sd-sample article A.D.A. Aniñon and others 41 SCIENCE DILIMAN (JANUARY-JUNE 2014) 26:1 41-52 Spectrophotometric Determination of Losartan Potassium in Tablets _______________ *Corresponding Author ABSTRACT I n t h e q u a l i t y c o n t r o l o f p h a r m a c e u t i c a l p r o d u c t s , i t i s o f u t m o s t i m p o r t a n c e t h a t v a l i d a t e d a n a l y t i c a l m e t h o d s a r e u s e d t o e n s u r e t h e cred i b i l i t y of t h e r e s u l t s g e n e r a ted . At t h e t i m e of t h e s t u d y, of f i c i a l monographs from the United States Pharmacopeia and National Formulary (USP-NF) for the quantif ication of Losar tan potassium in tablets were unavailable, denoting the need for a validated analytical procedure for the analysis of the drug. The study adapted direct and f irst-derivative UV spectrophotometry methods proposed by Bonf ilio and others (2010) for the assay of Losar tan potassium in Losar tan 50 mg. capsules, then m o d i f i ed a n d v a l i d a ted t h e s a i d p r o ced u r e s fo r t h e a s s a y of Lo s a r t a n p o t a s s i u m i n Lo s a r t a n 1 0 0 m g . t a b l e t s f o l l o w i n g t h e I n t e r n a t i o n a l Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines on method validation for accuracy, precision, specif icity, linearity, limit of detection, and limit o f q u a n t i t a t i o n . Re s u l t s d e m o n s t r a t e d t h a t a l l t h e p e r f o r m a n c e characteristics of both methods were highly satisfactory and conf irmed the possible application of the methods in routine analysis of Losar tan potassium tablets. Keywords: Lo s a r t a n Po t a s s i u m , U V s p ec t r o p h o t o m e t r i c d e te r m i n a t i o n , assay validation, direct and f irst-derivative spectra, hypertension ISSN 0115-7809 Print / ISSN 2012-0818 Online Arianne Diane A. Aniñon Richard Simon R. Binos Karen Mae M. Brizuela Marlyn C. Corpuz W ill ison John E. de Luna Regine Phill ine S. del Rosario Jesus John C. Dimalala T imothy Joseph P. Dueñas Isaac Ireneo B. Linatoc Juan Paolo D. Recto Melanie V. Sal inas West Kristian D. Paraiso* University of the Philippines Manila Spectrophotometric Determination of Losar tan 42 SPECTROPHOTOMETRIC DETERMINATION OF LOSARTAN POTASSIUM IN TABLETS Hypertension is a condition characterized by persistently elevated arterial blood pressure (Wells and others 2009). It is considered as one of the most signif icant risk factors in the development of heart disease (Wells and others 2009), which has been identif ied as the leading cause of mortality in the Philippines (World Health Organization 2010). Angiotensin II receptor blockers (ARB) are among the primary agents used as f irst line treatment for hypertension with compelling indications. ARBs potently and selectively inhibit angiotensin II generally by competitive binding to the AT 1 - receptor. Losartan, an example of ARB, is approved for stroke prophylaxis and is well tolerated in patients with heart failure (Jackson 2006). It is available in two dosage strengths (50 mg.- and 100 mg.-tablet), in different tablet preparations (core and f ilm-coated), and in combination with hydrochlorothiazide (Wai Fun and others 2008). Although available literature has described several analytical methods for the assay of Losar tan at the time of the study, no pharmacopeia has yet described a monograph for Losartan drug products. The absence of a universal procedure deprives the public of an assurance on safety, quality and eff icacy; thus, validated analytical procedures are needed to quantify Losartan potassium found in various drug preparations. Two methods are presented in this paper by the researchers for the analysis of Losartan drug products. This study aimed to validate the conditions and modify, if necessary, the direct and f irst-derivative UV spectrophotometry methods to quantify Losartan potassium in 100 mg. tablets with an appropriate level of conf idence. This study is of significance to the pharmaceutical industry for the routine analysis of Losartan potassium