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A New Analytical Q-Absorbance Ratio Method Development and Validation for Simultaneous Estimation of Lamivudine and Isoniazid

A New Analytical Q-Absorbance Ratio Method Development and Validation for Simultaneous Estimation... Hindawi Publishing Corporation ISRN Spectroscopy Volume 2013, Article ID 912376, 5 pages http://dx.doi.org/10.1155/2013/912376 Research Article A New Analytical Q-Absorbance Ratio Method Development and Validation for Simultaneous Estimation of Lamivudine and Isoniazid Gitu Pandey and Brahmeshwar Mishra Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi Uttar Pradesh 221005, India Correspondence should be addressed to Brahmeshwar Mishra; bmishrabhu@rediffmail.com Received 27 August 2013; Accepted 17 November 2013 Academic Editors: D. Chattopadhyay, A. A. Ensa,fi B. Kiran, R. Schneider, and A. Toncelli Copyright © 2013 G. Pandey and B. Mishra. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A new UV spectrophotometric absorption ratio method was developed and validated for the simultaneous estimation of lamivudine and isoniazid. The method involved Q-absorption ratio analysis using two wavelengths, with one being the𝜆 of lamivudine max (272 nm,𝜆 ) and the other being the isoabsorptive point of both drugs (246 nm,𝜆 ). Beer’s law was obeyed in the concentration 2 1 range between 5 and 30𝜇 g/mL for both lamivudine and isoniazid. The results of a nalysis have been validated statistically and by recovery studies as per ICH guidelines. The accuracy ranged between 99.65 and 101.91% and Sandell’s sensitivity ranged between 0.0229 and 0.0347𝜇 g/cm . eTh method was found to be simple, precise, reproducible, rapid, and economical. Hence, it could be used in the analysis of laboratory samples and marketed formulations containing these two drugs in the future. 1. Introduction is phosphorylated intracellularly and inhibits HIV reverse transcriptaseaswellashepatitis Bvirus DNApolymerase. A recent WHO report on tuberculosis (TB) in 2012 has Most human DNA polymerases are not affected and sys- shown that there were an estimated 8.7 million incident temic toxicity of 3TC is low [5]. Isoniazid (INH), a rfi st casesofTBin2011(13%coinfectedwithHIV). eTh re were line antitubercular, is chemically 4-pyridinecarboxylic acid also 1.4 million deaths from TB, 990,000 deaths among hydrazide or isonicotinic acid hydrazide (Figure 2), having HIV-negative individuals and 430,000 among people who molecular formula C H N O and molecular weight 137.14 6 7 3 were HIV-positive [1]. Many TB carriers who are infected [4]. It acts by inhibiting the synthesis of mycolic acids which with HIV are 30 to 50 times more likely to develop active get attached to arabinogalactan to form part of mycobacterial TB than those without HIV [2]. HIV infected individuals cell wall. It is an essential component of all antitubercular are not only at a greater risk for acquiring TB but also regimens, unless the patient is not able to tolerate it or bacilli reactivation of latent TB infection is greatly increased due are resistant [5, 6]. eTh simultaneous analysis of these two to the fact that the very cells that hold the latent TB in drugs INH and LAM is highly desirable as this will allow check(theCD4+Tlymphocytes) areprecisely thecells more efficient clinical data generation in the patients who are that are rendered dysfunctional in HIV-infected individuals. coinfected with tuberculosis and AIDS and for quantitative eTh re are evidences to believe that the main factor for the estimation of these drugs in combination formulations which resurgence of TB has been the human immunodeficiency maybemarketedinthe future.Theabsorbanceratio method virus (HIV) [3]. Lamivudine (LAM), a leading antiretroviral is a method for simultaneous estimation of two components drug, also known as 3TC, is chemically 2(1H)-pyrimidinone, depending upon the property that the ratio of absorbances 4-amino-1-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]-, (2R- at any two wavelengths is a constant value independent of concentration or pathlength [7, 8]. An extensive and cis) (Figure 1) with molecular formula C H N O Sand 8 11 3 3 molecular weight 229.26 [4]. This deoxycytidine analogue intensive literature survey has revealed that there is no 2 ISRN Spectroscopy O N were prepared from the standard stock solution by diluting NH aliquots of working stock solutions appropriately. O 2.5. Calibration Curve (Linearity). Acalibration curvewas plotted over a concentration range of 5–30𝜇 g/mL for both LAM and INH. Accurately measured working stock solution HO of LAM (2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mL) and working stock solution of INH (2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mL) Figure 1: Structure of lamivudine. were transferred to two separate series of 50 mL volumetric flask and diluted up to the mark with phosphate buffer (pH 7.4). eTh absorbance of both solutions was taken at their respective𝜆 and at isoabsorptive point. The calibration max curves were constructed by plotting concentration against NH N absorbance where each reading was an average of three determinations. 