Stability Indicating HPLC Method for Simultaneous Determination of Several Angiotensin-II-Receptor Antagonists in Their Dosage Forms

Angiotensin antagonists are the first major innovation in essential hypertension management as a first-line treatment. Angiotensin II receptor antagonists (ARA II) have been developed to specifically and selectively block the AT1 receptor of the rennin angiotensin system by displacing angiotensin II from it [1]. Losartan potassium, Telmisartan, Irbesartan and Valsartan are highly selective, non-peptide angiotensinII receptor antagonists (ARA-II). They are effective agents for the treatment of hypertension and heart failure either alone or together with diuretics [2].


Introduction
Angiotensin antagonists are the first major innovation in essential hypertension management as a first-line treatment. Angiotensin II receptor antagonists (ARA II) have been developed to specifically and selectively block the AT1 receptor of the rennin angiotensin system by displacing angiotensin II from it [1]. Losartan potassium, Telmisartan, Irbesartan and Valsartan are highly selective, non-peptide angiotensin-II receptor antagonists (ARA-II). They are effective agents for the treatment of hypertension and heart failure either alone or together with diuretics [2].
Information on the stability of the drug substance is an integral part of the systematic approach to stability evaluation. Stress testing (or forced degradation studies) is a critical component of the drug development process [3]. The ICH guideline indicates that stress testing is designed to help "determine the intrinsic stability of the molecule by establishing degradation pathways in order to identify the likely degradation products and to validate the stability indicating power of the analytical procedures used [4]. Stress testing also is becoming increasingly important in testing new molecules. Methods developed by stress testing and the stability information gained from those methods can have a significant effect on the actual compound selected for development [3]. Literature review revealed that several methods have been reported for analysis of the drug in pharmaceutical formulation alone or with other drugs in combination. USP described a HPLC method for assay and impurities of Losartan potassium, Valsartan and their impurities B, C (RP-HPLC), Valsartan impurity A (normal phase HPLC) and Irbesartan (ion pair HPLC), ion pair HPLC for Telmisartan [5]. BP described a potentiometric titration for assay of Losartan potassium, Irbesartan, Telmisartan and Valsartan. For impurities BP used a gradient RP-HPLC for Losartan potassium and ion pair HPLC for Telmisartan [6]. Some methods have been published for simultaneous determination of studied ARA-II-drugs including Spectrophotometry [7], HPLC-DAD [2], HPLC with fluorimetric detection [1] and capillary zone electrophoresis [8,9]. Some stability studies were reported for Losartan [10][11][12][13], Irbesartan [14,15], Valsartan [16][17][18] and Telmisartan [19]. But no published method depends on optimum stress test conditions [20] and it is the first stability study which comprises the four ARA-II compounds together. So, our scope is achieving a stress testing study of ARA-II drugs including development of a powerful stability indicating method. New study depends on previous study has been established to reach the optimum stress conditions in drug development process and time points for a detailed study (solid-state stress). Developed stability indicating method is accurate, sensitive, rapid and simple. The developed method was validated according to (ICH) guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness [21]. The method is proved to be robust with respect to change in flow rate, pH, organic phase composition and column temperature. It can be applied in majority of companies in the world.

Chemicals and reagents
All reagents are analytical or HPLC grade. Potassium dihydrogen phosphate, orthophosphoric acid and sodium hydroxide (NaOH) were supplied by (Merck, Darmstadt, and Germany), acetonitrile and methanol (HPLC grade) were supplied by (Fischer scientific, U.K.) and distilled water.
(Note: The water used in all the experiments was obtained from Milli-RO and Milli-Q systems (Millipore, Bedford, MA).
Irbesartan, Losartan potassium, Telmisartan and Valsartan working standard powders were kindly supplied by Egyptian international pharmaceutical industries company (EIPICO) (10 th Ramadan, Egypt) and they were used without further purification.