in tablet dosage form. The study also contributes to safeguarding the public against the presence of low-quality Losartan drug products in the market. Lastly, the study provides new and additional knowledge which can be used for future method development for the quantif ication of Losartan in tablet dosage forms. The paper of Bonf ilio and others (2010) described an analytical procedure for the quantitative analysis of Losar tan in the capsule but not in the tablet form. By determining the validity of their analytical procedure as it applies to the quantitative analysis of Losartan in tablets, and applying the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) validation parameters, the robustness of the methodology developed by Bonf ilio and others (2010) were fur ther tested. A.D.A. Aniñon and others 43 The study evaluated the following performance characteristics of the two methods: specif icity, accuracy, precision, linearity, range, limit of detection (LOD), and limit of quantitation (LOQ). Due to limitations in resources, the study only used the available spectrophotometer at the College of Pharmacy, University of the Philippines Manila and a single brand of Losartan potassium 100 mg. tablet (Cozaar®) preparation. The study did not include simultaneous determination of possible degradation products or impurities. METHODS Sample Losartan potassium working standard with a purity of 99.8%, Lot 2008-0111-06, and expiration date of October 2011 was used as reference standard and was generously provided by the Institute of Pharmaceutical Sciences, National Institutes of Health, University of the Philippines Manila. Losartan potassium (Cozaar®) tablets, labeled 100 mg. Losartan potassium, were obtained from a local distributor and were used as samples for the experiment. The tablets were described to contain excipients such as microcrystalline cellulose, lactose hydrous, pregelatinized starch, magnesium stearate, hydroxypropyl cellulose, hypromellose and titanium dioxide (Merck and Co. 2010). Starch, magnesium stearate, microcrystalline cellulose, lactose, hydroxyethycellulose, hydroxymethylpropylcellulose and titanium dioxide used in the preparation of the simulated standard of excipients (SSE) were of analytical grade and were provided by the Department of Industrial Pharmacy, College of Pharmacy, University of the Philippines Manila. Locally manufactured distilled water was used as solvent in both standard and sample solutions. Instrument A double-beam Genesys 10S UV – Visible Spectrophotometer unit, with a VISIONlite™ SE software and 1.8 nm bandwidth, was used for the spectrophotometric determination. Sartorius AG analytical balance was used to weigh all reagents. All solutions were placed in a 1 cm. quartz cuvette during measurement. Procedure The direct and f irst-derivative spectrophotometric procedures developed and validated by Bonf ilio and others (2010) for the assay of Losartan potassium in Losartan 50 mg. capsules were validated in the study for the assay of Losartan potassium in Losartan 100 mg. tablets. Twenty tablets were accurately weighed and powdered. One hundred milligrams (100 mg.) of Losartan potassium was Spectrophotometric Determination of Losar tan 44 weighed and placed on a 100 mL-volumetric flask to which 40 mL of distilled water was added. The solution was sonicated and the flask was f illed to volume. The absorbance of the standard solution (1 mg/mL) was measured from 200-300nm for both zero- and f irst-order derivative spectrophotometry to obtain the . Aliquots of the standard solution equivalent to 0.1-2 mg. Losartan potassium were transferred into 10 mL volumetric flasks. The analytical curve was produced by plotting drug concentration versus the absorbance obtained, for both zero- order and f irst-order derivative spectrophotometry. The calibration curve was plotted after measuring the concentration and recording the regression equation (Youssef and Taha 2007). The method validation was performed following ICH specif ications for specif icity, accuracy, precision, linearity, range, limit