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RP-HPLC-DAD method for determination of olmesartan medoxomil in bulk and tablets exposed to forced conditions

RP-HPLC-DAD method for determination of olmesartan medoxomil in bulk and tablets exposed to... Acta Pharm. 60 (2010) 13­24 10.2478/v10007-010-0010-2 Original research paper RITESH N. SHARMA SHYAM S. PANCHOLI S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva-382711 Gujarat, India A simple, sensitive and precise RP-HPLC-DAD method was developed and validated for the determination of esartan medoxomil (AT-II receptor blocker) in the presence of its degradation products. esartan medoxomil and all the degradation products were resolved on a C18 column with the mobile phase composed of methanol, acetonitrile and water (60:15:25, V/V/V, pH 3.5 by orthophosphoric acid) at 260 using a photodiode array detector. The method was linear over the concentration range of 1­18 mg mL­1 and precise with RSD < 1 % in intra- and inter-day study. Excellent recoveries of 99.3 ± 0.9 to 100.8 ± 1.2 % proved the accuracy of the method. Developed method was specific, as indicated by chromatographic resolution > 2.0 for each peak and sensitive with LOD 0.03 mg mL-1 and LOQ 0.1 mg mL­1. The method was used to study the drug degradation behavior under forced conditions. Four degradation products (, II, III, IV) were formed during the degradation study in 0.1 mol L­1 HCl whereas only , II and III were formed in water, 0.01 mol L­1 NaOH and 3 % H2O2. No significant thermal or photolytic degradation was observed in solid drug. The method was applied successfully for the assay of esartan medoxomil in the tablet dosage form. Keywords: esartan medoxomil, forced degradation, high performance liquid chromatography, stability Accepted January 14, 2010 Stability can be defined as the capacity of a drug substance or drug product to sustain its identity, strength, quality, and purity throughout the retest or expiration period (1). Stability testing of an active substance or finished product provides evidence of the quality of a drug substance or drug product to remain acceptable up to the stated period under storage conditions stated on the label. The International Conference on Harmonization (ICH) guidelines Q1A (R2) require the use of a validated stability-indicating assay method (SIAM) for stability testing of a drug substance or product (2). It also empha* Correspondence; e-mail: riteshn.sharma@gmail.com R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. sizes the conduct of a forced degradation study on the drug substance to generate information on degradation products that can form under the influence of hydrolytic, oxidative, thermal or photolytic degradation conditions. esartan medoxomil () (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxy-4- (1-hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)-phenyl]phenyl}methyl imidazo-5-carboxylate) (Fig. 1a) is a prodrug and rapidly hydrolyzes in plasma during absorption to form its active metabolite esartan (Fig. 1b) (3­5). It is a selective AT1 subtype angiotensin II receptor blocker (6, 7), which was recently approved by the US-FDA (8) to treat patients with hypertension. This reduces blood pressure by causing vasodilation and reducing peripheral resistance (9). is also reported to be effective in animal models of atherosclerosis, liver disorders and diabetic nephropathy (10). Methods of analyses of esartan medoxomil in biological fluids such as human plasma and urine by LC-MS and LC-MS-MS were reported previously (11­13). Use of capillary zone electrophoresis (CZE) for the determination of in pharmaceutical dosage form has also been reported (14). No stability indicating assay of in bulk and solid dosage form could be traced in the literature. However, the method that identified the main degradation products obtained during short-time storage using the hyphenated techniques has been reported (15). This method described the storage of tablets at 40 °C/75 % RH for 6 months and identification of the degradation product by complementary use of the HPLC hyphenated techniques (LC-IR, LC-R and LC-MS) without isolation or purification processes. In the present study, we aimed to develop and validate a stability indicating RP-HPLC-DAD assay method that allowed resolution, detection and quantitation of esartan medoxomil in the presence of degradation products obtained during the forced conditions in bulk substance and tablet dosage form. a) OH b) OH Suceptible linkage O N O O O O N H 3C N O OH N N N NH N NH esartan medoxomil (ester prodrug) esartan (free carboxylic form) Fig. 1. Chemical structure of esartan medoxomil and its free carboxylic acid form esartan. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. EXPERIMENTAL Chemicals and reagents esartan medoxomil was kindly provided as a gift a sample by Torrent Pharmaceutical Ltd. (India) with 99.94 % purity. Tablet dosage form of esartan medoxomil (ecip, Cipla Ltd. India, 20 mg) was purchased from a local pharmacy. HPLC grade methanol and acetonitrile were purchased from S.D. Fine Chemical, India. Hydrochloric acid, sodium hydroxide pellets, hydrogen peroxide solution, and orthophosphoric acid were also purchased from S.D. Fine Chemical and were of analytical grade. Water for RP-HPLC was prepared by triple distillation in a glass still and filtered through a nylon 0.45 mm membrane filter (Gelman Laboratory, India). Instruments The Shimadzu (Japan) LC system (LC-2010c HT) was equipped with a diode array detector (SPD-M20A), auto sampler (SIL-10ADvp) and column oven CTO-10A(C) vp. Chromatographic