CHAPTER VI TITRIMETRIC, SPECTROPHOTOMETRIC AND CHROMATOGRAPHIC ASSAY OF EPROSARTAN

Transcription

1 CHAPTER VI TITRIMETRIC, SPECTROPHOTOMETRIC AND CHROMATOGRAPHIC ASSAY OF EPROSARTAN

2 Section 6.0 DRUG PROFILE AND LITERATURE SURVEY DRUG PROFILE Eprosartan mesylate (EPR) is chemically known as monomethane sulfonate of (E)-2-butyl-1-(p-carboxybenzyl)-α-2-thienylmethylimidazole-5-acrylic acid. The molecular formula of EPR is C24H28N2O7S2 and its formula weight is g mol-1. Physically, pure EPR is a white crystalline and odorless powder [1]. It has the following chemical structure: O O O S O O HO N S N EPR is soluble in dimethyl sulfoxide, dimethyl formimide, methanol, ethanol and acetic acid. It is practically insoluble in water, chloroform, dichloromethane, toluene, ethyl acetate and acetone. EPR is a highly selective, nonpeptide angiotensin-ii receptor (ATII) antagonist used for the treatment of high blood pressure [2]. It has been shown to inhibit angiotensin-ii induced vasoconstriction and to reduce systolic and diastolic blood pressure [3]. The drug acts on the renin-angiotensin system in two ways to decrease total peripheral resistance. First, it blocks the binding of angiotensin II to ATI receptors in vascular smooth muscle, causing vascular dilatation. Second, it inhibits sympathetic norepinephrine production; further reducing blood pressure [4]. Its oral bioavailability is approximately 13% and is not significantly affected by food [5]. It was launched by the pharmaceutical company Smithkline Beckman corporation in the year 1990 [6]. EPR has an official United States Pharmacopoeial monograph for its quantification assay [7]. The report describes a liquid chromatographic method using acetonitrile, phosphoric acid, and water (27: 0.2: 73) as mobile phase. 214

3 6.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR EPROSARTAN MESYLATE Titrimetric and visible spectrophotometric methods Literature survey revealed that no titrimetric or visible spectrophotometric methods have ever been reported for EPR UV-spectrophotometric methods Only a few UV-spectrophotometric methods have been reported for EPR when it is present in tablets. Two methods [8] one based on the measurement of the absorbance at 233 nm, and the other, a first-derivative method where the absorbance was measured at 222 nm. Both the methods obey Beer s law range in the 1-70 µg ml -1 range with excellent linearity (r 2 =0.999). Measurement of the absorbance of methanolic solution of drug at 232 nm in the range 2-30 µg ml -1 has served as the basis of a method reported by Kamila et al [9]. The excipients were not found to interfere in the assay when applied to tablets. A difference spectrophotometric method [10] in which the difference in absorptivities in 0.1 M NaOH and 0.1 M HCl was measured at nm, thus facilitating a method for tablets without interference by the tablet excipients has also been developed. Simultaneous determination of EPR and hydrochlorothiazide by two different approaches is found in the literature. A mixed hydrotropy-solubilisation approach in which, the poorly water soluble drugs were dissolved in a mixture of 2 M sodium acetate and 8 M urea (50:50) and absorbance measured at nm for EPR and nm for hydrochlorothiazide has been described by Jain et al [11]. In a slightly different approach [12], EPR was assayed by first derivative zero-crossing technique while hydrochlorothiazide was determined by second derivative zero-crossing technique after extraction with ethyl acetate. The method was proved to be accurate and was able to cope with the strong spectral interference of the binary mixtures. El- Yazbi et al [13] have described a kinetic UV-spectrophotometric method for the drug in formulations. The method is based on the oxidation of EPR by cerium (IV) in HClO 4 medium on the subsequent measurement of the absorbance at 326 nm using a fixed time procedure with UV-detection. 215

4 Chromatographic methods Several RP-HPLC methods with UV-detection [7, 14-18] have been described for the determination of EPR in bulk drug and tablets. HPLC methods for the simultaneous determination of EPR and hydrochlorothiazide have also been reported many workers [19-23]. Simultaneous determination of EPR and hydrochlorothiazide in tablets by HPTLC with UV-absorption densitometry has been accomplished by Patel et al [24]. There are only few reports concerning the application of UPLC for the determination of EPR. Patel et al [25] have described a stability-indicating assay procedure by UPLC. The drug was subjected to acid, neutral, alkali hydrolytic conditions at 80 0 C and to oxidative decomposition at room temperature besides photo-degradation. Separation of the drug and the degradation products was achieved on a BEH C18 column (1.7 µm, 2.1 mm 150 mm) with gradient elution of wateracetonitrile as mobile phase, with a flow rate of 0.1 ml min -1 and UV- detection at 232 nm. The authors were able to determine the drug in 5-25 µg ml -1 range with good accuracy and precision. A method for the simultaneous determination of EPR and hydrochlorothiazide and their related impurities in tablets has been described [26]. In this method, separation was performed on Acquity HSS C 18 column (1.7 µm, 2.1 mm 150 mm) by adopting gradient elution using acetonitrile and disodium acetate buffer (0.01M; ph 5.5) at a flow rate of 0.3 ml min -1 with a total runtime of 20 min, 2 main compounds and ten other unknown impurities were separated Other methods Differential pulse polorography [13], capillary electrophoresis [27] and micellar electrokinetic chromatography [28] are the other methods reported for EPR in pharmaceuticals Methods for body fluids In addition to the above methods for the determination of EPR in pharmaceuticals, HPLC [21,29], solid-phase elution-hplc [30,31] and LC-MS [32] methods have been described for body fluids such as blood plasma [21,29-31] and blood plasma and urine [32]. 216