of detection, and limit of quantitation. Specificity Specif icity was determined by comparing the sample solution to a simulated sample of excipients (SSE). The sample solution (1 mg/mL) was spiked with a SSE concentration of 25 ug/mL. Using the Handbook of Pharmaceutical Excipients as reference (Rowe and others 2006), the percentages of the inactive components of the Losartan tablet were approximated (Appendix 1). The SSE mixture, with an amount equal to the weight of one tablet, was mixed with 100 mg. Losartan standard and dissolved in distilled water. Solutions were subsequently prepared following the procedure of Bonf ilio and others (2010) to obtain a f inal concentration of 5 mg/L and 10 mg/L for the direct and f irst-derivative spectra, respectively, using distilled water as diluent. Accuracy Accuracy was determined by recovery of known amounts of Losar tan potassium standard added to the sample solution. Sample solutions of 5.0 mg/L and 10 mg/L for the two spectra were mixed with adequate Losartan standard solutions using serial dilutions to obtain a f inal concentration of 4.0, 5.0 and 6.0 mg/L for the direct spectrophotometry and of 8.0, 10, and 12 mg/L for the f irst-derivative spectrophotometry. All measurements were done in triplicates. Precision Precision was evaluated in terms of repeatability and intermediate precision. For repeatability, six standard solutions of the same concentration were measured twice λmax λmax A.D.A. Aniñon and others 45 = 3 SD α (1) = 10 SD α (2) on the same day. For intermediate precision, six standard solutions of the same concentration were measured on a different day by a different analyst.The concentrations used were 5.0 mg/L and 10 mg/L solutions for the direct and f irst- derivative spectra, respectively. Linearity and Range Linearity and range were evaluated by using Losartan sample solutions of 0, 4.0, 5.0, 6.0 and 7.0 mg/L for the direct derivative spectra, and 6.0, 8.0, 10, 12, 14 mg/L for the f irst-derivative spectra to produce an analytical curve. Limits of Detection and Limits of Quantitation The limits of detection (LOD) and quantitation (LOQ) were calculated using Equations (1) and (2): (1) (2) where SD is the standard deviation of the 10 blank readings (distilled water and mobile phase) and is the calibration curve slope obtained in the linearity. Data Analysis All data were entered and analyzed using Microsoft® Off ice Excel® 2007. RESULTS AND DISCUSSION Validation of the assay procedure developed by Bonf ilio and others (2010) started with the determination of the wavelength at which absorbance for the direct and f irst-derivative spectrophotometry was to be measured. The wavelength of maximum absorbance of the zero-order spectrum (Appendix 2) obtained using 5.0 mg/L as the 100% nominal concentration was found to be at 205.4 nm. On the other hand, the f irst-derivative spectrum, calculated and graphed according to the absorbances measured by the instrument, showed an intense negative peak at 234 nm (Appendix 3). At this wavelength, the absorbances of the Losartan potassium standard solution of 4.0, 8.0, 12, 16, and 20 mg/L showed absorbance values of 0.201, 0.393, 0.564, 0.762, and 0.951, respectively. For the subsequent analyses, 10 mg/L was set at the 100% level of the analytical curve in the f irst- derivative spectrophotometric method so that the analytical signal corresponds to the direct spectrophotometric method. Based on the results, the wavelengths determined for both methods closely corresponded the f indings of Bonf ilio and α (λmax) Spectrophotometric Determination of Losar tan 46 others (2010) of 205 nm for the direct spectrophotometry and 234 nm for the f irst-derivative spectrophotometry. Thus, subsequent absorbance for the direct spectrophotometry was done at 205.4 nm while absorbance was set at 229 and 239 nm to obtain the data for the f irst-derivative spectrophotometry. Following the ICH guidelines on analytical method validation, the adapted procedure from Bonf ilio and others (2010) was validated for the assay