2.6. Application of the Proposed Method for Estimation in Figure 2: Structure of isoniazid. Standard Laboratory Mixture. eTh absorptivity coefficient of both drugs was determined and the individual concentration of LAM and INH was determined using the following equations: absorption ratio method for simultaneous analysis of LAM and INH in pharmaceutical preparations. However it has 𝑄 −𝑄 𝐴 𝑀 𝑌 1 been used for simultaneous analysis of prednisolone and 𝐶 = × , INH 𝑄 −𝑄 𝑎 𝑋 𝑌 𝑋1 5-amino salicylic acid, valsartan, and hydrochlorothiazide, (1) metformin hydrochloride, and fenofibrate [ 9–11]. The present 𝑄 −𝑄 𝐴 𝑀 𝑋 1 𝐶 = × , work describes a simple, accurate, and precise absorption LAM 𝑄 −𝑄 𝑎 𝑌 𝑋 𝑌1 ratio method for simultaneous determination of these two drugs. The method was validated as per the current ICH where𝑄 =𝐴 /𝐴 ,𝑄 =𝑎 /𝑎 ,and𝑄 =𝑎 /𝑎 ;𝐴 and 𝑀 2 1 𝑋 𝑋2 𝑋1 𝑌 𝑌2 𝑌1 1 guidelines [12–14]. 𝐴 are the absorbance, of the mixture at 246 nm and 272 nm, respectively;𝑎 and𝑎 are absorptivities of INH and LAM, 𝑋1 𝑌1 respectively, at 246 nm;𝑎 and𝑎 are absorptivities of INH 𝑋2 𝑌2 2. Materials and Methods and LAM, respectively, at 272 nm. 2.1. Reagents and Apparatus. Lamivudine and isoniazid were obtained as gift samples from Mylan Laboratories, Nashik, 3. Method Validation Maharashtra, India, and Lupin Pharma Ltd., Pune, Maha- 3.1. Linearity and Range. Linearity, consisting of the basic rashtra, India, respectively. All other chemicals used were of elements input→ converter → output, is the assumption analytical grade. A double beam UV-Visible spectrophotome- that there is a straight line relationship between the input (𝑥 ) ter, model 1700, Shimadzu, Japan, with software UV Probe 2.10 and 1 cm quartz cell, was used for all analysis. and output (𝑦 ) variables that can be written mathematically by the expression𝑦=𝑓(𝑥) if the straight line crosses through the origin or by the expression𝑦=𝑓(𝑥)+𝛿 if the straight 2.2. Preparation of Standard Stock Solutions. Standard stock line does not cross through the origin. eTh linear range solution (1000𝜇 g/mL) of LAM and INH was prepared sep- corresponds to the valid interval of functional dependence arately by dissolving carefully weighed 100 mg of drug in of the signal on concentration or mass which assumes 100 mL volumetric flask and diluting up to the mark with homoscedasticity of the measurements over the linear range. phosphate bueff r (pH 7.4). Ten mL of this solution was diluted The linear response of LAM and INH was determined by up to 100 mL with phosphate buffer (pH 7.4) to get working analyzing vfi e independent levels of the calibration curve in stock solution (100𝜇 g/mL). the range of 5–30𝜇 g/mL. 2.3. Determination of Isoabsorptive Point and Wavelength 3.2. Precision. The term precision is defined by the ISO of Maximum Absorbance (𝜆 ). Solutions of 10𝜇 g/mL of max International Vocabulary of Basic and General Terms in both drugs were prepared from working stock solution and Metrology (ISO-VIM) and ICH as the closeness of agreement scannedinthe rangeof200nm to 400nmagainst phosphate between quantity values obtained by replicate measurements bueff r (pH 7.4) as blank. The overlaying spectrum was also of a quantity under specified conditions [ 11]. Assessing the obtained to determine isoabsorptive point. precision implies expressing numerically the random error or thedegreeofdispersionofasetofindividualmeasurements 2.4. Preparation of Sample Solutions from Standard Stock by means of the standard deviation, the variance, or the Solution. The sample solutions of various concentrations coefficient of variation. ISRN Spectroscopy 3 3.3. Repeatability (Within-Run Precision). It is the concor- dance of a series of measurements of the same quantity when the experiments are conducted under same conditions (ana- lyst,apparatus,instrument, andday)inarapidsuccession. 0.8 For this experiment, standard solution of LAM and INH (15+15𝜇 g/mL) was prepared and analyzed six times as per the proposed method. 