5 From the literature survey presented in the above paragraphs, it is clear that no titrimetric and visible spectrophotometric methods have ever been reported for the assay of EPR. The reported UV-spectrophotometric methods [8-10] are not stability indicating and there is only one UPLC method [25] which is stability indicating. Keeping the above in view, two titrimetric, two UV-spectrophotometric, two visible spectrophotometric, one HPLC and one UPLC methods were developed, optimized and validated as per the ICH guidelines. These details are present in their chapter. 217

6 Section 6.1 IODOMETRIC DETERMINATION OF MILLIGRAM AMOUNTS OF EPROSARTAN MESYLATE IN PHARMACEUTICALS BY TITRIMETRY INTRODUCTION The well-known reaction between iodide and iodate in the presence of acid solution has been used for the determination of acids; this is used as an alternative, often more accurate than the usual titration with alkali [33]. IO3-+5I-+6H+ 3I2+H2O Iodide present is oxidized, in amounts equivalent to the acid present, to iodine which is titrated with thiosulphate. This reaction has earlier been exploited for the assay of a number of organic acids [34-38]. The acidic property of EPR is due to the presence of two carboxylic acid moieties and a mesylate group present. As it is clear from the literature survey presented in Section 6.0.2, the acidic property of EPR was not exploited for its quantification. The author developed and validated two methods for the determination of EPR in both bulk and dosage form. The methods exploit the well-known analytical reaction between iodide and iodate in the presence of acid solution. Iodide present is oxidized by iodate in an amount equivalent to the carboxylic groups and mesylate group present in EPR to iodine and the liberated iodine is determined by two titrimetric procedures and involve either direct titration of iodine by thiosulphate (method A) or potentiometric determination of liberated iodine by thiosulphate (method B). Both the methods have a reaction stoichiometry of 1:3 (EPR: liberated iodine), inferring the participation of two carboxylic acid groups and one mesylate group EXPERIMENTAL Apparatus An Elico LI 120 digital potentiometer meter with platinum-sce system was used for potentiometric titration Reagents All chemicals used were of analytical reagent grade and distilled water was used throughout the study. 218

7 Potassium iodate: A saturated solution of potassium iodate (Merck, Mumbai, India) was prepared by stirring approximately 20 g of the chemical in a beaker containing 100 ml water with the help of magnetic stirrer for 60 minutes. The solution was decanted and filtered using quantitative filter paper. Potassium iodide: A saturated solution of potassium iodide (Merck, Mumbai, India) was prepared just before use in order to prevent atmospheric oxidation to iodine. Sodium thiosulphate: A 0.01 M sodium thiosulphate solution was prepared by dissolving g of the chemical (Lobachemie, Mumbai, India) in 250 ml of water. The solution was standardized against 0.01M potassium dichromate. 1 % starch: A paste of 1 g of the chemical (potato starch, loba chemie, Mumbai, India) in cold water was added to 100 ml of boiling water, boiled for 1 min and cooled. It was prepared afresh every day. Preparation of standard EPR solution Pure EPR (Pharmaceutical grade) sample was kindly provided by Jubiliant Life Sciences Ltd, Nanjangud, Mysore, India, as a gift and used as received. A stock standard solution equivalent to 1.5 mg ml-1 was prepared by dissolving 375 mg of pure drug in methanol and diluting to 250 ml in a calibrated flask with methanol PROCEDURES Method A A 10 ml aliquot of pure EPR solution containing mg of EPR was taken in an Erlenmeyer flask. Five ml each of saturated solution of KIO3 and of KI were added and the flask was stopperred and let stand for 10 min with occasional swirling. Finally, 1 ml of 1% starch indicator was added and liberated iodine was titrated against standardized solution of 0.01 N Na 2S2O3 until the decoloration of blue color. The amount of EPR was computed from the following formula: Amount ( mg ) (B S ) M r C n where B = volume of thiosulphate consumed in the blank titration S = volume of thiosulphate consumed in the sample titration Mr = relative molecular mass of drug C = concentration of titrant, moles l-1 n = number of moles of titrant reacting with per mole of EPR 219

8 Method B An aliquot of the standard drug solution equivalent to mg of EPR was measured accurately and transferred into a clean 100 ml beaker. Five ml each of saturated solutions of KI and KIO3 were added. The content was stirred magnetically for 10 min and the titrant (0.01 N Na2S2O3) was added from a microburette. Near the equivalence point, titrant was added in 0.1 ml increments. After each addition of titrant, the solution was stirred magnetically for 30 s and the steady emf was noted. The addition of titrant was continued until there was no significant change in emf on further addition of titrant. The equivalence point was determined by applying the graphical method. The amount of the drug in the measured aliquot was calculated as described under visual titration. Procedure for tablets Ten tablets were weighed accurately and ground into a fine powder. An accurately weighed amount of the powdered tablet equivalent to 150 mg of EPR was transferred into a 100 ml calibrated flask. 60 ml of methanol was added and the content was shaken thoroughly for min to extract the drug into the liquid phase; the volume was finally diluted to the mark with the methanol, mixed well and filtered using a Whatman No. 42 filter paper. An aliquot of the filtrate (1.5 mg ml-1 in EPR) was used for assay by following the general procedures. Procedure for the analysis of placebo blank and synthetic mixture A placebo blank of the composition: starch (20 mg), acacia (30 mg), hydroxyl cellulose (20 mg), sodium citrate (20 mg), talc (40 mg), magnesium stearate (30 mg) and sodium alginate (20 mg) was prepared. A 50 mg of the placebo blank was accurately weighed and its solution was prepared as described under Procedure for tablets, and then subjected to analysis by following the general procedures. To 50 mg of the placebo blank of the composition described above, 75 mg of EPR was added and homogenized, transferred to a 50 ml calibrated flask and the solution was prepared as described under Procedure for tablets, and then subjected to analysis by the procedure described above. This analysis was performed to study the interference by excipients normally present in tablet preparation. 220