of Losartan potassium in Losartan 100-mg tablet preparation. To reiterate, the performance characteristics tested were specif icity, accuracy, precision (repeatability and intermediate precision), linearity, range, LOQ, and LOD. Results are shown in Table 1 together with the set acceptance criteria. Specificity* No significant No significant Passed No significant Passed difference difference difference between between between spiked and spiked and spiked and unspiked unspiked unspiked absorbance absorbance absorbance curves curves curves Accuracy 95-105% Conc % Passed Conc % Passed recovery per recovery# recovery concentration level* 80% 99.2 80% 104.3 (90-110%***) 100% 98.8 100% 98.5 120% 96.9 120% 101.1 Precision** RSD<5% Passed Passed Overall 2.21% repeatab. 3.28% Intermed. 3.17% 3.77% precision Linearity R2> 0.99**** 1.00 Passed 1.00 Passed P-value of y- 0.66 Passed 0.54 Passed intercept >0.05***** Range* 80-120% % Conc Passed % Conc Passed nominal nominal (mg/L) nominal (mg/L) concentration conc conc 80 4.00 80 8.00 100 5.00 100 10.00 120 6.00 120 12.00 LOD* Variable 0.03 mg/L Passed 0.61 mg/L Passed LOQ* Variable 0.10 mg/L Passed 1.86 mg/L Passed * Adapted from ICH 2006 * * Adapted from Brazil, as cited in Bonfilio and others 2010 * * * Adapted from Brazilian Pharmacopeia **** Adapted from Bryan 2009 * * * * * Adapted from Chan and others 2004 #- %recovery formula: % = (actual concentration/theoretical concentration) x 100 Parameter Acceptance Criteria Direct Spectrophotometry Result Remark First-Derivative Spectrophotometry Result Remark Table 1. Summary of performance characteristics of direct and first-derivative spectrophotometry A.D.A. Aniñon and others 47 In both methods, none of the excipients displayed any absorbance upon being spiked to a standard solution of known concentration. The excipients also did not interfere with the measurement of the Losartan potassium contained in the solution as shown in the zero-order spectrum (Appendix 4) and f irst-order spectrum (Appendix 5) of the unspiked and spiked standard solutions. These f indings indicate that absorbance at 205.4 nm for the direct spectrophotometry, and at 229 and 239 nm for the f irst- derivative spectrophotometry, is specif ic for Losartan potassium. Both methods exhibited good accuracy with a mean recovery range of 96.9-104.3 for the direct spectrophotometric method and 98.5-101.1 for the f irst-derivative method. Being specif ic and accurate, both direct and f irst-derivative methods are therefore suitable for the determination of Losartan potassium in tablets. The results of the precision study conformed to the acceptance criteria, with the overall repeatability value of 2.2120% and 3.2837%, for the direct and f irst- derivative spectrophotometry, respectively, and intermediate precision of 3.1740% and 3.7659%, respectively, for the two methods. The methods were therefore expected to be insensitive to small changes in conditions (i.e. , sample preparation, weighing, dilution, time, operator). Linearity was evaluated by linear regression. The coeff icient of determination (R2) values obtained from direct spectrophotometric method over the range of 4.0-6.0 mg/L was 1.00 and the equation produced was A=0.0949C-0.0068. For the f irst- derivative spectrophotometric method, the R2 value obtained over the range of 8.0-12.0 mg/L was 1.00 and the calibration equation was dA/dW = -0.0018C- 0.0002. The method was found to be linear, with the absorbance response being directly proportional to the concentration of Losartan. Using the formula for LOD and LOQ, the limits of quantitation and detection were calculated to be 0.10 and 0.03 mg/L, respectively, for direct spectrophotometry whereas limits of quantitation and detection for f irst-derivative spectrophotometry were calculated to be 1.86 and 0.61 mg/L, respectively. The results indicate that the analyses were performed beyond the quantitation limit. In summary, all performance characteristics were found to be highly satisfactory. These results conf irmed the suitability of both methods for