0.6 3.4. Intermediate Precision (Between-Run Precision). It is the concordance of a series of measurements of the same quan- tity when the experiments are conducted within the same 0.4 laboratory under different conditions (analyst, apparatus, instrument, and day). Standard solution of LAM and INH (15 + 15𝜇 g/mL) was prepared and analyzed as per the 0.2 proposed method. 3.5. Accuracy (% Recovery). eTh accuracy of an analytical procedure expresses the closeness of agreement between 200 300 400 the value which is accepted either as a conventional true Wavelength (nm) valueoranaccepted referencevalue andthe valuefound. The recovery experiments were carried out in triplicate by Figure 3: UV scan of LAM and INH showing isoabsorptive points. spiking previously analyzed samples with three different concentrations of standards. 1.4 3.6. Limit of Detection (LOD) and Limit of Quanticfi ation 1.2 (LOQ). The detection limit of an individual analytical proce- dure is the lowest amount of analyte in the sample which can be detected but not necessarily quantitated as an exact value. eTh quantitation limit of an individual analytical procedure 0.8 is the lowest amount of analyte in the sample which can 0.6 be quantitatively determined with suitable precision and y = 0.0405x + 0.0191 accuracy.TheLOD andLOQ of theproposedmethodwere R = 0.9989 0.4 determined by using calibration curve: y = 0.0266x + 0.0065 0.2 R = 0.999 3.3𝜎 10𝜎 LOD= , LOQ= , (2) 𝑆 𝑆 010 20 30 40 where 𝜎 is the standard deviation of the response (Y- Concentration (𝜇 g/mL) intercept) and S is the slope of the calibration curve. Abs. of LAM at 272 nm Abs. of INH at 272 nm 3.7. Sandell’s Sensitivity. Sandell’s sensitivity, the concentra- Figure 4: Calibration curve of LAM and INH at 272 nm. tion of the analyte (in𝜇 g/mL or𝜇 g/cm ) which will give an absorbance of 0.001 in a cell of path length 1 cm, was calculated. It gives valuable information regarding sensitivity of the method. both wavelengths 246 nm and 272 nm. eTh representative linear equationswerecalculatedbythe leastsquares method and the correlation coefficients have indicated very good 4. Results and Discussion linearity (Table 1). Evaluation of repeatability and interme- The solutions of 10 𝜇 g/mL of both LAM and INH were diate precision was done and coefficients of variation (CV) analyzed and the𝜆 was found to be 272 nm and 262 nm, or percent relative standard deviation (%RSD) values were max respectively.Threeisoabsorptive points:246nm,257nm,and calculated. es Th e values were found to be less than two (CV < 292 nm were found in overlaying spectra (Figure 3)and the 2), indicating good precision (Table 2). Good accuracy of isoabsorptive point 246 nm was selected for further analysis. the proposed method was proved by good percent recovery The calibration curve of LAM and INH individually in standard addition method. It ranged between 99.65 and andthe mixtureofbothdrugs at 272nm(𝜆 )and 246nm 101.91% for LAM and 101.26 and 100.12% for INH (Table 3). (𝜆 ) were plotted (Figures 4, 5,and 6). The relationship The limit of detection of LAM and INH at isoab- between the absorbance and the concentration of LAM and sorptive point (246 nm) was found to be 0.106𝜇 g/mL INH was found to be linear in the range of 5–30𝜇 g/mL at and 0.078𝜇 g/mL. The LOD at 272 nm was found to be Absorbance Absorbance 4 ISRN Spectroscopy Table 1: Calibration points of standard curve with standard deviation (SD) and %RSD. At 246 nm At 272 nm Concentration LAM INH LAM INH of the solution Mean Mean Mean Mean (𝜇 g/mL) absorbance± %RSD absorbance± %RSD absorbance± %RSD absorbance± %RSD SD (𝑛=3 ) SD (𝑛=3 ) SD (𝑛=3 ) SD (𝑛=3 ) 5 0.152±0.0015 1.007159 0.149±0.0020 1.400224 0.222±0.0025 1.132 0.144±0.0026 1.83733 10 0.288±0.0026 0.918664 0.287±0.0025 0.875851 0.435±0.0026 0.608 0.281±0.0049 1.75756 15 0.429±0.0040 0.941332 0.419±0.0040 0.965315 0.645±0.0035 0.544 0.409±0.0040 0.98894 20 0.569±0.0035 0.617565 0.538±0.0036 0.670177 0.836±0.007 0.837 0.525±0.0045 0.85945 25 0.715±0.0046 0.64092 0.687±0.0057 0.828093 1.038±0.0060 0.581 0.661±0.0065 0.98383 30 0.834±0.0055 0.660116 0.802±0.0060 0.751273 1.214±0.0095 0.786 0.813±0.0035 0.43214 0.9 Table 2: Intraday and intermediate precision study. 0.8 % Estimation % Estimation of 0.7 Precision of LAM± SD %RSD INH± SD %RSD 0.6 (𝑛=6 ) (𝑛=6 ) 0.5 Intraday 99.88±0.56 0.562 99.92±0.84 0.844 precision 0.4 y = 0.0279x + 0.0079 Intermediate 0.3 99.79±0.53 0.531 99.82±0.86 0.861 R = 0.9994 precision 0.2 y = 0.0267x + 0.0118 R = 0.999 0.1 Table 3: Results of recovery studies of LAM and INH. 0 10203040 Concentration (𝜇 g/mL) Amount Amount Amount %Recovery± Drug taken added found SD (𝑛=3 ) Abs. of LAM (𝜇 g/mL) (𝜇 g/mL) (𝜇 g/mL) Abs. of INH 10 1 11.14 101.26±0.40 Figure 5: Calibration curve of LAM and INH at 246 nm. INH 10 2 12.02 100.17±0.87 10 3 13.02 100.12±0.98 15 1 16.31 101.91±0.68 2.5 LAM 15 2 17.15 100.90±0.57 15 3 17.93 99.65±1.23 1.5 0.563𝜇 g/mL and 0.639𝜇 g/mL for LAM and INH, respec- tively. Sandell’s sensitivity of LAM and INH at 246 nm was y = 0.0554x + 0.003 found to be 0.0347 and 0.0348𝜇 g/cm .At272nm it was R = 0.9998 0.5 0.0229 and 0.0356𝜇 g/cm for LAM and INH, respectively, y = 0.0675x + 0.0118 R = 0.9998 which indicates good sensitivity of the method. Various 0 validation parameters have been summarized in Table 4. 