9 6.1.3 RESULTS AND DISCUSSION Preliminary experiments showed that EPR is sufficiently acidic to release iodine from iodate-iodide mixture allowing the titrimetric determination of drug where the liberated iodine was titrated with thiosulphate and the end point being located visually to the starch end point in method A, and potentiometric end point in method B (Figure 6.1.1) emf, mv ΔE/ΔV Na2S2O3 added,ml Na2S2O3 added, ml Figure Potentiometric titration curves for 7.5 mg EPR vs 0.01 M Na2S2O3 Optimization of experimental variables such as the amount of iodate-iodide mixture and contact time, revealed that for the range of EPR studied, 5 ml each of saturated solutions of KIO3 and KI and 10 min contact time were sufficient for quantitative liberation of iodine Method Validation Intra-day and inter-day accuracy and precision The precision of the methods was evaluated in terms of intermediate precision (intra-day and inter-day). Three different amounts of EPR within the range of study in each method were analyzed in seven and five replicates in method A and method B, respectively, during the same day (intra-day precision) and five consecutive days (inter-day precision). For inter-day precision, each day analysis was performed in triplicate and pooled-standard deviation was calculated. The RSD (%) values of intraday and inter-day studies for EPR showed that the precision of the methods was good (Table 6.1.1). The accuracy of the methods was determined by the percent mean deviation from known concentration, and results are presented in Table

10 Robustness and ruggedness of the methods Method ruggedness was expressed as the RSD (%) of the same procedure applied by four different analysts as well as using four different burettes. The interanalysts RSD (%) were within 1.72% whereas the inter-burettes RSD (%) for the same EPR amounts was less than about 1.84% suggesting that the developed methods were rugged. The robustness was studied by slightly altering the ml of KIO3 and reaction time and studying their effect on the results. The results are presented in Table Table Results of intra-day and inter-day accuracy and precision study Intra-day (n=7) Method EPR taken, mg EPR found, mg A B Inter-day (n=5) RE, % RSD, % EPR found, mg RE, % RSD, % RE.relative error, RSD. relative standard deviation. Table Results of robustness and ruggedness study expressed as intermediate precision (% RSD) Method EPR taken, mg A B a Robustness Volume of Reaction timea KIO3 (% RSD): (%RSD) (n=3) Ruggedness Inter-analysts Inter-burettes (% RSD): (% RSD): (n=4) (n=4) Reaction times employed were 10 ± 1min; volumes of KIO3 were 5± 0.5 ml 222

11 Selectivity Placebo blank and synthetic mixture were prepared as described in the previous section and analyzed. In the analysis of matrix, the volume of Na2S2O3 consumed was the same as that of the indicator blank suggesting the non-interference by the inactive ingredients added to prepare the matrix substances. The assay of EPR in synthetic mixture was performed by following the general procedures (n = 5). The percentage recovery of EPR was calculated by using the amount of active substance taken and found, which yielded a % recovery values in the range from to 103.2% with standard deviation value of less than 1.94%. The results obtained which are close to 100% complements the findings of matrix (placebo) analysis with respect to selectivity. Application to tablets The described titrimetric procedures were successfully applied to the determination of EPR in its pharmaceutical formulations (Teveten tablet of 600 mg EPR/tablet). The obtained results (Table 6.1.3) were statistically compared with those obtained by the official chromatographic method [7]. The reference method describes chromatographic detection of EPR using UV-detector at 235 nm. The results obtained by the proposed methods agree well with those of reference method and with the label claim. The results were also compared statistically by a Student s t-test for accuracy and by a variance F-test for precision [39] with those of the reference method at 95 % confidence level as summarized in Table The results showed that the calculated t-and F-values did not exceed the tabulated values inferring that proposed methods are as accurate and precise as the reference method. Table Results of assay in tablets and comparison with the reference method. Tablet brand name Nominal amount (mg/tablet) Teveten Found* (Percent of label claim ± SD) Reference Proposed methods method Method A Method B 102.3± ± ±0. 76 t=2.38 t=0.39 F=2.53 F=1.70 *Average of five determinations. 223

12 Recovery Study To a fixed amount of drug in formulation (pre-analysed): pure drug at three different levels was added, and the total was found by the proposed methods. Each test was repeated three times. The recoveries were in the range from to 102.4% with the relative standard deviations of % indicating that commonly added excipients to tablets did not interfere in the determination. These results are compiled in Table Table Results of recovery study using standard addition method. Method A EPR in Pure EPR tablet Tablet studied added, extract, mg mg Tevaten Total EPR found, mg EPR in Pure EPR tablet recovered* % extract, mg 102.2± ± ± Mean value of three determinations.* 224 Method B Pure Total EPR EPR Pure EPR added, found, recovered* % mg mg ± ± ±1.65