Stock standard solutions
Stock standard solutions containing (1.5, 0.5, 0.8, 0.8 mg/ml) of Irbesartan, Losartan potassium, Telmisartan and Valsartan were prepared by dissolving (150, 50, 80, 80 mg) of them in methanol in 100 ml volumetric flask respectively. Solutions were sonicated for 15 minutes and the final volume of solutions was made up to 100 ml with methanol to get stock standard solutions.

Preparation of calibration plot (working standard solutions)
To construct calibration plots, The stock standard solutions were diluted with the mobile phase (freshly prepared) to prepare working standard solutions in the concentration ranges (30-180,10-60,16-96, 16-96 μl/ml) for Irbesartan, Losartan potassium, Telmisartan, Valsartan respectively. Each solution (n=5) was injected in triplicate and analyzed under the mentioned conditions above. Linear relationships were obtained when average drug standard peak area were plotted against the corresponding concentrations for each drug. Regression equation was computed.

Sample preparation
A composite of ten X-tension tablets, Losar Mepha tablets, Disartan capsules and Micardis tablets were prepared by grinding each type of tablets separately to a fine, uniform size powder, triturated using mortar and pestle. After calculating the average tablet weight, amounts of powder equivalent to 150, 50, 80 and 80 mg for Irbesartan, Losartan potassium, Telmisartan, and Valsartan respectively were accurately weighed and transferred separately of each type of tablets to 100 ml volumetric flasks respectively then complete with methanol up to 100 ml. The solutions were sonicated for 15 min and the solutions were then filtered through 0.45 μm Nylon membrane filters (Millipore, Milford, MA, USA). Aliquots of appropriate volume (10 ml) were transferred to 100 ml calibrated flasks and diluted to volume with mobile phase to obtain the mentioned concentration above. The diluted solutions were analyzed under optimized chromatographic conditions and chromatogram is showed in Figure 2.  To determine the proposed method as a stability-indicating method, Irbesartan, Losartan potassium, Telmisartan, and Valsartan respectively bulk powders were stressed under different conditions in forced degradation studies.
Acidic and alkaline degradation: Hydrochloric acid (HCl) (1 M, 10 ml) and sodium hydroxide (NaOH) (1 M, 10 ml) were separately added to 10 ml methanolic stock solutions of Irbesartan, Losartan potassium, Telmisartan, and Valsartan respectively. These mixtures were separately heated at 70°C for 14 days in the dark (to exclude the possible degradative effect of light). The solutions (1 ml) were then transferred to 10 ml volumetric flasks, neutralized by addition of 1 M NaoH or 1 M HCl, and diluted to final volume with mobile phase [4,20].
Oxidation: Hydrogen peroxide (H 2 O 2 ; 3%, v/v, 10 ml) was added to 10 ml methanolic stock solutions of Irbesartan, Losartan potassium, Telmisartan, and Valsartan respectively. These solutions were separately set aside for 7 days at ambient temperature in the dark. The solutions (1 ml) obtained were then transferred to 10 ml volumetric flasks and diluted to final volume with mobile phase [4,20].

Neutral degradation (Thermal degradation):
Methanolic stock solutions of Irbesartan, Losartan potassium, Telmisartan, and Valsartan respectively were heated at 70°C for 14 days in the dark to study the effect of thermal stress. Also the experiment was performed on solidstate samples which could be stressed under previous condition and then diluted with a known amount of mobile phase. The experiment was performed in the dark to exclude the possible degradative effect of light. The solutions (1 ml) obtained were then transferred to 10 ml volumetric flasks and diluted to final volume with mobile phase [4,20].
Photo stability: Methanolic stock solutions of Irbesartan, Losartan potassium, Telmisartan, and Valsartan respectively (10 ml) were exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 watt hours/square meter. Also the experiment was performed on solid-state samples which could be stressed under previous condition and then diluted with a known amount of mobile phase. The solutions (1 ml) obtained were then transferred to 10 ml volumetric flasks and diluted to final volume with mobile phase [4,22].