0 102030 40 Concentration (𝜇 g/mL) 5. Conclusion Abs. of LAM + INH at 246 nm Abs. of LAM + INH at 272 nm The UV spectrophotometric Q-absorption ratio method was developed and validated for the simultaneous analysis of Figure 6: Calibration curve of LAM + INH at 272 nm and 246 nm. LAM and INH. eTh results together established that the method is simple, accurate, precise, reproducible, rapid, and sensitive. The method could be applied successfully and 0.186𝜇 g/mL and 0.211𝜇 g/mL for LAM and INH, respec- economically for the simultaneous estimation of LAM and tively. eTh limit of quanticfi ation of LAM and INH at INH in laboratory samples for efficient data generation and isoabsorptive point (246 nm) was found to be 0.321𝜇 g/mL for combination formulations of these two drugs in the and 0.238𝜇 g/mL. The LOQ at 272 nm was found to be future. Absorbance Absorbance ISRN Spectroscopy 5 Table 4: Summary of regression characteristics and validation [9] G.Singh,D.Kumar,D.Sharma, M. Singh, andS.Kaur, “Q- parameters. Absorbance ratio spectrophotometric method for the simulta- neous estimation of prednisolone and 5-Amino salicylic acid in LAM INH tablet dosage form,” Journal of Applied Pharmaceutical Science, Parameters vol. 2, no. 6, pp. 222–226, 2012. 246 nm 272 nm 246 nm 272 nm [10] A. B. Chaudhary, R. K. Patel, S. A. Chaudhary, and K. V. Beer’s law limit 5–30 5–30 5–30 5–30 Gadhvi, “Estimation of valsartan and hydrochlorothiazide in (𝜇 g/mL) pharmaceutical dosage forms by absorption ratio method,” Absorptivity 287 435 288 276 International Journal of Applied Biology and Pharmaceutical Regression equation Technology,vol.1,no. 2, pp.455–464,2010. (𝑦=𝑚𝑥+𝑐 ) [11] P. C. Bhamare, S. B. Bari, S. Natarajan, A. A. Patil, S. H. Slope (𝑚 ) 0.0279 0.0405 0.0267 0.0266 Patil, and P. T. Shirode, “A new analytical method development Intercept (𝑐 ) 0.0079 0.0191 0.0118 0.0065 and validation of metformin hydrochloride and fenob fi rate Correlation coefficient by absorbance ratio UV spectrophotometric method,” Asian 0.9994 0.9989 0.999 0.999 (𝑟 ) Journal of Biochemical and Pharmaceutical Research,vol.1,no. Standard deviation 2, pp. 115–128, 2011. 0.0009 0.0023 0.0006 0.0017 (SD) [12] ICH, “Q2A, Validation of analytical methods: definition and terminology,” 1994. LOD (𝜇 g/mL) 0.106 0.186 0.078 0.211 [13] Validation of Analytical Procedures: Text and Methodology Q2 LOQ (𝜇 g/mL) 0.321 0.563 0.238 0.639 (R1), ICH Harmonised Tripartite Guideline, London, UK, 2005. Sandell’s sensitivity 0.0347 0.0229 0.0348 0.0356 [14] ISO-VIM, International Vocabulary of Basic and General Terms (𝜇 g/cm ) in Metrology, International Organisation for Standardization, Conflict of Interests eTh authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments eTh authors are thankful to Mylan Laboratories, Nashik, Maharashtra, India, and Lupin Pharma Ltd., Pune, Maha- rashtra, India, for providing gift samples of pure drug lamivu- dine and isoniazid, respectively, and Department of Phar- maceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi, UP, for research support. References [1] http://apps.who.int/iris/bitstream/10665/75938/1/97892415645 02 eng.pdf. [2] Global Aids Response Progress Reporting,UNAIDS, Geneva, Switzerland, 2012. [3] D. Aerts and R. Jobim, “eTh epidemiological profile of tubercu- losis in southern Brazil in times of AIDS,” International Journal of Tuberculosis and Lung Disease,vol.8,no.6,pp.785–791,2004. [4] United States Pharmacopoeia 30/National Formulary 25, United States Pharmacopoeial Convention, Rockville, Md, USA, 2007. [5] K. D. Tripathi, Essentials of Medical Pharmacology,Jaypee Brothers Medical, New Delhi, India, 6th edition, 2009. [6] L.L.Brunton,J.S.Lazo,andK.L.Parker, Goodman and Gilman’s the Pharmacological Basis of eTh rapeutics , McGraw Hill, New York,NY, USA, 11thedition,2006. [7] A.H.Beckett andJ.B.Stenlake, Practical Pharmaceutical Chemistry—Part Two, CBS, New Delhi, India, 4th edition, 2005. [8] R. C. Hirt, F. T. King, and R. G. Schmitt, “Graphical absorbance- ratio method for rapid two-component spectrophotometric analysis,” Analytical Chemistry,vol.26, no.8,pp. 1270–1273, 1954. 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A New Analytical Q-Absorbance Ratio Method Development and Validation for Simultaneous Estimation of Lamivudine and Isoniazid