13 Section 6.2 IODOMETRIC DETERMINATION OF MICROGRAM AMOUNTS OF EPROSARTAN MESYLATE IN PHARMACEUTICALS BY SPECTROPHOTOMETRY INTRODUCTION The reaction of iodate-iodide reagent in presence of acidic functional groups has been utilized as described under section From the literature survey presented in Section 6.0.2, the iodimetric method for the spectrophotometric determination of EPR has not been reported. This section describes the development and optimization of two iodimetric spectrophotomeric methods for the quantification of EPR. The methods involve spectrophotometric determination of liberated iodine at 360 nm (method A) or the iodine-starch complex at 570 nm (method B) EXPERIMENTAL Apparatus The instrument is the same that was described in Section Reagents All chemicals used were of analytical reagent grade and distilled water was used throughout the study. Potassium iodate: A 1% solution of potassium iodate (Merck, Mumbai, India) was prepared by stirring approximately 1 g of the chemical in a beaker containing 100 ml water with the help of magnetic stirrer for 60 minutes. Potassium iodide: Potassium iodide solution (Merck, Mumbai, India) was prepared just before use in order to prevent atmospheric oxidation to iodine. A 5% KI solution was prepared by dissolving 2.5 g of the chemical in 50 ml of water. Saturated Borax: Approximately 50 g of borax (S.d. Fine Chem., Mumbai, India) was dissolved in 100 ml water and stirred with the help of magnetic stirrer for 15 minutes. The solution was decanted and filtered. The ph of the solution was between 8 and

14 Preparation of standard EPR solution A stock standard solution equivalent to 200 µg ml-1 EPR was prepared by dissolving 20 mg of pure drug in methanol and diluting to 100 ml in a calibrated flask with methanol. This stock solution was appropriately diluted to get 20 µg ml-1 and 60 µg ml-1 for the use in method A and method B, respectively, with the same solvent. Assay procedures Method A A ml of 20 µg ml-1 EPR was added in to a series of 10 ml calibrated flasks by means of microburette. To each flask, 1 ml each of 1% KIO3 and 5% KI solutions were added, flasks stoppered, content mixed and let stand for 10 min. Then 1 ml of saturated borax solution was added and made up to the mark with water. Absorbance of each solution was measured at 360 nm against reagent blank. Method B Different volumes ( ml) of 60 µg ml-1 EPR were taken in a series of 10 ml calibrated flasks. One ml each of 1% KIO3 and 5% KI solutions were added, flasks stoppered and content mixed. The flasks were let stand for 10 min before adding 1 ml each of saturated borax and 1% starch to each flask; and made up to 10 ml with water. Absorbance of each solution was measured at 570 nm against reagent blank. Standard graph was prepared by plotting the absorbance versus drug concentration, and the concentration of the unknown was read from the calibration graph or computed from the respective regression equation. Procedure for tablets Ten tablets were weighed accurately and ground into a fine powder. An accurately weighed amount of the powdered tablet equivalent to 20 mg of EPR was transferred into a 100 ml calibrated flask. 60 ml methanol was added and the content was shaken thoroughly for min to extract the drug into the liquid phase; the volume was finally diluted to the mark with the methanol, mixed well and filtered using a Whatman No. 42 filter paper. An aliquot of the filtrate (20 µg ml-1 and 60 µg ml-1 in EPR) was used for method A and method B as described above. 226

15 Procedure for the analysis of placebo blank and synthetic mixture A placebo blank was prepared as described under Section A 20 mg of the placebo blank was accurately weighed and its solution was prepared as described under tablets, and then subjected to analysis by following the general procedures. To the placebo blank of the composition described above, 10 mg of EPR was added and homogenized, transferred to a 50 ml calibrated flask and the solution was prepared as described under Procedure for tablets, and then appropriately diluted and subjected to analysis by the procedure described above RESULTS AND DISCUSSION The acidic property of EPR owing to the presence of carboxyl and mesylate functional groups present in its molecule was found to react in a stiochiometric amount with KIO 3 -KI mixture and thereby liberating stiochiometric amount of iodine. Iodine liberated in the above reaction was quantitatively determined using two spectrophotometric methods which involve the measurement of absorbance of either liberated iodine at 360 nm or the iodine-starch complex at 570 nm. Method development Absorbance of the liberated iodine or starch-iodine complex was measured at 360 or 570 nm as deduced from the absorption spectra of the colored species (Figure 6.2.1). The blanks had no absorption. In both the methods, the reaction was relatively fast in the beginning and iodine continued to be liberated even after 10 min. Since most of the iodine was liberated within 10 min, the reaction was stopped by adding borax to the reaction mixture after a standing time of 10 min. The absorbance remained constant for 45 and 60 min in method A and method B, respectively, after the reaction was ceased. Attempts to hasten the reaction by heating were unsuccessful owing to the volatility of iodine and dissociation of iodine-starch complex at elevated temperature. For quantitative liberation of iodine, 1 ml each of 1 % KIO 3 and 5% KI solutions were found adequate. 227