separations were performed using the Phenomenex (Torrance, USA) C18 column (250 mm ´ 4.6 mm id, 5 mm particle size) at 35 °C column oven temperature and analyzed by LC solution software Shimad. For photolytic degradation, the UV cabinet producing short wavelength (254 ) produced by Acemas Technocracy Prt. Ltd, India was used. High precision water bath and hot air oven (Narang Scientific Works, India) capable of controlling the temperature within ± 1 and ± 2 °C, respectively, were used for the hydrolytic and thermal degradation studies. Preparation of standard solutions A stock solution of esartan medoxomil was prepared by dissolving the drug in methanol to get a concentration of 1.0 mg mL­1. Fresh stock solution was prepared every day during the experiment. Working solution containing 100 mg mL­1 was prepared from this stock solution as well as seven calibration standards (1­18 mg mL­1) and quality control samples (4, 10 and 14 mg mL­1) by diluting appropriate aliquots of standard solutions. Preparation of sample solution Twenty tablets were weighed, transferred to a clean, dry mortar and well ground. Powder equivalent to 50 mg drug was then transferred to a 100-mL volumetric flask containing 50 mL methanol. The flask was attached to a rotary shaker for 10 min to disperse the material completely. The mixture was then sonicated for 20 min and centrifuged at 3,000 rpm for 5 min. An aliquot of supernatant solution was diluted appropriately to give a solution of 500 mg mL­1 and 10 mL of this solution was used for forced degradation studies. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Forced degradation studies Forced degradation of drug substances and drug products was carried out under acid/neutral/basic hydrolytic, oxidative, thermolytic, and photolytic stress conditions. For hydrolytic and oxidative degradation, drug solutions were prepared with a concentration of 500 mg mL­1. After degradation, aliquots were diluted with methanol to achieve a concentration of 10 mg mL­1. Methanol (60 %, V/V) was used for drug solubilization in acidic, neutral and oxidative media. Hydrolytic degradation studies were carried out under acid (0.1 mol L­1 HCl, pH 1.03), neutral (water, pH 7.12), and basic (0.01 mol L­1 NaOH, pH 12.05) conditions at 60 °C as well as at room temperature over 12 to 48 h. Oxidative degradation was carried out in a 3 % H2O2 solution at room temperature over 48 h. Thermal and photo degradation of drug substances and drug products was carried out in the solid state. For thermal degradation, the drug was spread in a borosilicate glass Petri dish and placed in the hot-air oven maintained at 60 °C for 10 days. Also, photolytic studies were carried out by exposing a thin layer of the solid drug in a Petri dish in the UV chamber for 10 days during which time the total light exposure equaled to 1.2 ´ 106 lx h. After degradation, stock solutions were prepared by dissolving the samples in methanol to achieve a concentration of 1 mg mL­1. From these solutions, aliquots were diluted with methanol to get the final concentration of 10 mg mL­1 of . Samples were withdrawn initially, subsequently at prefixed time intervals and stored at 2­8 °C until analysis for all forced conditions. Method development Detection wavelength for the HPLC study was selected as 260 after recording the UV spectrum from 190 to 400 of the drug and representative sample from each forced condition. The maximum area and peak selectivity of was observed at this wavelength. The chromatographic conditions were optimized for resolution of the peak of the drug and degradation products under each forced condition by varying the stationary phase, proportion of methanol/acetonitrile/water in the mobile phase and the flow rate using representative samples from each forced condition. Several trials using various proportions of methanol and water as mobile phase were carried out. However, to attain the selective resolution of and its degradation products, acetonitrile was introduced as the third solvent; apparent pH 3.5 was adjusted by orthophosphoric acid. Subsequently, a mixture of different stress conditions was used to optimize the chromatographic conditions for resolving and all the degradation products in a single run. An appropriate blank was injected before the analysis of all forced samples. Such an optimized method was then used to study the forced degradation behavior of and was also applied in the stability indicating assay of tablets. Validation of the method The optimized method was validated in accordance with the ICH guidelines (16). Linearity was determined by analyzing, in triplicate, standard drug solutions of concentrations 1, 2, 4, 6, 10, 14 and 18 mg mL­1 using 20 mL of the injection volume. For intra- R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. -day precision, three quality control drug concentrations (4, 10 and 14 mg mL­1) were analyzed seven times on the same day whereas the same drug concentrations were analyzed on three different days for inter-day precision. Accuracy/recovery was evaluated by spiking the mixture of degradation samples with three known drug concentrations and calculating the percent recovery from the differences between the peak areas obtained for concentrated and diluted solutions. Signal-to-noise ratios were employed to estimate limits of detection (3:1) and limits of quantitation (10:1). The specificity of a method is its suitability for analysis of a substance in the presence of potential impurities. Specificity of the method was established through the study of the resolution (Rs) of samples. Overall selectivity was