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Hindawi Publishing Corporation ISRN Spectroscopy Volume 2013, Article ID 912376, 5 pages http://dx.doi.org/10.1155/2013/912376 Research Article A New Analytical Q-Absorbance Ratio Method Development and Validation for Simultaneous Estimation of Lamivudine and Isoniazid Gitu Pandey and Brahmeshwar Mishra Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi Uttar Pradesh 221005, India Correspondence should be addressed to Brahmeshwar Mishra; bmishrabhu@rediffmail.com Received 27 August 2013; Accepted 17 November 2013 Academic Editors: D. Chattopadhyay, A. A. Ensa,fi B. Kiran, R. Schneider, and A. Toncelli Copyright © 2013 G. Pandey and B. Mishra. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A new UV spectrophotometric absorption ratio method was developed and validated for the simultaneous estimation of lamivudine and isoniazid. The method involved Q-absorption ratio analysis using two wavelengths, with one being the𝜆 of lamivudine max (272 nm,𝜆 ) and the other being the isoabsorptive point of both drugs (246 nm,𝜆 ). Beer’s law was obeyed in the concentration 2 1 range between 5 and 30𝜇 g/mL for both lamivudine and isoniazid. The results of a nalysis have been validated statistically and by recovery studies as per ICH guidelines. The accuracy ranged between 99.65 and 101.91% and Sandell’s sensitivity ranged between 0.0229 and 0.0347𝜇 g/cm . eTh method was found to be simple, precise, reproducible, rapid, and economical. Hence, it could be used in the analysis of laboratory samples and marketed formulations containing these two drugs in the future. 1. Introduction is phosphorylated intracellularly and inhibits HIV reverse transcriptaseaswellashepatitis Bvirus DNApolymerase. A recent WHO report on tuberculosis (TB) in 2012 has Most human DNA polymerases are not affected and sys- shown that there were an estimated 8.7 million incident temic toxicity of 3TC is low [5]. Isoniazid (INH), a rfi st casesofTBin2011(13%coinfectedwithHIV). eTh re were line antitubercular, is chemically 4-pyridinecarboxylic acid also 1.4 million deaths from TB, 990,000 deaths among hydrazide or isonicotinic acid hydrazide (Figure 2), having HIV-negative individuals and 430,000 among people who molecular formula C H N O and molecular weight 137.14 6 7 3 were HIV-positive [1]. Many TB carriers who are infected [4]. It acts by inhibiting the synthesis of mycolic acids which with HIV are 30 to 50 times more likely to develop active get attached to arabinogalactan to form part of mycobacterial TB than those without HIV [2]. HIV infected individuals cell wall. It is an essential component of all antitubercular are not only at a greater risk for acquiring TB but also regimens, unless the patient is not able to tolerate it or bacilli reactivation of latent TB infection is greatly increased due are resistant [5, 6]. eTh simultaneous analysis of these two to the fact that the very cells that hold the latent TB in drugs INH and LAM is highly desirable as this will allow check(theCD4+Tlymphocytes) areprecisely thecells more efficient clinical data generation in the patients who are that are rendered dysfunctional in HIV-infected individuals. coinfected with tuberculosis and AIDS and for quantitative eTh re are evidences to believe that the main factor for the estimation of these drugs in combination formulations which resurgence of TB has been the human immunodeficiency maybemarketedinthe future.Theabsorbanceratio method virus (HIV) [3]. Lamivudine (LAM), a leading antiretroviral is a method for simultaneous estimation of two components drug, also known as 3TC, is chemically 2(1H)-pyrimidinone, depending upon the property that the ratio of absorbances 4-amino-1-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]-, (2R- at any two wavelengths is a constant value independent of concentration or pathlength [7, 8]. An extensive and cis) (Figure 1) with molecular formula C H N O Sand 8 11 3 3 molecular weight 229.26 [4]. This deoxycytidine analogue intensive literature survey has revealed that there is no 2 ISRN Spectroscopy O N were prepared from the standard stock solution by diluting NH aliquots of working stock solutions appropriately. O 2.5. Calibration Curve (Linearity). Acalibration curvewas plotted over a concentration range of 5–30𝜇 g/mL for both LAM and INH. Accurately measured working stock solution HO of LAM (2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mL) and working stock solution of INH (2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mL) Figure 1: Structure of lamivudine. were transferred to two separate series of 50 mL volumetric flask and diluted up to the mark with phosphate buffer (pH 7.4). eTh absorbance of both solutions was taken at their respective𝜆 and at isoabsorptive point. The calibration max curves were constructed by plotting concentration against NH N absorbance where each reading was an average of three determinations. 