16 a Absorbance Absorbance b Wavelength, nm Wavelength, nm Figure Absorption spectra of : a. reaction product of method A (5µg ml-1epr) and b. reaction product of method B (12 µg ml-1epr) Method validation Linearity, sensitivity, limits of detection and quantification A linear correlation (Figure 6.2.2) was found between absorbance at max and concentration of EPR in the ranges given in Table Regression analysis of the Beer s law data using the method of least squares was made to evaluate the slope (b), intercept (a) and correlation coefficient (r) for each system and the values obtained from this investigations are presented in Table A plot of absorbance versus concentration yielded a straight line in both the cases with slope equal to and for method A and method B, respectively. The optical characteristics such as Beer s law limits, molar absorptivity and Sandell sensitivity values [40] of both the methods are also given in Table The limits of detection (LOD) and quantitation (LOQ) calculated according to ICH guidelines [41]. The high values of ε and low values of Sandell sensitivity and LOD indicate the high sensitivity of the proposed Absorbance Absorbance methods Concentration of EPR, µg ml Method A Concentration of EPR, µg ml-1 Method B Figure Calibration curves. 228

17 Table Regression and analytical parameters. Parameter max, nm Beer s law limits (µg ml-1) Molar absorptivity (l mol-1 cm-1) Sandell sensitivity* (µg cm-2) Limit of detection (µg ml-1) Limit of quantification (µg ml-1) Regression equation, Y** Intercept,(a) Slope,(b) Correlation coefficient (r) Standard deviation of intercept (Sa) Standard deviation of slope (Sb) Method A Method B * Limit of determination as the weight in µg ml-1of solution, which corresponds to an absorbance of A = measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. ** Y a bx, where Y is the absorbance, a is the intercept, b is the slope and X is the concentration in µg ml-1. Precision and accuracy To check the repeatability and system suitability of the proposed methods, the assays described under general procedures were repeated seven times within the day (intra-day precision) and five times on five different days (inter-day precision). These assays were performed for three levels of analyte. The RSD values were 2.26% (intra-day) and 2.50% (inter-day) indicating high precision of the methods. The accuracy of the methods was evaluated as percentage relative error, RE (%), between the measured mean concentrations and taken concentrations for EPR. RE (%) values of 2.50% demonstrate the high accuracy of the proposed methods. The results of this study are summarized in Table Robustness and ruggedness The robustness of the methods was evaluated by making small incremental changes in the reaction time and the effect of the changes was studied on the absorbance of reaction product. The changes had negligible influence on the results as revealed by small intermediate precision values expressed as RSD (%) ( 1.91%). Method ruggedness was demonstrated having the analysis done by four analysts, and also by a single analyst performing analysis on four different cuvettes. Intermediate 229

18 precision values (%RSD) in both instances were in the range % indicating acceptable ruggedness. The results are presented in Table Table Results of intra-day and inter-day accuracy and precision study Method EPR taken (µg ml-1) A B a Intra-day (n = 5) EPR %RSDb %REc founda (µg ml-1) Inter-day (n = 5) EPR %RSDb founda (µg ml-1) %REc Mean value of five determinations; b. Relative standard deviation (%); c. Relative error (%). Table Results of method Robustness and ruggedness study expressed as intermediate precision A Method robustness Parameter altered Reaction time, mina RSD, % (n = 3) B Method a EPR taken, µg ml-1 Method ruggedness Inter-analysts RSD, % (n = 4) Inter-cuvettes RSD, % (n = 4) Reaction time employed was 10 ± 1.0 min. Selectivity A placebo blank solution as described in the previous sections was prepared and then subjected to analysis. The absorbance of the placebo solution in each case was almost equal to the absorbance of the blank which revealed no interference. To assess the role of the inactive ingredients on the assay of EPR, a synthetic mixture was separately prepared by adding 10 mg of EPR to the placebo mentioned above in a 50 ml calibrated flask. The drug was extracted and solution prepared as described under the general procedure for tablets. The appropriately diluted solutions were 230

19 analyzed by following the recommended procedures. The percentage recovery values of EPR obtained from this study were in the range from to This unequivocally demonstrated the non-interference of the inactive ingredients in the assay of EPR. Further, the slopes of the calibration plots prepared from the synthetic mixture solutions were about the same as those prepared from pure drug solutions. Application to tablets The proposed methods were applied for the quantification of EPR in commercial tablets. The results obtained were compared with those obtained using a reference chromatographic method [7]. The reference method describes chromatographic detection of EPR using UV-detector at 235 nm. Statistical analysis of the results did not detect any significant difference in the performance of the proposed method to the reference method with respect to accuracy and precision as revealed by the Student s t-value and variance ratio F-value. The results of this study are given in Table Table Results of analysis of tablets by the proposed methods. Tablet brand name Label claim mg/tablet Teveten Found (Percent of label claim ±SD)a Proposed methods Reference Method A Method B method ± ± 1.18 t = 0.92 F= ± 1.23 t = 2.72 F = 2.62 a Mean value of five determinations. Recovery study Recovery experiment was performed by applying the standard-addition technique. The recovery was assessed by determining the agreement between the measured standard concentration and added known concentration to the sample. The test was done by spiking the pre-analysed tablet powder with pure EPR at three different levels (50, 100 and 150 % of the content present in the tablet powder (taken) and the total was found by the proposed method. Each test was repeated three times. From this test the percentage recovery values were found in the range of to 231

20 103.3 with standard deviation values from %. Closeness of the results to 100% showed the fairly good accuracy of the method. These results are shown in Table Table Results of recovery study by standard addition method. Method A EPR Pure Total in EPR Tablets tablets, added, found,-1 studied µg ml-1 µg ml-1 µg ml Teveten Method B Pure Pure EPR EPR in Total EPR recovered*, tablets, found, added, -1-1 Percent±SD µg ml -1 µg ml µg ml 103.3± ± ± *Mean value of three determinations. 232 Pure EPR recovered*, Percent±SD 99.67± ± ±0.98