Method Validation Specificity
The Specificity of the method was evaluated by assessing whether    [23]. A placebo for each tablet was prepared by mixing the respective excipients. Solutions were prepared by following the procedure described in the section on sample preparation. The commonly used tablet excipients did not interfere with the method. The diluent chromatogram shows that the tablet diluent has negligible contribution after the void volume at the method detection wavelength of 220 nm. The method were also evaluated by assessing whether degradation products present in the pharmaceutical formulations interfered with the analysis, obtained from stress studies involving acid, base, peroxide, and heat as well as analysis of samples stored under ICH stability conditions (Figure 3).

Linearity and range
According to ICH recommendations [Q2R1] [21], at least five concentrations must be used. In this study five concentrations were chosen in the ranges (30-180, 10-60, 16

Precision
The precision of the method was investigated by measurement of both repeatability and Intermediate precision [21].
Repeatability: Repeatability is also termed intra-assay precision. Repeatability was investigated by injecting 6 determinations at 100% of the test concentration. RSD were calculated (Table 2).
Intermediate precision: In the inter-day studies, standard and sample solutions prepared as described above, were analyzed in triplicate on three consecutive days at 100% of the test concentration. RSD were calculated (Table 3).

Accuracy
Accuracy was assessed using 9 determinations over 3 concentration levels covering the specified range (80,100 and120%). Accuracy was reported as percent recovery by the assay of known added amount of analyte in the sample ( Table 4).

Limits of detection and Limits of quantitation
In accordance with ICH recommendations [21], determination of limits of detection and quantitation was based on the standard deviation of the y-intercepts of regression lines (n=3) and the slope of the calibration plots. The values obtained are given in table 5.

Robustness
Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness was assessed by studying the effect of changing mobile phase pH by ± 0.1, the amount of acetonitrile in the mobile phase by ± 2%, temperature ± 2°C, different column and flow rate ± 0.05 ml/min. Variations had no significant effect on the chromatographic resolution of the method.

Stability of analytical solution
Also as part of evaluation of robustness, Solution stability was evaluated by monitoring the peak area response. Standard solutions in methanol were analyzed after its preparation 1, 2 and 3 days after at 5°C and for a day at room temperature. The change in standard solution peak area response over 3 days was (0.56, 0.74, 0.53and 0.97%) for Irbesartan, Losartan potassium, Telmisartan and Valsartan respectively and no significant degradation was observed during this period. Their solutions were found to be stable for 3 days at 5°C (in the refrigerator) and for a day at room temperature at least.

Application on pharmaceutical Preparation
The proposed methods were successfully used to determine Irbesartan, Losartan potassium, Telmisartan and Valsartan respectively in their dosage forms e.g. X-tension tablets, LosarMepha tablets, Micardis tablets and Disartan capsules respectively.

Drug name
Average μg/ml Average % RSD     Five replicate determinations were performed. Satisfactory results were obtained for each compound in good agreement with label claims (Table 6 and 7). The results obtained were compared statistically with those from published methods [15,[23][24][25] by using Student's t-test (for accuracy) and the variance ratio F-test (for precision). The results in (Table 8) showed that the t and F values were smaller than the critical values. So, there were no significant differences between the results obtained from this method and published methods.

Optimization of parameters of HPLC method
To establish and validate an accurate method for analysis of these drugs in pharmaceutical formulations, preliminary tests were performed with the objective of selecting optimum conditions. The main problems encountered during these investigations were lack of resolution between of Irbesartan and Valsartan and excessive retention of Telmisartan. To solve these problems, ACE column (25 cm), Hypersil C18 (15 cm), Hypersil cyano (CN) and Hypersil CPS columns were tried for simultaneous determination of the drugs.
The effect of mobile phase composition were also studied (a) aqueous phase e.g. ammonium acetate buffer, citrate buffer and phosphate buffer (b) organic modifier e.g. acetonitrile and methanol (c) pH of aqueous phase e.g. 2.5, 3.5, 4.5, 6.0.
Changing pH of mobile phase from 3.5 to 6.0 affects on eluting of Valsartan because It is ionizable compound containing carboxylic group (COOH), pH of mobile phase greater than pKa of Valsartan (4.9) by more one unit and at pH's above the pKa of the analyte, the acidic analyte carries a negative charge and behaves as an extremely polar molecule [26].
The optimum wavelength for detection was 220 nm at which much better detector responses for four drugs were obtained. The best resolution with reasonable retention time was obtained at 65% phosphate buffer pH 6.0 and 35% acetonitrile as organic modifier. A major reason for using a concentration of 25 mM was achieving maximum sensitivity of UV detection at low wavelengths.