2.6. Application of the Proposed Method for Estimation in Figure 2: Structure of isoniazid. Standard Laboratory Mixture. eTh absorptivity coefficient of both drugs was determined and the individual concentration of LAM and INH was determined using the following equations: absorption ratio method for simultaneous analysis of LAM and INH in pharmaceutical preparations. However it has 𝑄 −𝑄 𝐴 𝑀 𝑌 1 been used for simultaneous analysis of prednisolone and 𝐶 = × , INH 𝑄 −𝑄 𝑎 𝑋 𝑌 𝑋1 5-amino salicylic acid, valsartan, and hydrochlorothiazide, (1) metformin hydrochloride, and fenofibrate [ 9–11]. The present 𝑄 −𝑄 𝐴 𝑀 𝑋 1 𝐶 = × , work describes a simple, accurate, and precise absorption LAM 𝑄 −𝑄 𝑎 𝑌 𝑋 𝑌1 ratio method for simultaneous determination of these two drugs. The method was validated as per the current ICH where𝑄 =𝐴 /𝐴 ,𝑄 =𝑎 /𝑎 ,and𝑄 =𝑎 /𝑎 ;𝐴 and 𝑀 2 1 𝑋 𝑋2 𝑋1 𝑌 𝑌2 𝑌1 1 guidelines [12–14]. 𝐴 are the absorbance, of the mixture at 246 nm and 272 nm, respectively;𝑎 and𝑎 are absorptivities of INH and LAM, 𝑋1 𝑌1 respectively, at 246 nm;𝑎 and𝑎 are absorptivities of INH 𝑋2 𝑌2 2. Materials and Methods and LAM, respectively, at 272 nm. 2.1. Reagents and Apparatus. Lamivudine and isoniazid were obtained as gift samples from Mylan Laboratories, Nashik, 3. Method Validation Maharashtra, India, and Lupin Pharma Ltd., Pune, Maha- 3.1. Linearity and Range. Linearity, consisting of the basic rashtra, India, respectively. All other chemicals used were of elements input→ converter → output, is the assumption analytical grade. A double beam UV-Visible spectrophotome- that there is a straight line relationship between the input (𝑥 ) ter, model 1700, Shimadzu, Japan, with software UV Probe 2.10 and 1 cm quartz cell, was used for all analysis. and output (𝑦 ) variables that can be written mathematically by the expression𝑦=𝑓(𝑥) if the straight line crosses through the origin or by the expression𝑦=𝑓(𝑥)+𝛿 if the straight 2.2. Preparation of Standard Stock Solutions. Standard stock line does not cross through the origin. eTh linear range solution (1000𝜇 g/mL) of LAM and INH was prepared sep- corresponds to the valid interval of functional dependence arately by dissolving carefully weighed 100 mg of drug in of the signal on concentration or mass which assumes 100 mL volumetric flask and diluting up to the mark with homoscedasticity of the measurements over the linear range. phosphate bueff r (pH 7.4). Ten mL of this solution was diluted The linear response of LAM and INH was determined by up to 100 mL with phosphate buffer (pH 7.4) to get working analyzing vfi e independent levels of the calibration curve in stock solution (100𝜇 g/mL). the range of 5–30𝜇 g/mL. 2.3. Determination of Isoabsorptive Point and Wavelength 3.2. Precision. The term precision is defined by the ISO of Maximum Absorbance (𝜆 ). Solutions of 10𝜇 g/mL of max International Vocabulary of Basic and General Terms in both drugs were prepared from working stock solution and Metrology (ISO-VIM) and ICH as the closeness of agreement scannedinthe rangeof200nm to 400nmagainst phosphate between quantity values obtained by replicate measurements bueff r (pH 7.4) as blank. The overlaying spectrum was also of a quantity under specified conditions [ 11]. Assessing the obtained to determine isoabsorptive point. precision implies expressing numerically the random error or thedegreeofdispersionofasetofindividualmeasurements 2.4. Preparation of Sample Solutions from Standard Stock by means of the standard deviation, the variance, or the Solution. The sample solutions of various concentrations coefficient of variation. ISRN Spectroscopy 3 3.3. Repeatability (Within-Run Precision). It is the concor- dance of a series of measurements of the same quantity when the experiments are conducted under same conditions (ana- lyst,apparatus,instrument, andday)inarapidsuccession. 0.8 For this experiment, standard solution of LAM and INH (15+15𝜇 g/mL) was prepared and analyzed six times as per the proposed method. 0.6 3.4. Intermediate Precision (Between-Run Precision). It is the concordance of a series of measurements of the same quan- tity when the experiments are conducted within the same 0.4 laboratory under different conditions (analyst, apparatus, instrument, and day). Standard solution of LAM and INH (15 + 15𝜇 g/mL) was prepared and analyzed as per the 0.2 proposed method. 