21 Section 6.3 SIMPLE UV-SPECTROPHOTOMETRIC METHODS FOR THE DETERMINATION OF EPROSARTAN MESYLATE IN INTRODUCTION PHARMACEUTICALS The importance and applications of UV-spectrophotometry in pharmaceutical analysis as well as the degradation profile under different conditions as recommended by the International Conference on Harmonization (ICH) guidelines are described in Section From the literature survey presented in Section it is evident that a few UV-spectrophotometric methods have been reported for quantification of EPR either alone or in combination with hydrochlorothiazide [8-13], but none of them is stability-indicating. This prompted the author to develop two simple and rapid UVspectrophotometric methods for EPR in bulk drug as well as in tablets dosage form and to study its degradation under various forced stress conditions. The methods are based on the measurement of the absorbance of EPR in H 2 SO 4 (0.1 M) at 234 nm (method A) and in acetonitrile: water at 232 nm (method B). The drug (EPR) was subjected to acid and alkali hydrolysis, oxidation, dry heat treatment and photo degradation as a part of the stability-indicating study EXPERIMENTAL Apparatus in Section Materials The instrument used for absorbance measurement is same as described All chemicals used were of analytical reagent grade. Doubly-distilled water was used to prepare solutions wherever required. Hydrogen peroxide (H 2 O 2 ), Sulphuric acid (H 2 SO 4 ), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Merck (Mumbai, India). Pure EPR (Pharmaceutical grade) sample was kindly provided by Jubiliant Life Sciences Ltd, Nanjangud, Mysore, India, as a gift and used as received. 233

22 Sulphuric acid (H2SO4, 0.1 M): Prepared by diluting concentrated acid (Merck, Mumbai, India, specific gravity 1.84) with water. Hydrochloric acid (2M), hydrogen peroxide (5%), sodium hydroxide (2M) solutions required for stress study were prepared as described under Section Standard drug solution Standard drug solution of 500 µg ml-1 EPR was prepared by dissolving accurately weighed 50 mg of pure drug in 100 ml calibrated flask in 0.1 M H2SO4 for method A, and in acetonitrile: water (40:60) for method B, and subsequently diluted to 50 µg ml-1 working standards Recommended procedures Calibration curves Method A Varying aliquots (0.2, 0.5, 1.0, 2,0, 3.0, and 4.0 ml) of working standard solution corresponding to 1-20 µg ml-1 EPR were taken in a series of 10 ml volumetric flasks and volume was made up to mark with 0.1 M H2SO4. The absorbance of each solution was measured at 234 nm against 0.1 M H2SO4. Method B Into a series of 10 ml calibration flasks, aliquots of EPR standard solution (50 µg ml-1) equivalent to 1-20 µg ml-1 EPR were accurately transferred and volume was made upto mark with acetonitrile: water (40:60). The absorbance of each solution was measured at 232 nm versus acetonitrile: water (40:60). In both the cases, calibration curves were prepared and the concentration of the unknown was read from the respective calibration curve or computed from the regression equation derived using the Beer s law data Procedure for tablets Weighed amount of tablet powder equivalent to 5 mg of EPR was transferred into a 100 ml volumetric flask. The content was shaken well with about 50 ml of 0.1 M H2SO4 or acetonitrile-water mixture for 20 min. The mixture was diluted to the mark with the respective solvent. It was filtered using Whatman No 42 filter paper. First 10 ml portion of the filtrate was discarded and a subsequent portion was subjected to analysis by following the procedures described earlier. 234

23 Placebo blank analysis A placebo blank was prepared as described under section A 20 mg of the placebo blank was accurately weighed and its solution was prepared as described under Procedure for tablets, and then subjected to analysis by following the general procedures. Procedure for synthetic mixture analysis To 10 mg of the placebo blank of the composition described above, 5 mg of EPR was added and homogenized, transferred to 100 ml calibrated flask and the solution was prepared as described under procedure for tablets Then the resulting solution was subjected to analysis using the procedure described above. Forced degradation study (Stability study) In both the methods, a 2 ml aliquot of the standard 50 µg ml-1 EPR was taken (in triplicate) in a 10 ml volumetric flask and mixed with 5 ml of 2M HCl (acid hydrolysis) or 2M NaOH (alkaline hydrolysis) or 5% H2O2 (oxidative degradation) and boiled for 2 h at 80 C in a hot water bath. The solution was cooled to room temperature and diluted to the mark with 0.1 M H2SO4 in method A and with acetonitrile: water in method B after neutralization. In thermal degradation, solid drug was kept in Petri dish in oven at 100 C for 24 h. After cooling to room temperature, 5 mg of EPR was weighed and transferred to a 100 ml volumetric flask, dissolved in and diluted up to the mark with 0.1 M H2SO4 and acetonitrile: water for method A and method B, respectively. For UV degradation study, the stock solution of the drug (50 µg ml-1) was exposed to UV radiation of wavelength 254 nm and of 1.2K flux intensity for 48 h in a UV chamber. Finally, the absorbance of all the resulting solutions (50 µg ml-1 in EPR) obtained from acid and alkaline hydrolysis, oxidative degradation, thermal and UV degradation of EPR, was measured at 234 nm against 0.1 M H2SO4 in method A and at 232 nm against acetonitrile: water in method B RESULTS AND DISCUSSION Spectral characteristics The absorption spectra of 10 µg ml-1 EPR solution in 0.1 M H2SO4 (method A) and 10 µg ml-1 EPR solution in acetonitrile: water (method B) were recorded between 200 and 400 nm and showed maximum absorption at 234 and 232 nm, for 235