the assay of Losartan potassium in Losartan 100 mg. tablet preparation. No modifications to the procedure developed by Bonf ilio and others (2010) were necessary to conform to the set acceptance criteria. Robustness and inter-laboratory studies are recommended to provide further evidence for the applicability of both methods for routine analysis of Losartan potassium in various Losartan tablet preparations. Spectrophotometric Determination of Losar tan 48 APPENDICES Appendix 1. Composition of the simulated sample of excipients Table 2. Simulated Sample of Excipients* * Composition based on Rowe RC, Sheskey PJ, Quinn ME 2009 Ingredients Amount Starch 0.6000 g Magnesium stearate 0.1500 g Microcrystalline cellulose 0.6000 g Lactose 1.3500 g Hydroethylcellulose 0.1922 g Hydroxymethylpropylcellulose 0.0600 g Titanium dioxide 0.0481 g TOTAL 3.0003 g Appendix 2. Losartan potassium zero-order absorption spectrum Figure 1. Losartan potassium zero-order absorption spectrum at 5 mg/L using distilled water as solvent. A.D.A. Aniñon and others 49 Appendix 3. Losar tan potassium f irst-order absorption spectrum Figure 2. Losartan potassium f irst-order absorption spectrum at 10 mg/L using distilled water as solvent. Appendix 4. Comparison of the zero-order spectrum of unspiked and spiked solutions Figure 3. Zero-order absorption spectra of the unspiked and spiked Losartan potassium aqueous solution at 5 mg/L (n=1). Spectrophotometric Determination of Losar tan 50 ACKNOWLEDGMENT The authors would like to thank the Department of Industrial Pharmacy, College of Pharmacy and the Institute of Pharmaceutical Sciences, National Institutes of Health, University of the Philippines Manila for providing some of the reagents needed for the experiments. REFERENCES B o n f i l i o R , F a vo r e t t o L B , Pe r e i r a G R , d e Ca s s i a R , Azev ed o P, d e A r a u j o M . 2 0 1 0 . C o m p a r a t i v e s t u d y o f a n a l y t i c a l m e t h o d s b y d i r e c t a n d f i r s t - d e r i v a t i v e U V spectrophotometry for evaluation of Losar tan potassium in capsules. Brazilian Journal of Pharmaceutical Sciences 46(1): 147-155. Bryan PD. 2009. What acceptance criteria and specif ications should be used for non- clinical dose formulation analysis (NCFDA)? Available from: http: //mediaserver.aaps pharmaceutica.com/meetings/09AM/Slides/11.12.09_Thu/406%20AB/0700/Peter%20 Bryan.pdf. Chan CC, Lam H, Lee YC, Zhang X. 2004. Analytical method validation and instrument performance verif ication. New Jersey: John Wiley & Sons, Inc. Appendix 5. Comparison of the f irst-order spectrum of unspiked and spiked solutions Figure 4. First-order absorption spectra of the spiked and unspiked losartan potassium aqueous solution at 10 mg/L (n=1). A.D.A. Aniñon and others 51 Ermer J, Miller, JH, editors. 2005. Method validation in pharmaceutical analysis. Germany: Wiley-VCH. ( I C H ) I n te r n a t i o n a l Co n f e r e n ce o n H a r m o n i z a t i o n . 2 0 0 6 . Va l i d a t i o n of a n a l y t i c a l p r o ced u r e s : Tex t a n d m e t h o d o l o g y Q 2 ( R 1 ) . Av a i l a b l e f r o m : h t t p : / / p r i v a te . i c h . o r g / M ed i a S e r ve r. j s e r ? @ _ I D = 4 1 7 & @ _ M O D E = G L B . Jackson EK, Brunton LL, Lazo JS, Parker KL. 2006. Renin and angiotensin. In: Goodman and Gilman’s The pharmacological basis of therapeutics, 11thed. New York: McGraw-Hill Companies, Inc. p 810, 813-814. (MIMS) Medical Information Management System. 2014. Concise prescribing information of Cozaar. Available from: http://www.mims.com/PHILIPPINES/drug/info/Cozaar/. Merck & Co. , Inc. 2010. Cozaar: Prescribing information, June 2010 ed. NJ: Merck & Co. , Inc. Merck & Co. , Inc. 2010.The Merck index, 13th ed. [CD-ROM]. NJ: Cambridge Soft. Rowe RC, Sheskey PJ, Owen SC, American Pharmacists Association. 