established through determination of drug purity and Rs peak each time. Reproducibility of the method was established through separate studies on a mixture of degradation samples by different persons on the same chromatographic system as well as on different chromatographic systems in different laboratories on different days by another analyst. Various system suitability parameters were also evaluated on a mixture sample on six different days using freshly prepared mobile phase each time. RESULTS AND DISCUSSION Method development and optimization The peak of pure obtained using 50 % methanol and 50 % acetonitrile (V/V) suggested to employ 70 % methanol and 30 % water (V/V), pH 3.5 adjusted with orthophosphoric acid, as the mobile phase. Forced degradation samples were then analyzed using the same mobile phase flowing at a rate of 1.0 mL min­1 on a C18 column employing DAD detection. The method resolved the drug and degradation products under neutral, basic and oxidative conditions, but it could not resolve the cluster of peaks observed under acidic conditions. To separate the degradation products formed under acidic conditions, acetonitrile was introduced in the mobile phase and all other variables were kept the same. However, degradation products still remained unresolved under acidic conditions. The alteration in the stationary phase from C18 to C8 and the flow rate of the mobile phase did not furnish promising results either. Many compositions of different mobile phase strength were tried and, finally, mobile phase composed of methanol, acetonitrile and water (60:15:25, V/V/V) pH 3.5 adjusted with orthophosphoric acid, at a flow rate of 1.0 mL min­1 on a C18 column, was used. This mobile phase could optimally resolve the and all the degradation products formed under different conditions in the mixture sample in a single run (Fig. 2a). The relative retention time (tR) of each peak of the degradation product with respect to esartan medoxomil is given in Table I. Validation of the method Linearity. ­ Peak area and concentrations were subjected to the least square linear regression analysis to calculate the calibration equations and correlation coefficients. The calibration plot for assay was linear over the calibration range 1­18 mg mL­1, and the regression coefficient, slope and intercept were 0.998, 541214 ± 253.1 and 340.9 ± 6.2, R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. respectively. These results demonstrate an excellent correlation between the peak area and analyte concentration. a) b) I I V II V II Time (min) c) Time (min) d) I I II V II Time (min) e) Time (min) I II Time (min) Fig. 2. HPLC chromatograms showing resolution of esartan medoxomil and degradation products in: a) mixture of forced degradation samples in a single run, b) 0.1 mol L­1 HCl at 60 °C after 24 h, c) 0.01 mol L­1 NaOH at RT after 4 h, d) water at 60 °C after 48 h, e) 3 % H2O2 at RT after 48 h. 18 R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Table I. Relative retention time, peak purity data and system suitability parameters of esartan medoxomil and its degradation products Peak Parameter Relative retention time (tR)a Peak purity index Purity threshold Asymmetry (As) Tailing factor (T) Chromatographic resolution (Rs) Capacity factor (k') Selectivity (á)b 0.740 0.789 0.842 1.11 1.044 1.91 4.93 1.11 I 0.809 1.000 0.998 1.28 ­ 1.24 5.48 1.18 3164.63 II 0.932 0.999 0.883 1.23 ­ 1.99 6.47 1.08 3195.41 V 1.154 0.999 0.974 1.32 ­ 2.05 7.02 1.17 4215.38 2688.93 1.000 1.000 0.999 1.06 1.058 1.16 8.26 Number of theoretical plates (N) 2725.85 a b With respect to . With respect to succeeding peak. Limits of detection and quantification. ­ LOD was 0.03 mg mL­1 for at a signal-to-noise ratio of 3:1 and the limit of quantification was determined as 0.1 mg mL­1 for at a signal-to-noise ratio of 10:1. Precision. ­ Intra-day precision was expressed throught relative standard deviation of seven repeated assays of samples at three concentration levels. Inter-day precision was determined by analyzing the same set of samples on five different days. RSD in the precision study for the assay was less than 1.0 % and confirmed that the method was highly precise. Results of the precision study for by the proposed RP-HPLC-DAD method are given in Table II. Recovery. ­ Standard addition method was used to examine the recovery of the RP-HPLC-DAD method (17). Recovery of from bulk drug samples ranged from 99.3 ± 0.9 to 100.7 ± 1.0 % and that from tablet dosage forms ranged from 99.5 ± 0.8 to 100.8 ± 1.2 % (Table III). Table II. Precision data of the proposed RP-HPLC-DAD method Measured concentration (mg mL­1)a Interday 4.0 ± 0.1 9.8 ± 0.3 14.1 ± 0.2 Intraday 3.9 ± 0.4 10.2 ± 0.5 13.8 ± 0.8 Actual conc. (mg mL­1) 4.0 10.0 14.0 Mean ± SD, n = 7. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Table III. Recovery for bulk drug substance and drug product Conc. of drug taken (mg mL­1) Bulk drug substance 10.0 10.0 10.0 Drug product 10.0 10.0 10.0 Mean ± SD, n = 3. Conc. of standard added (mg mL­1) 5.0 10.0 15.0 5.0 10.0 15.0 Conc. found (mg mL­1) 14.9 ± 0.2 19.9 ± 0.4 25.1 ± 0.3 15.0 ± 0.5 20.0 ± 0.3 24.9 ± 0.2 Recovery (%) 99.6 ± 0.8 99.3 ± 0.9 100.7 ± 1.0 100.8 ± 1.2 100.3 ± 0.6 99.5 ± 0.8 Specificity. ­ There was no interference due to placebo and sample diluents and degradation products. Resolution between closely eluting degradation products, i.e., between , I, and between II, , were greater than 2.0, illustrated the chromatographic selectivity of the method. Stability of stock solution. ­ During solution stability and mobile phase stability experiments, RSD for the assay