24 method A and method B, respectively. At these wavelengths, 0.1 M H2SO4 and acetonitrile: water had insignificant absorbance. Therefore, the analysis of EPR was carried out at 234 and 232 nm, for method A and method B, respectively (Fig ). (a) (b) Figure Absorption spectra of: (a) EPR in 0.1 M H2SO4 ( 10 µg ml-1 EPR, method A); (b) EPR in acetonitrile: water (10 µg ml-1 EPR, method B) Forced degradation study The extent of degradation was evaluated based on the comparison of the UV spectra of stressed EPR samples with that of the standard EPR solution [42]. The resulting UV spectrum of stress EPR solution (10 µg ml-1 in acetonitrile: water) subjected to acid hydrolysis showed the same spectrum (Fig ) of the standard solution which indicated that EPR does not undergo degradation under this condition. Under alkaline conditions EPR solution in acetonitrile: water medium undergoes significant degradation (Fig ). These results are summed up in Table Figure Degradation study of EPR solution treated with 2M HCl 236

25 Figure Degradation study of EPR solution treated with 2M NaOH. Treatment with hydrogen peroxide also caused significant degradation (Figure 6.3.4). Exposure to dry heat and UV-light had no effect (Figure 6.3.5). Figure Degradation study of EPR solution treated with 5% H2O2. (a) (b) Figure Degradation study of EPR solution treated with a. dry heat at 105oC for 4 hr and b. UV radiation for lux hr. 237

26 Table Results of degradation study Degradation condition % Assay* Observation Control sample 99.9 Not applicable Acid hydrolysis (2M HCl, 80 C, 2 hours) 99.7 Slightly degraded Base hydrolysis (2M NaOH, 80 C, 2 hours) - Extensively degraded Oxidation (5% H2O2, 80 C, 2 hours) - Extensively degraded Thermal (105 C, 3 hours) 99.5 Photolytic (1.2 million lux hours) 99.4 No degradation observed No degradation observed * Percentage against standard EPR Method validation The proposed methods were validated for linearity, sensitivity, accuracy, precision, robustness, ruggedness, selectivity, interference and recovery. Linearity and sensitivity Linear correlation was obtained between the absorbance and concentration of EPR in the range of µg ml-1 for both methods as shown in Figure The calibration graphs are described by the equation: Y=a+bX (Where Y= absorbance, a= intercept, b= slope and X= concentration in µg ml-1) obtained by the method of least squares. Correlation coefficient, intercept and slope for the calibration data are summarized in Table Sensitivity parameters such as apparent molar absorptivity and Sandell sensitivity values, the limits of detection and quantification are calculated as per the current ICH guidelines [41] are compiled in the same table, speak of the excellent sensitivity of the proposed method. The limits of detection (LOD) and quantification (LOQ) were calculated using the formulae: LOD = 3.3σ/b and LOQ = 10σ/b where σ is the standard deviation of five reagent blank determinations and b is the slope of the calibration curve. 238

27 1.2 Absorbance Concentration of EPR, µg ml-1 25 Figure Calibration curve Table Regression and analytical parameters. Parameter max, nm Beer s law limits (µg ml-1) Molar absorptivity (l mol-1 cm-1) Sandell sensitivity (µg cm-2) Limit of detection (µg ml-1) Limit of quantification (µg ml-1) Regression equation, Y Intercept,(a) Slope,(b) Correlation coefficient (r) Standard deviation of intercept (Sa) Standard deviation of slope (Sb) Method A Method B Accuracy and precision Intra-day accuracy and precision of the proposed method were evaluated by replicate analysis (n = 7) of calibration standards at three different concentration levels on the same day. Inter-day accuracy, expressed as percentage relative error (% RE) and precision expressed as percentage relative standard deviation (%RSD) were determined by assaying the calibration standards at the same concentration levels on five consecutive days. The %RE and %RSD of the proposed method were calculated. These results reveal fairly good accuracy and precision of the proposed methods (Table 6.3.3). 239

28 Ruggedness To evaluate method ruggedness analysis was performed by four different analysts, and also with four different cuvettes by a single analyst. The intermediate precision, expressed as percent RSD, which is a measure of ruggedness, was within the acceptable limits as shown in the Table Table Results of intra-day and inter-day accuracy and precision study. Method A B a. EPR taken (µg ml-1) Intra-day (n = 5) EPR %RSDb %REc founda (µg ml-1) Inter-day (n = 5) EPR %RSDb founda (µg ml-1) %REc Mean value of five determinations; b. Relative standard deviation (%); c. Relative error (%). Selectivity The absorbance of the placebo solution prepared in each case was almost equal to the absorbance of the blank which revealed no interference. The synthetic mixture solutions were analyzed by following the recommended procedures. The percentage recovery values of EPR obtained from this study were in the range from to This unequivocally demonstrated the non-interference of the inactive ingredients in the assay of EPR. Analysis of tablet When the methods were applied to commercial tablets, the results were comparable with those of official method [7]. The assay was performed for one brand of tablet containing 600 mg of active ingredient (Teveten-600) as described earlier. Statistical analysis of the results did not detect any significant difference between the performance of the proposed methods and reference method with respect to accuracy and precision as revealed by the Student s t-value and variance ratio F-value. The results of this study are presented in Table