2006. Handbook of pharmaceutical excipients. London: Pharmaceutical Press. Schmauser B. 2008. Pharmaceutical development with focus on paediatric formulations. A v a i l a b l e f r o m : h t t p : / / a p p s . w h o . i n t / p r e q u a l / t r a i n i n g r e s o u r c e s / p q _ p r e s / w o r k s h o p _ India-AprilMay08/Day_3/Analy_Meth_Dev.ppt . Wells, BG, DiPiro, Jt, Schwinghammer, TL, DiPiro, CV. 2009. Pharmacotherapy Handbook, 7th ed. New York: McGraw-Hill Companies, Inc. p 111. (WHO) The World Health Organization. 2010. WHO Western Pacif ic Region – Philippines – Health situation and trends. Available from: http://www.wpro.who.int/countries/2010/ phl/health_situation.htm. Youssef NY, Taha EA . 2007. Development and validation of spectrophotometric, TLC and H P LC m e t h o d s fo r t h e d e te r m i n a t i o n of l a m o t r i g i n e i n p r e s e n c e of i t s i m p u r i t y. Chemistry and Pharmacy Bulletin 55(4): 541-545. _______________ Arianne Diane A. Aniñon is currently working as a Quality Assurance and Regulatory Pharmacist at a local distributing company. She previously worked as a research assistant for the technical team that prepared the Country Situational Analysis on Antimicrobial Resistance of the Philippines and as an associate to several pharmacy practice research studies. She is also currently taking her Master's degree program in Health Policy Studies at the University of the Philippines Manila alongside her regulatory work. Richard Simon R. Binos is currently working at the Product Research and Standards Development Division, Center for Drug Regulation and Research, Food and Drug Spectrophotometric Determination of Losar tan 52 Administration of the Department of Health, Philippines. He is tasked with the formulation of technical guidelines and requirements for the registration of drug products, licensing and monitoring of establishments, and other related regulations. Karen Mae M. Brizuela is currently a student at the University of Manitoba in Winnipeg City, Canada under the Chemistry program – specif ically geared towards its application towards pharmacological studies. She was a previous Instructor at the Department of Pharmaceutical Chemistry at the College of Pharmacy, University of the Philippines Manila. She is also currently working on her pharmacist license registration in Manitoba and is interning at a local clinical retail pharmacy. W ill ison John E. de Luna is currently working at the Licensing and Registration Division, Center for Drug Regulation and Research, Food and Drug Administration of the Department of Health, Philippines. He is assigned in the evaluation and processing of applications of pharmaceutical products, particularly prescription and over-the-counter drugs, herbal medicines and traditionally-used herbal products. Juan Paolo D. Recto is a registered pharmacist who currently works in the Quality Assurance team of a multinational company with a corporate site in the Philippines. He provides end-to-end quality oversight and ensures that local contract manufacturers, laboratories, and distributors comply with local and international quality and regulatory requirements. On top of routine operations, he also assists in cGMP audits and inspections. Melanie V. Sal inas is currently working on the completion of her Master’s Degree in Pharmacology at the College of Medicine, University of the Philippines Manila. She is particularly interested in investigating the mechanisms of action of herbal medicines. She was formerly an Instructor at the Department of Pharmaceutical Chemistry of the College of Pharmacy, University of the Philippines Manila where she had been conducting research on Pharmaceutical Analysis. West Kristian D. Paraiso is currently a Research Student at the Faculty of Pharmaceutical Sciences, Hokkaido University in Sapporo City, Japan. He was an Assistant Professor at the Depar tment of Pharmaceutical Chemistry, College of Pharmacy, University of the Philippines Manila where he taught and performed research supervision in Pharmaceutical Assay Validation. Marlyn C. Corpuz, Regine Phill ine S. del Rosario, Jesus John C. Dimalala, T imothy Joseph P. Dueñas, and Isaac Ireneo B. Linatoc were former UP Pharmacy students and were part of the class that performed this project.