was within 1 % for three replicates. Results of the solution stability and mobile phase stability experiments confirmed that standard solutions and solutions in the mobile phase were stable for up to 48 h during the assay. Forced degradation During optimization of the hydrolytic degradation process, drug samples were initially placed in 0.1 mol L­1 HCl, 0.1 mol L­1 NaOH and in water at 60 °C. However, after 24 and 48 h, the sample in HCl and water showed 31 and 12 % degradation, respectively, while in 0.1 mol L­1 NaOH was completely degraded. Thus, we decided to carry out degradation at room temperature for the 0.1 mol L­1 NaOH sample. The compound was completely degraded to instantly at room temperature. When NaOH concentration was lowered to 0.01 mol L­1, the sample containing was 90 % degraded at room temperature after 4 h. Degradation pattern under all forced conditions showed the presence of , which is the major degradation product of formed during every degradation study. As illustrated in Fig. 2b for acidic hydrolysis all the degradation products (, II, III, IV) were observed in reasonable amounts. The was degraded after 24 h at 60 °C in 0.1 mol L­1 HCl; here there was more I than in other stress condition samples. For hydrolysis in basic medium, 0.1 mol L­1 NaOH, almost all the drug had degraded at room temperature; however, 0.01 mol L­1 NaOH at room temperature was appropriate for significant degradation. Three degradation products (, I and II) were present in these forced samples, being the major one (Fig. 2c). Degradation in neutral hyd- R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. a) b) mAU 3.36 0.9938 D:\Ritesh\ Cal\10ugNew.jcm 80 I Wavelength () c) mAU 3.87 0.9944 D:\Ritesh\ Cal\10ugNew.jcm 8 Wavelength () d) mAU 25.0 4.79 0.9954 D:\Ritesh\ Cal\10ugNew.jcm 22.5 201 II V Wavelength () Wavelength () Fig. 3. UV spectra of esartan medoxomil () and degradation products: a) , b) I, c) II, d) V. rolytic medium at 60 °C (Fig. 2d) occurred at a slower rate than under other hydrolytic conditions. Exposure to light and dark did not exhibit any significant difference in the degradation pattern under hydrolytic conditions. Results proved that the degradation of was more pronounced under basic hydrolytic condition than under acidic and neutral conditions. degradation was 12 % under oxidative conditions (3 % H2O2) at room temperature for 48 h. Oxidative degradation produced , II and III, but and II were formed in minor amounts (Fig. 2e). No degradation was seen in solid drug kept at 60 °C for 10 days and no degradation was observed upon exposure to light intensity of 1.2 ´ 106 lx h either. Summary of the data for all forced degradations is given in Table IV. Peak purity test suggested that the peak as well as the peaks of degradation products were pure for all the forced samples analyzed (Table I). No additional peak was observed after 5 min in chromatograms obtained for the extended runtime of 25 min in a sample study. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Table IV. Summary of forced degradation study results Stress condition Acidic hydrolysis (0.1 mol L­1 HCl, 60 °C) Basic hydrolysis (0.01 mol L­1 NaOH, RT) Aqueous hydrolysis (60 °C) Oxidation (3% H2O2, RT) Thermal (60 °C) Photo (UV 254 ) Time 24 h 4h 48 h 48 h 10 days 10 days (%) 68.7 10.3 88.1 54.3 99.4 99.8 Remarks (major degradation product) and II and II No degradation product formed No major degradation product observed ­ esartan modoxomil; DP ­ degradation product The UV spectrum of pure was compared with the spectrum of the drug subjected to the different forced conditions; all the spectra showed slight changes in the absorption pattern. Purity of all degradation products was confirmed by comparing their spectra with the spectrum of (Figs. 3a-d). Comparison of the spectra of two major degradation products, and II, with the spectrum (Figs. 3a and b) suggested the absence of the ester moiety of in both the and II, since the absorption maximum characteristic of the 5-methyl-2-oxo-1,3-dioxolen-4-yl-methyl group (ester moiety) in at 260 was not visible. esartan medoxomil is an ester prodrug and easily de-esterifies to its active metabolite esartan under hydrolytic conditions (4). Analogously to degradation products reported to be formed in losartan tablets stored at 40 °C and 75 % relative humidity (18), the major degradation products are , likely to be the esartan free carboxylic acid form (Fig. 1b) and I, the dimer of its free carboxylic acid form of esartan. The goal of determining in the presence of degradation products by the proposed stability indicating RP-HPLC-DAD method was successfully achieved but the method can be also used for routine quality control of tablets. CONCLUSIONS The RP-HPLC-DAD method developed for quantitative analysis of esartan medoxomil in both bulk drug and pharmaceutical dosage forms is precise, accurate and specific. Satisfactory results were obtained by validation of the method. No attempt was made to quantify the degradation products; quantitation is possible after isolation of degradation products in pure form. This method can be used for the estimation of esartan medoxomil in the presence of degradation products obtained under different forced conditions. Acknowledgements. ­ Author would like to thank Dr. B. G. Choudhary, Assistant Professor, S. K. Patel College of Pharmaceutical Education, Ganpat University, for his needfull suggestions during the research work. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Pharmaceutica de Gruyter

RP-HPLC-DAD method for determination of olmesartan medoxomil in bulk and tablets exposed to forced conditions