3.5. Accuracy (% Recovery). eTh accuracy of an analytical procedure expresses the closeness of agreement between 200 300 400 the value which is accepted either as a conventional true Wavelength (nm) valueoranaccepted referencevalue andthe valuefound. The recovery experiments were carried out in triplicate by Figure 3: UV scan of LAM and INH showing isoabsorptive points. spiking previously analyzed samples with three different concentrations of standards. 1.4 3.6. Limit of Detection (LOD) and Limit of Quanticfi ation 1.2 (LOQ). The detection limit of an individual analytical proce- dure is the lowest amount of analyte in the sample which can be detected but not necessarily quantitated as an exact value. eTh quantitation limit of an individual analytical procedure 0.8 is the lowest amount of analyte in the sample which can 0.6 be quantitatively determined with suitable precision and y = 0.0405x + 0.0191 accuracy.TheLOD andLOQ of theproposedmethodwere R = 0.9989 0.4 determined by using calibration curve: y = 0.0266x + 0.0065 0.2 R = 0.999 3.3𝜎 10𝜎 LOD= , LOQ= , (2) 𝑆 𝑆 010 20 30 40 where 𝜎 is the standard deviation of the response (Y- Concentration (𝜇 g/mL) intercept) and S is the slope of the calibration curve. Abs. of LAM at 272 nm Abs. of INH at 272 nm 3.7. Sandell’s Sensitivity. Sandell’s sensitivity, the concentra- Figure 4: Calibration curve of LAM and INH at 272 nm. tion of the analyte (in𝜇 g/mL or𝜇 g/cm ) which will give an absorbance of 0.001 in a cell of path length 1 cm, was calculated. It gives valuable information regarding sensitivity of the method. both wavelengths 246 nm and 272 nm. eTh representative linear equationswerecalculatedbythe leastsquares method and the correlation coefficients have indicated very good 4. Results and Discussion linearity (Table 1). Evaluation of repeatability and interme- The solutions of 10 𝜇 g/mL of both LAM and INH were diate precision was done and coefficients of variation (CV) analyzed and the𝜆 was found to be 272 nm and 262 nm, or percent relative standard deviation (%RSD) values were max respectively.Threeisoabsorptive points:246nm,257nm,and calculated. es Th e values were found to be less than two (CV < 292 nm were found in overlaying spectra (Figure 3)and the 2), indicating good precision (Table 2). Good accuracy of isoabsorptive point 246 nm was selected for further analysis. the proposed method was proved by good percent recovery The calibration curve of LAM and INH individually in standard addition method. It ranged between 99.65 and andthe mixtureofbothdrugs at 272nm(𝜆 )and 246nm 101.91% for LAM and 101.26 and 100.12% for INH (Table 3). (𝜆 ) were plotted (Figures 4, 5,and 6). The relationship The limit of detection of LAM and INH at isoab- between the absorbance and the concentration of LAM and sorptive point (246 nm) was found to be 0.106𝜇 g/mL INH was found to be linear in the range of 5–30𝜇 g/mL at and 0.078𝜇 g/mL. The LOD at 272 nm was found to be Absorbance Absorbance 4 ISRN Spectroscopy Table 1: Calibration points of standard curve with standard deviation (SD) and %RSD. At 246 nm At 272 nm Concentration LAM INH LAM INH of the solution Mean Mean Mean Mean (𝜇 g/mL) absorbance± %RSD absorbance± %RSD absorbance± %RSD absorbance± %RSD SD (𝑛=3 ) SD (𝑛=3 ) SD (𝑛=3 ) SD (𝑛=3 ) 5 0.152±0.0015 1.007159 0.149±0.0020 1.400224 0.222±0.0025 1.132 0.144±0.0026 1.83733 10 0.288±0.0026 0.918664 0.287±0.0025 0.875851 0.435±0.0026 0.608 0.281±0.0049 1.75756 15 0.429±0.0040 0.941332 0.419±0.0040 0.965315 0.645±0.0035 0.544 0.409±0.0040 0.98894 20 0.569±0.0035 0.617565 0.538±0.0036 0.670177 0.836±0.007 0.837 0.525±0.0045 0.85945 25 0.715±0.0046 0.64092 0.687±0.0057 0.828093 1.038±0.0060 0.581 0.661±0.0065 0.98383 30 0.834±0.0055 0.660116 0.802±0.0060 0.751273 1.214±0.0095 0.786 0.813±0.0035 0.43214 0.9 Table 2: Intraday and intermediate precision study. 0.8 % Estimation % Estimation of 0.7 Precision of LAM± SD %RSD INH± SD %RSD 0.6 (𝑛=6 ) (𝑛=6 ) 0.5 Intraday 99.88±0.56 0.562 99.92±0.84 0.844 precision 0.4 y = 0.0279x + 0.0079 Intermediate 0.3 99.79±0.53 0.531 99.82±0.86 0.861 R = 0.9994 precision 0.2 y = 0.0267x + 0.0118 R = 0.999 0.1 Table 3: Results of recovery studies of LAM and INH. 0 10203040 Concentration (𝜇 g/mL) Amount Amount Amount %Recovery± Drug taken added found SD (𝑛=3 ) Abs. of LAM (𝜇 g/mL) (𝜇 g/mL) (𝜇 g/mL) Abs. of INH 10 1 11.14 101.26±0.40 Figure 5: Calibration curve of LAM and INH at 246 nm. INH 10 2 12.02 100.17±0.87 10 3 13.02 100.12±0.98 15 1 16.31 101.91±0.68 2.5 LAM 15 2 17.15 100.90±0.57 15 3 17.93 99.65±1.23 1.5 0.563𝜇 g/mL and 0.639𝜇 g/mL for LAM and INH, respec- tively. Sandell’s sensitivity of LAM and INH at 246 nm was y = 0.0554x + 0.003 found to be 0.0347 and 0.0348𝜇 g/cm .At272nm it was R = 0.9998 0.5 0.0229 and 0.0356𝜇 g/cm for LAM and INH, respectively, y = 0.0675x + 0.0118 R = 0.9998 which indicates good sensitivity of the method. Various 0 validation parameters have been summarized in Table 4. 