS, Weshahy SA, Shehata MA, Hassan NY, Pauwels J, [13] Pimpodkar NV, Nalawade RS, Kuchekar BS, Mahajan NS and Jadhav Hoogmartens J, Van Schepdael A. Separation and determination of RL. New spectrophotometric method for the estimation of Aspirin in clopidogrel and its impurities by capillary electrophoresis. J bulk and pharmaceutical formulation. Inter J Chem Sci. 2008; 6(2): Pharmaceut Biomed. 2009; 49(2): 193-200. 993-999. [11] Kalaichelvi R, Fatima Rose M, Vadivel K, Jayachandran E. Simple [14] Challa BR, Boddu SH, Awen BZ, Chandu BR, Bannoth CK, Khagga M, extractivecolorimetric determination of Aspirin sodium by acid dye Kanala K, Shaik RP.Development and validation of a sensitive complexation method in solid dosage form. Int J Chem Res. 2010; bioanalytical method for the quantitative estimation of Aspirin in 1(1): 6-8. human plasma samples by LC-MS/MS: application to bioequivalence [12] Kakde RB, Gedam SN, Chaudhary NK, Barsagade AG, Kale DL study. J Chromatogr B.2010; 878(19):1499-1505. Kasture AV. Three-wavelength spectrophotometric method for [15] International Conference on Harmonization (ICH) of Technical simultaneous estimation of Aspirin and domperidone in Requirements for the registration of Pharmaceuticals for Human pharmaceutical preparations. Inter J Pharm Tech Research. 2009; use, Validation of Analytical Procedures Methodology; ICH- 1(2): 386-389. Q2(R1), Geneva, 1996,1-8.