Acta Pharmaceutica , Volume 60 (1) – Mar 1, 2010

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Abstract

Acta Pharm. 60 (2010) 13­24 10.2478/v10007-010-0010-2 Original research paper RITESH N. SHARMA SHYAM S. PANCHOLI S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva-382711 Gujarat, India A simple, sensitive and precise RP-HPLC-DAD method was developed and validated for the determination of esartan medoxomil (AT-II receptor blocker) in the presence of its degradation products. esartan medoxomil and all the degradation products were resolved on a C18 column with the mobile phase composed of methanol, acetonitrile and water (60:15:25, V/V/V, pH 3.5 by orthophosphoric acid) at 260 using a photodiode array detector. The method was linear over the concentration range of 1­18 mg mL­1 and precise with RSD < 1 % in intra- and inter-day study. Excellent recoveries of 99.3 ± 0.9 to 100.8 ± 1.2 % proved the accuracy of the method. Developed method was specific, as indicated by chromatographic resolution > 2.0 for each peak and sensitive with LOD 0.03 mg mL-1 and LOQ 0.1 mg mL­1. The method was used to study the drug degradation behavior under forced conditions. Four degradation products (, II, III, IV) were formed during the degradation study in 0.1 mol L­1 HCl whereas only , II and III were formed in water, 0.01 mol L­1 NaOH and 3 % H2O2. No significant thermal or photolytic degradation was observed in solid drug. The method was applied successfully for the assay of esartan medoxomil in the tablet dosage form. Keywords: esartan medoxomil, forced degradation, high performance liquid chromatography, stability Accepted January 14, 2010 Stability can be defined as the capacity of a drug substance or drug product to sustain its identity, strength, quality, and purity throughout the retest or expiration period (1). Stability testing of an active substance or finished product provides evidence of the quality of a drug substance or drug product to remain acceptable up to the stated period under storage conditions stated on the label. The International Conference on Harmonization (ICH) guidelines Q1A (R2) require the use of a validated stability-indicating assay method (SIAM) for stability testing of a drug substance or product (2). It also empha* Correspondence; e-mail: riteshn.sharma@gmail.com R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. sizes the conduct of a forced degradation study on the drug substance to generate information on degradation products that can form under the influence of hydrolytic, oxidative, thermal or photolytic degradation conditions. esartan medoxomil () (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxy-4- (1-hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)-phenyl]phenyl}methyl imidazo-5-carboxylate) (Fig. 1a) is a prodrug and rapidly hydrolyzes in plasma during absorption to form its active metabolite esartan (Fig. 1b) (3­5). It is a selective AT1 subtype angiotensin II receptor blocker (6, 7), which was recently approved by the US-FDA (8) to treat patients with hypertension. This reduces blood pressure by causing vasodilation and reducing peripheral resistance (9). is also reported to be effective in animal models of atherosclerosis, liver disorders and diabetic nephropathy (10). Methods of analyses of esartan medoxomil in biological fluids such as human plasma and urine by LC-MS and LC-MS-MS were reported previously (11­13). Use of capillary zone electrophoresis (CZE) for the determination of in pharmaceutical dosage form has also been reported (14). No stability indicating assay of in bulk and solid dosage form could be traced in the literature. However, the method that identified the main degradation products obtained during short-time storage using the hyphenated techniques has been reported (15). This method described the storage of tablets at 40 °C/75 % RH for 6 months and identification of the degradation product by complementary use of the HPLC hyphenated techniques (LC-IR, LC-R and LC-MS) without isolation or purification processes. In the present study, we aimed to develop and validate a stability indicating RP-HPLC-DAD assay method that allowed resolution, detection and quantitation of esartan medoxomil in the presence of degradation products obtained during the forced conditions in bulk substance and tablet dosage form. a) OH b) OH Suceptible linkage O N O O O O N H 3C N O OH N N N NH N NH esartan medoxomil (ester prodrug) esartan (free carboxylic form) Fig. 1. Chemical structure of esartan medoxomil and its free carboxylic acid form esartan. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. EXPERIMENTAL Chemicals and reagents esartan medoxomil was kindly provided as a gift a sample by Torrent Pharmaceutical Ltd. (India) with 99.94 % purity. Tablet dosage form of esartan medoxomil (ecip, Cipla Ltd. India, 20 mg) was purchased from a local pharmacy. HPLC grade methanol and acetonitrile were purchased from S.D. Fine Chemical, India. Hydrochloric acid, sodium hydroxide pellets, hydrogen peroxide solution, and orthophosphoric acid were also purchased from S.D. Fine Chemical and were of analytical grade. Water for RP-HPLC was prepared by triple distillation in a glass still and filtered through a nylon 0.45 mm membrane filter (Gelman Laboratory, India). Instruments The Shimadzu (Japan) LC system (LC-2010c HT) was equipped with a diode array detector (SPD-M20A), auto sampler (SIL-10ADvp) and column oven CTO-10A(C) vp. Chromatographic separations were performed using the Phenomenex (Torrance, USA) C18 column (250 mm ´ 4.6 mm id, 5 mm particle size) at 35 °C