0 102030 40 Concentration (𝜇 g/mL) 5. Conclusion Abs. of LAM + INH at 246 nm Abs. of LAM + INH at 272 nm The UV spectrophotometric Q-absorption ratio method was developed and validated for the simultaneous analysis of Figure 6: Calibration curve of LAM + INH at 272 nm and 246 nm. LAM and INH. eTh results together established that the method is simple, accurate, precise, reproducible, rapid, and sensitive. The method could be applied successfully and 0.186𝜇 g/mL and 0.211𝜇 g/mL for LAM and INH, respec- economically for the simultaneous estimation of LAM and tively. eTh limit of quanticfi ation of LAM and INH at INH in laboratory samples for efficient data generation and isoabsorptive point (246 nm) was found to be 0.321𝜇 g/mL for combination formulations of these two drugs in the and 0.238𝜇 g/mL. The LOQ at 272 nm was found to be future. Absorbance Absorbance ISRN Spectroscopy 5 Table 4: Summary of regression characteristics and validation [9] G.Singh,D.Kumar,D.Sharma, M. Singh, andS.Kaur, “Q- parameters. Absorbance ratio spectrophotometric method for the simulta- neous estimation of prednisolone and 5-Amino salicylic acid in LAM INH tablet dosage form,” Journal of Applied Pharmaceutical Science, Parameters vol. 2, no. 6, pp. 222–226, 2012. 246 nm 272 nm 246 nm 272 nm [10] A. B. Chaudhary, R. K. Patel, S. A. Chaudhary, and K. V. Beer’s law limit 5–30 5–30 5–30 5–30 Gadhvi, “Estimation of valsartan and hydrochlorothiazide in (𝜇 g/mL) pharmaceutical dosage forms by absorption ratio method,” Absorptivity 287 435 288 276 International Journal of Applied Biology and Pharmaceutical Regression equation Technology,vol.1,no. 2, pp.455–464,2010. (𝑦=𝑚𝑥+𝑐 ) [11] P. C. Bhamare, S. B. Bari, S. Natarajan, A. A. Patil, S. H. Slope (𝑚 ) 0.0279 0.0405 0.0267 0.0266 Patil, and P. T. Shirode, “A new analytical method development Intercept (𝑐 ) 0.0079 0.0191 0.0118 0.0065 and validation of metformin hydrochloride and fenob fi rate Correlation coefficient by absorbance ratio UV spectrophotometric method,” Asian 0.9994 0.9989 0.999 0.999 (𝑟 ) Journal of Biochemical and Pharmaceutical Research,vol.1,no. Standard deviation 2, pp. 115–128, 2011. 0.0009 0.0023 0.0006 0.0017 (SD) [12] ICH, “Q2A, Validation of analytical methods: definition and terminology,” 1994. LOD (𝜇 g/mL) 0.106 0.186 0.078 0.211 [13] Validation of Analytical Procedures: Text and Methodology Q2 LOQ (𝜇 g/mL) 0.321 0.563 0.238 0.639 (R1), ICH Harmonised Tripartite Guideline, London, UK, 2005. Sandell’s sensitivity 0.0347 0.0229 0.0348 0.0356 [14] ISO-VIM, International Vocabulary of Basic and General Terms (𝜇 g/cm ) in Metrology, International Organisation for Standardization, Conflict of Interests eTh authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments eTh authors are thankful to Mylan Laboratories, Nashik, Maharashtra, India, and Lupin Pharma Ltd., Pune, Maha- rashtra, India, for providing gift samples of pure drug lamivu- dine and isoniazid, respectively, and Department of Phar- maceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi, UP, for research support. References [1] http://apps.who.int/iris/bitstream/10665/75938/1/97892415645 02 eng.pdf. [2] Global Aids Response Progress Reporting,UNAIDS, Geneva, Switzerland, 2012. [3] D. Aerts and R. Jobim, “eTh epidemiological profile of tubercu- losis in southern Brazil in times of AIDS,” International Journal of Tuberculosis and Lung Disease,vol.8,no.6,pp.785–791,2004. [4] United States Pharmacopoeia 30/National Formulary 25, United States Pharmacopoeial Convention, Rockville, Md, USA, 2007. [5] K. D. Tripathi, Essentials of Medical Pharmacology,Jaypee Brothers Medical, New Delhi, India, 6th edition, 2009. [6] L.L.Brunton,J.S.Lazo,andK.L.Parker, Goodman and Gilman’s the Pharmacological Basis of eTh rapeutics , McGraw Hill, New York,NY, USA, 11thedition,2006. [7] A.H.Beckett andJ.B.Stenlake, Practical Pharmaceutical Chemistry—Part Two, CBS, New Delhi, India, 4th edition, 2005. [8] R. C. Hirt, F. T. King, and R. G. Schmitt, “Graphical absorbance- ratio method for rapid two-component spectrophotometric analysis,” Analytical Chemistry,vol.26, no.8,pp. 1270–1273, 1954. 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