29 Table Results of ruggedness study expressed as intermediate precision (%RSD) Method ruggedness Inter-analysts Inter-cuvettes RSD, % RSD, % (n = 4) (n = 4) EPR taken, µg ml-1 Method A B Table Results of analysis of tablets by the proposed methods. Tablet Brand name Label claim mg/tablet Teveten Found (Percent of label claim ±SD)a Proposed methods Reference Method A Method B method ± ± ± 0.76 t = 2.63 t = 0.82 F= 3.02 F = 1.31 a Mean value of five determinations. Recovery study The recovery test was done by spiking the pre-analyzed tablet powder with pure EPR at three different levels (50, 100 and 150 % of the content present in the tablet powder (taken) and the total was found by the proposed methods. Each test was repeated three times. The recovery percentage values ranged between and 102.8% with relative standard deviation in the range %. Closeness of the results to 100% showed the fairly good accuracy of the methods. The results are shown in Table

30 Table Results of recovery study by standard addition method. Method A EPR in tablets, µg ml Pure EPR added, µg ml Teveten Tablets studied Method B Total found, µg ml-1 Pure EPR recovered*, Percent±SD EPR in tablets, µg ml ± Pure EPR added, µg ml ± ± *Mean value of three determinations. 242 Total found, µg ml-1 Pure EPR recovered*, Percent±SD ± ± ±0.91

31 Section 6.4 DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE DETERMINATION OF EPROSARTAN MESYLATE IN TABLETS AND ITS STABILITY STUDY INTRODUCTION In the literature survey presented in Section various chromatographic techniques have been cited for the assay of EPR in pharmaceuticals. However, few reported HPLC methods have narrow linear concentration ranges [17,18]. United States pharmacopeia describes a HPLC method for the determination of EPR using a mobile phase (acetonitrile, phosphoric acid, and water) at 235 nm [7]. The author, under slightly altered chromatographic conditions, has been able to develop a method which is characterized by improved sensitivity and linear dynamic range. The details are contained in this Section EXPERIMENTAL Chemicals and Reagents HPLC-grade acetonitrile, triethyl amine and formic acid were purchased from Merk Ltd., Mumbai, India. Bi-distilled water was used to prepare all solutions. All other chemicals and reagents used were of analytical reagent grade and purchased from S.D Fine Chemicals, Mumbai, India. A buffer of ph 3.0 was prepared by adjusting the ph of triethyl amine with formic acid. The mobile phase was prepared by mixing the buffer and acetonitrile in 70:30 (v/v) ratio. The mobile phase was used as diluent. HCl (1M), NaOH (1M) and H 2 O 2 (5%) solutions for degradation study were prepared as described previously. A stock solution of EPR (1000 µg ml -1 ) was prepared in diluent. Standard solutions were prepared by dilution of the stock solution with the diluent to get solution in the concentration range, 5 to 150 µg ml -1 EPR HPLC instrumentation and chromatographic conditions HPLC analysis was performed with a Waters HPLC system equipped with Alliances 2695 series low pressure quaternary gradient pump, a programmable variable wavelength UV-visible detector and autosampler. Data were collected and processed using Waters Empower 2 software. 243

32 Chromatographic separation was achieved on a Symmetry C8 ( mm, 3.5u) column. The mobile phase was a 70:30 (v/v) mixture of triethyl amine with formic acid (ph 3.0)-acetonitrile, at a flow rate of 1.0 ml min-1; and UV-detection was performed at 233 nm. Before use, the mobile phase was filtered through 0.2 µm filter. The column temperature was maintained at 30 C Procedures calibration graph Working standard solutions (5-150 µg ml-1 EPR) were injected automatically onto the column in triplicate and the chromatograms were recorded. The calibration graph was prepared by plotting the mean peak area versus concentration of EPR in µg ml-1. A standard curve prepared with known solution or regression equation derived using mean peak area-concentration data allowed the calculation of unknown concentration. Procedure for tablets Ten tablets were accurately weighed and crushed into a fine powder and mixed using a mortar and pestle. A quantity of tablet powder equivalent to 100 mg of EPR was weighed accurately into a 100 ml calibrated flask, 50 ml of diluent solution added and was sonicated for 20 min to complete dissolution of the EPR, and the solution was then diluted to the mark with the diluent and mixed well. A small portion of the tablet solution (say 10 ml) was withdrawn and filtered through a 0.2 µm filter to ensure the absence of particulate matter. The filtrate was appropriately diluted with the diluent before injection onto the column Stress study All stress decomposition studies were performed at an initial drug concentration of 100 µg ml-1 in mobile phase. Acid hydrolysis was performed in 1 M HCl at 80 C for 2 h. The study in alkaline condition was carried out in 1 M NaOH at 80 C for 2 h. Oxidative studies were carried out at 80 C in 5% hydrogen peroxide for 2 h. For photolytic degradation studies, pure drug in solid state was exposed to 1.2 million flux hours in a photo stability chamber. Additionally, the drug powder was exposed to dry heat at 105 C for 3 h. Samples were withdrawn at appropriate time, cooled and neutralized by adding base or acid and subjected to HPLC analysis after suitable dilution or preparing solution of appropriate concentration in the diluent. 244