column oven temperature and analyzed by LC solution software Shimad. For photolytic degradation, the UV cabinet producing short wavelength (254 ) produced by Acemas Technocracy Prt. Ltd, India was used. High precision water bath and hot air oven (Narang Scientific Works, India) capable of controlling the temperature within ± 1 and ± 2 °C, respectively, were used for the hydrolytic and thermal degradation studies. Preparation of standard solutions A stock solution of esartan medoxomil was prepared by dissolving the drug in methanol to get a concentration of 1.0 mg mL­1. Fresh stock solution was prepared every day during the experiment. Working solution containing 100 mg mL­1 was prepared from this stock solution as well as seven calibration standards (1­18 mg mL­1) and quality control samples (4, 10 and 14 mg mL­1) by diluting appropriate aliquots of standard solutions. Preparation of sample solution Twenty tablets were weighed, transferred to a clean, dry mortar and well ground. Powder equivalent to 50 mg drug was then transferred to a 100-mL volumetric flask containing 50 mL methanol. The flask was attached to a rotary shaker for 10 min to disperse the material completely. The mixture was then sonicated for 20 min and centrifuged at 3,000 rpm for 5 min. An aliquot of supernatant solution was diluted appropriately to give a solution of 500 mg mL­1 and 10 mL of this solution was used for forced degradation studies. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Forced degradation studies Forced degradation of drug substances and drug products was carried out under acid/neutral/basic hydrolytic, oxidative, thermolytic, and photolytic stress conditions. For hydrolytic and oxidative degradation, drug solutions were prepared with a concentration of 500 mg mL­1. After degradation, aliquots were diluted with methanol to achieve a concentration of 10 mg mL­1. Methanol (60 %, V/V) was used for drug solubilization in acidic, neutral and oxidative media. Hydrolytic degradation studies were carried out under acid (0.1 mol L­1 HCl, pH 1.03), neutral (water, pH 7.12), and basic (0.01 mol L­1 NaOH, pH 12.05) conditions at 60 °C as well as at room temperature over 12 to 48 h. Oxidative degradation was carried out in a 3 % H2O2 solution at room temperature over 48 h. Thermal and photo degradation of drug substances and drug products was carried out in the solid state. For thermal degradation, the drug was spread in a borosilicate glass Petri dish and placed in the hot-air oven maintained at 60 °C for 10 days. Also, photolytic studies were carried out by exposing a thin layer of the solid drug in a Petri dish in the UV chamber for 10 days during which time the total light exposure equaled to 1.2 ´ 106 lx h. After degradation, stock solutions were prepared by dissolving the samples in methanol to achieve a concentration of 1 mg mL­1. From these solutions, aliquots were diluted with methanol to get the final concentration of 10 mg mL­1 of . Samples were withdrawn initially, subsequently at prefixed time intervals and stored at 2­8 °C until analysis for all forced conditions. Method development Detection wavelength for the HPLC study was selected as 260 after recording the UV spectrum from 190 to 400 of the drug and representative sample from each forced condition. The maximum area and peak selectivity of was observed at this wavelength. The chromatographic conditions were optimized for resolution of the peak of the drug and degradation products under each forced condition by varying the stationary phase, proportion of methanol/acetonitrile/water in the mobile phase and the flow rate using representative samples from each forced condition. Several trials using various proportions of methanol and water as mobile phase were carried out. However, to attain the selective resolution of and its degradation products, acetonitrile was introduced as the third solvent; apparent pH 3.5 was adjusted by orthophosphoric acid. Subsequently, a mixture of different stress conditions was used to optimize the chromatographic conditions for resolving and all the degradation products in a single run. An appropriate blank was injected before the analysis of all forced samples. Such an optimized method was then used to study the forced degradation behavior of and was also applied in the stability indicating assay of tablets. Validation of the method The optimized method was validated in accordance with the ICH guidelines (16). Linearity was determined by analyzing, in triplicate, standard drug solutions of concentrations 1, 2, 4, 6, 10, 14 and 18 mg mL­1 using 20 mL of the injection volume. For intra- R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. -day precision, three quality control drug concentrations (4, 10 and 14 mg mL­1) were analyzed seven times on the same day whereas the same drug concentrations were analyzed on three different days for inter-day precision. Accuracy/recovery was evaluated by spiking the mixture of degradation samples with three known drug concentrations and calculating the percent recovery from the differences between the peak areas obtained for concentrated and diluted solutions. Signal-to-noise ratios were employed to estimate limits of detection (3:1) and limits of quantitation (10:1). The specificity of a method is its suitability for analysis of a substance in the presence of potential impurities. Specificity of the method was established through the study of the resolution (Rs) of samples. Overall selectivity was established through determination of drug purity and Rs peak each time. Reproducibility of the method was established through