Optimization of parameters of stability indicating method
According to current good manufacturing practice (cGMP), all drugs must be tested under a stability-indicating method before release. Stress testing of drug substance can help in identifying the likely degradation products which can establish the degradation pathways and the intrinsic stability of the molecule. The nature of the stress testing will depend on the individual drug substance. These studies provide valuable information on drugs inherent stability and help in the validation of analytical methods to be used in stability studies. Because of the special nature of separation requirement during analysis of stability samples, chromatographic methods have taken precedence over the conventional methods of analysis. Other than separation of multiple components, the merits of chromatographic methods are that possess greater accuracy and sensitivity for even small quantities of degradation products produced [4,20].
Degradation studies: The chromatograms obtained from samples treated with acid, base, hydrogen peroxide, heat and photo degradation were examined. The chromatograms showed that both Losartan potassium and Valsartan were degradated under acidic conditions (Figure 4 and 5). Irbesartan also was degradated to large extent under basic conditions ( Figure 6) unlike Telmisartan wasn't degradated under all previous condition (Figure 7). In this study, thermal degradation had no effect and acidic and alkaline degradation had some extent effect but if acidic and alkaline degradation undergoes under effect of heat at 70°C, the extent of degradation increased for Valsartan, Losartan and Irbesartan (Figure 8-10).
The ratio of degradation of Losartan potassium, Valsartan and Irbesartan respectively had been calculated by normalization method (The percentage content of a component of the substance to be examined is calculated by determining the area of the corresponding peak as a percentage of the total area of all the peaks, excluding those due to solvents or reagents or arising from the mobile phase or the sample matrix and those at or below the disregard limit) [5] (Table 9).
Losartan potassium was degradated by acidic hydrolysis (1M HCl at 70°C) to give impurity E and F [12]. The impurity E was the major. It was found that many compounds will undergo dimerization reactions: those containing olefins, alcohols, and carboxylic acids (or other carbonyl chemistry e.g. aldol condensation reactions). Indoles have been shown to dimerize under acidic conditions. The dimerization is presumed to occur via protonation and nucleophilic attack of a second indole ( Figure 11).            Impurity E and F are isomer resulting from dimerization of two molecules of Losartan potassium by formation of a bond between nitrogen atom in tetrazole ring and carbon atom in 5-methanol in imidazole ring producing dimer and water.
It was found that carboxylic acids typically have pKa's in the range of 2-5.5 (depending on the nature of the substituents). Below the pKa (as in acidic hydrolysis), the carboxylic group is protonated and therefore the carbonyl carbon is more electrophilic. The carbonyl can undergo nucleophilic attack to form esters, amides, thioesters, etc.
In the case of attack by an alcohol, the reaction product is an ester and the reaction is called an esterification reaction. This can occur as an artifact reaction when acid/base hydrolysis reactions are performed using an alcohol co-solvent system such as methanol. Esters of the parent compound can also be observed as process-related impurities especially when alcohol solvents are used in the re-crystallization step.
Above the pKa, the carboxylate is anionic and the charge is resonance stabilized; the group is therefore less electrophilic and does not have a good leaving group. Therefore, reactions with nucleophiles are significantly suppressed when compared to the protonated form. Carboxylic acids are not prone to oxidative degradation ( Figure 15).  the three drugs subjected to liquid tandem mass analysis by using full scan and selective ion monitoring (SIM) and the results from HPLC method were confirmed by that of LC-MS