separate studies on a mixture of degradation samples by different persons on the same chromatographic system as well as on different chromatographic systems in different laboratories on different days by another analyst. Various system suitability parameters were also evaluated on a mixture sample on six different days using freshly prepared mobile phase each time. RESULTS AND DISCUSSION Method development and optimization The peak of pure obtained using 50 % methanol and 50 % acetonitrile (V/V) suggested to employ 70 % methanol and 30 % water (V/V), pH 3.5 adjusted with orthophosphoric acid, as the mobile phase. Forced degradation samples were then analyzed using the same mobile phase flowing at a rate of 1.0 mL min­1 on a C18 column employing DAD detection. The method resolved the drug and degradation products under neutral, basic and oxidative conditions, but it could not resolve the cluster of peaks observed under acidic conditions. To separate the degradation products formed under acidic conditions, acetonitrile was introduced in the mobile phase and all other variables were kept the same. However, degradation products still remained unresolved under acidic conditions. The alteration in the stationary phase from C18 to C8 and the flow rate of the mobile phase did not furnish promising results either. Many compositions of different mobile phase strength were tried and, finally, mobile phase composed of methanol, acetonitrile and water (60:15:25, V/V/V) pH 3.5 adjusted with orthophosphoric acid, at a flow rate of 1.0 mL min­1 on a C18 column, was used. This mobile phase could optimally resolve the and all the degradation products formed under different conditions in the mixture sample in a single run (Fig. 2a). The relative retention time (tR) of each peak of the degradation product with respect to esartan medoxomil is given in Table I. Validation of the method Linearity. ­ Peak area and concentrations were subjected to the least square linear regression analysis to calculate the calibration equations and correlation coefficients. The calibration plot for assay was linear over the calibration range 1­18 mg mL­1, and the regression coefficient, slope and intercept were 0.998, 541214 ± 253.1 and 340.9 ± 6.2, R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. respectively. These results demonstrate an excellent correlation between the peak area and analyte concentration. a) b) I I V II V II Time (min) c) Time (min) d) I I II V II Time (min) e) Time (min) I II Time (min) Fig. 2. HPLC chromatograms showing resolution of esartan medoxomil and degradation products in: a) mixture of forced degradation samples in a single run, b) 0.1 mol L­1 HCl at 60 °C after 24 h, c) 0.01 mol L­1 NaOH at RT after 4 h, d) water at 60 °C after 48 h, e) 3 % H2O2 at RT after 48 h. 18 R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Table I. Relative retention time, peak purity data and system suitability parameters of esartan medoxomil and its degradation products Peak Parameter Relative retention time (tR)a Peak purity index Purity threshold Asymmetry (As) Tailing factor (T) Chromatographic resolution (Rs) Capacity factor (k') Selectivity (á)b 0.740 0.789 0.842 1.11 1.044 1.91 4.93 1.11 I 0.809 1.000 0.998 1.28 ­ 1.24 5.48 1.18 3164.63 II 0.932 0.999 0.883 1.23 ­ 1.99 6.47 1.08 3195.41 V 1.154 0.999 0.974 1.32 ­ 2.05 7.02 1.17 4215.38 2688.93 1.000 1.000 0.999 1.06 1.058 1.16 8.26 Number of theoretical plates (N) 2725.85 a b With respect to . With respect to succeeding peak. Limits of detection and quantification. ­ LOD was 0.03 mg mL­1 for at a signal-to-noise ratio of 3:1 and the limit of quantification was determined as 0.1 mg mL­1 for at a signal-to-noise ratio of 10:1. Precision. ­ Intra-day precision was expressed throught relative standard deviation of seven repeated assays of samples at three concentration levels. Inter-day precision was determined by analyzing the same set of samples on five different days. RSD in the precision study for the assay was less than 1.0 % and confirmed that the method was highly precise. Results of the precision study for by the proposed RP-HPLC-DAD method are given in Table II. Recovery. ­ Standard addition method was used to examine the recovery of the RP-HPLC-DAD method (17). Recovery of from bulk drug samples ranged from 99.3 ± 0.9 to 100.7 ± 1.0 % and that from tablet dosage forms ranged from 99.5 ± 0.8 to 100.8 ± 1.2 % (Table III). Table II. Precision data of the proposed RP-HPLC-DAD method Measured concentration (mg mL­1)a Interday 4.0 ± 0.1 9.8 ± 0.3 14.1 ± 0.2 Intraday 3.9 ± 0.4 10.2 ± 0.5 13.8 ± 0.8 Actual conc. (mg mL­1) 4.0 10.0 14.0 Mean ± SD, n = 7. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Table III. Recovery for bulk drug substance and drug product Conc. of drug taken (mg mL­1) Bulk drug substance 10.0 10.0 10.0 Drug product 10.0 10.0 10.0 Mean ± SD, n = 3. Conc. of standard added (mg mL­1) 5.0 10.0 15.0 5.0 10.0 15.0 Conc. found (mg mL­1) 14.9 ± 0.2 19.9 ± 0.4 25.1 ± 0.3 15.0 ± 0.5 20.0 ± 0.3 24.9 ± 0.2 Recovery (%) 99.6 ± 0.8 99.3 ± 0.9 100.7 ± 1.0 100.8 ± 1.2 100.3 ± 0.6 99.5 ± 0.8 Specificity. ­ There was no interference due to placebo and sample diluents and degradation products. Resolution between closely eluting degradation products, i.e., between , I, and between II, , were greater than 2.0, illustrated the chromatographic selectivity of the method. Stability of stock solution. ­ During solution stability and mobile phase stability experiments, RSD for the assay was within 1 % for three replicates. Results of the solution stability and mobile phase stability experiments confirmed that standard solutions and solutions in the mobile phase were stable for up to 48 h during the assay. Forced degradation During optimization of the hydrolytic degradation process, drug samples were initially placed in 0.1 mol L­1 HCl, 0.1 mol L­1 NaOH and in water at 60 °C. However, after 24 and 48 h, the sample in HCl and water showed 31 and 12 % degradation, respectively, while in 0.1 mol L­1 NaOH was completely degraded. Thus, we decided to carry out degradation at room temperature for the 0.1 mol L­1 NaOH sample. The compound was completely degraded to instantly at room temperature. When NaOH concentration was lowered to 0.01 mol L­1, the sample containing was 90 % degraded at room temperature after 4 h. Degradation pattern under all forced conditions showed the presence of , which is the major degradation product of formed during every degradation study. As illustrated in Fig. 2b for acidic hydrolysis all the degradation products (, II, III, IV) were observed in reasonable amounts. The was degraded after 24 h at 60 °C in 0.1 mol L­1 HCl; here there was more I than in other stress condition samples. For hydrolysis in basic medium, 0.1 mol L­1 NaOH, almost all the drug had degraded at room temperature; however, 0.01 mol L­1 NaOH at room temperature was appropriate for significant degradation. Three degradation products (, I and II) were present in these forced samples, being the major one (Fig. 2c). Degradation in neutral hyd- R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. a) b) mAU 3.36 0.9938 D:\Ritesh\ Cal\10ugNew.jcm 80 I Wavelength () c) mAU 3.87 0.9944 D:\Ritesh\ Cal\10ugNew.jcm 8 Wavelength () d) mAU 25.0 4.79 0.9954 D:\Ritesh\ Cal\10ugNew.jcm 22.5 201 II V Wavelength () Wavelength () Fig. 3. UV spectra of esartan medoxomil () and degradation products: a) , b) I, c) II, d) V. rolytic medium at 60 °C (Fig. 2d) occurred at a slower rate than under other hydrolytic conditions. Exposure to light and dark did not exhibit any significant difference in the degradation pattern under hydrolytic conditions. Results proved that the degradation of was more pronounced under basic hydrolytic condition than under acidic and neutral conditions. degradation was 12 % under oxidative conditions (3 % H2O2) at room temperature for 48 h. Oxidative degradation produced , II and III, but and II were formed in minor amounts (Fig. 2e). No degradation was seen in solid drug kept at 60 °C for 10 days and no degradation was observed upon exposure to light intensity of 1.2 ´ 106 lx h either. Summary of the data for all forced degradations is given in Table IV. Peak purity test suggested that the peak as well as the peaks of degradation products were pure for all the forced samples analyzed (Table I). No additional peak was observed after 5 min in chromatograms obtained for the extended runtime of 25 min in a sample study. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24. Table IV. Summary of forced degradation study results Stress condition Acidic hydrolysis (0.1 mol L­1 HCl, 60 °C) Basic hydrolysis (0.01 mol L­1 NaOH, RT) Aqueous hydrolysis (60 °C) Oxidation (3% H2O2, RT) Thermal (60 °C) Photo (UV 254 ) Time 24 h 4h 48 h 48 h 10 days 10 days (%) 68.7 10.3 88.1 54.3 99.4 99.8 Remarks (major degradation product) and II and II No degradation product formed No major degradation product observed ­ esartan modoxomil; DP ­ degradation product The UV spectrum of pure was compared with the spectrum of the drug subjected to the different forced conditions; all the spectra showed slight changes in the absorption pattern. Purity of all degradation products was confirmed by comparing their spectra with the spectrum of (Figs. 3a-d). Comparison of the spectra of two major degradation products, and II, with the spectrum (Figs. 3a and b) suggested the absence of the ester moiety of in both the and II, since the absorption maximum characteristic of the 5-methyl-2-oxo-1,3-dioxolen-4-yl-methyl group (ester moiety) in at 260 was not visible. esartan medoxomil is an ester prodrug and easily de-esterifies to its active metabolite esartan under hydrolytic conditions (4). Analogously to degradation products reported to be formed in losartan tablets stored at 40 °C and 75 % relative humidity (18), the major degradation products are , likely to be the esartan free carboxylic acid form (Fig. 1b) and I, the dimer of its free carboxylic acid form of esartan. The goal of determining in the presence of degradation products by the proposed stability indicating RP-HPLC-DAD method was successfully achieved but the method can be also used for routine quality control of tablets. CONCLUSIONS The RP-HPLC-DAD method developed for quantitative analysis of esartan medoxomil in both bulk drug and pharmaceutical dosage forms is precise, accurate and specific. Satisfactory results were obtained by validation of the method. No attempt was made to quantify the degradation products; quantitation is possible after isolation of degradation products in pure form. This method can be used for the estimation of esartan medoxomil in the presence of degradation products obtained under different forced conditions. Acknowledgements. ­ Author would like to thank Dr. B. G. Choudhary, Assistant Professor, S. K. Patel College of Pharmaceutical Education, Ganpat University, for his needfull suggestions during the research work. R. N. Sharma and S. S. Pancholi: RP-HPLC-DAD method for determination of esartan medoxomil in bulk and tablets exposed to forced conditions, Acta Pharm. 60 (2010) 13­24.

Journal

Acta Pharmaceuticade Gruyter

Published: Mar 1, 2010

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