Development of a Stability-Indicating HPLC Method for Simultaneous Determination of Amlodipine Besylate and Atorvastatin Calcium in Bulk and Pharmaceutical Dosage Form

Copyright: © 2014 Hafez HM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Development of a Stability-Indicating HPLC Method for Simultaneous Determination of Amlodipine Besylate and Atorvastatin Calcium in Bulk and Pharmaceutical Dosage Form


Introduction
Hypertension and dyslipidemia are two most commonly cooccurring cardiovascular risk factors. Coronary artery disease (CAD) is the leading cause of morbidity and mortality worldwide accounting for in excess of 930,000 deaths. It is a multifactorial disease, emphasis is to treat overall cardiovascular risk, rather than single risk factors in isolation .The third National Health and Nutrition Examination Survey (NHANES) estimated that more than 64% of patients with hypertension also have dyslipidemia; conversely, approximately 47% of patients with dyslipidemia have hypertension. Antihypertensive and lipid lowering medications substantially reduce the risk of CAD, stroke, and death in patients with cardiovascular risk factors.
The fixed dose combination containing the antihypertensive agent amlodipine and the statin, atorvastatin, is the first combination of its kind designed to treat two risk factors for cardiovascular disease (CVD). The pharmacokinetic and pharmacodynamic properties of amlodipine and atorvastatin make them well suited for combination in a single pill to manage cardiovascular risk. The half lives of both agents facilitate once daily dosing, and both can be administered at any time of day with or without food. These drugs have no adverse effects on the other's efficacy or tolerability [1].
The preparation of new combinations of drugs in pharmaceuticals for pharmacological activity development, as well as the requirements of modern industrial-scale pharmaceutical analysis, encourages researchers to develop new and efficient methods for multi-quantification with separation procedures. High performance liquid chromatography is a dominant separation technique, especially in pharmaceutical analysis [3].
Literature survey indicated that several analytical methods have been described for the determination of active ingredients (Amlodipine Besylate and Atorvastatin Calcium) each alone [4,5], in combination with other compound [6,7]. Several method were reported for simultaneous determination of them together like Spectrophotometry [8][9][10], HPTLC [11] Spectrofluorimetry, HPLC  Coupled with Fluorescence Detection [12] Capillary Electrophoresis [3] HPLC Coupled with UV Detection [13][14][15][16][17], HPLC-MS-MS [18][19][20], RP-UPLC [21,22]. Stability testing and stress testing (forced degradation studies) are critical components of drug development strategy. The studies help us understand the mechanism of a drug's decomposition, which further helps in obtaining information on physical and chemical factors that result in instability. These factors are then controlled in order to stabilize the drug or drug formulation, resulting in increased shelf-life or improved efficacy [23]. Stress testing is defined as the stability testing of drug substances and drug products under conditions exceeding those used for accelerated testing. These studies are undertaken to elucidate the intrinsic stability of the drug substance. According to International Conference on Harmonization (ICH) guideline Q1A (R2), the stability testing of drug substances should be carried out under different stress conditions (hydrolysis, oxidation, photolysis, and thermal degradation) to validate the stability-indicating supremacy of analytical methods used for the analysis of stability samples [24]. The standard conditions for photo stability testing are described in ICH guideline Q1B [25]. These tests allow accurate and precise quantification of drugs and their degradation and interaction products. Few analytical methods have been reported as stability indicating methods for simultaneous determination of amlodipine and atorvastatin in presence of their degradation products [26][27][28][29]. The focus of the present study was to develop a simple, rapid, precise, and accurate isocratic reversed-phase stability-indicating HPLC method for the simultaneous determination of amlodipine and atorvastatin and their impurities in tablet dosage form.

Experimental Instrumentation
Analysis was performed on a chromatographic system of WATERS 2695 separation module connected to WATERS 2487 UV/VIS detector. The system equipped by Empower PC program. The chromatographic separation was achieved on Phenomenex (100×4.6 mm)

Chemicals and reagents
All reagents used were of analytical grade or HPLC grade. Potassium dihydrogen phosphate and ortho-phosphoric acid were supplied by (Merck, Darmstadt, 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).
Amlodipine Besylate and Atorvastatin Calcium working standard powders were kindly supplied by Egyptian international pharmaceutical industries company (EIPICO) (10 th Ramadan, Egypt), and were used without further purification.
Potassium dihydrogen phosphate (pH 5.5, 0.03M) was prepared by dissolving 4.08 g Potassium dihydrogen phosphate in approximately 950 ml distilled water. The pH was adjusted to 5.5 with sodium hydroxide. Water was added to 1000 ml. Mobile phase was filtered through a 0.45 µm Nylon membrane filter (Millipore, Milford, MA, USA) under vacuum and degassed by ultrasonication (Cole Palmer, Vernon Hills, USA) before usage. Mixture of acetonitrile and distilled water (50:50 V/V) was prepared to be used as diluent.

Preparation of stock standard solutions
Stock standard solutions containing 1 mg/ml of Amlodipine (as Besylate) (actual weight =103.6 mg ) and 1 mg/ml of Atorvastatin (as Calcium) (actual weight=105.2 mg ) were prepared by dissolving 100 mg of each in 40 ml methanol in 100 ml volumetric flask respectively. It was sonicated for 5 minutes and the final volume of solutions was made up to 100 ml with diluent to get stock standard solutions.

Preparation of calibration plot (working standard solutions)
To construct calibration plots, the stock standard solutions were diluted with diluent to prepare working solutions in the concentration ranges (5 -15 µg/ml) for Amlodipine Besylate and Atorvastatin Calcium. Each solution (n=5) was injected in triplicate and chromatographed 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 Caduet10/10 tablets was prepared by grinding it to a fine, uniform size powder, triturated using mortar and pestle. After calculating the average tablet weight, amounts of powder equivalent to 10 mg for both drugs was accurately weighed and transferred to 100 ml volumetric flasks then complete with diluent 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 diluent to obtain the mentioned concentration above (10 µg/ml). The diluted solutions were analyzed under optimized chromatographic conditions and chromatogram is showed in (Figure 2A).

Forced degradation of amlodipine besylate and atorvastatin calcium
To determine the proposed method as a stability-indicating method for Amlodipine Besylate, Atorvastatin Calcium and Caduet tablets bulk powders were stressed under different conditions in forced degradation studies. Stock solutions of Amlodipine Besylate, Atorvastatin Calcium and Caduet tablets bulk powders-used to forced degradation studieswere prepared by dissolving it in diluent [30].
Acidic degradation: Hydrochloric acid (HCl) (1 M, 10 ml) was added to 10 ml prepared stock solutions of Amlodipine Besylate, Atorvastatin Calcium and Caduet tablets respectively. These solutions were separately heated at 70°C for 6 hours in the dark (to exclude the possible degradative effect of light). The solutions (2 ml) were then transferred to 25 ml volumetric flasks, neutralized by addition of1ml of 1 M NaOH and diluted to final volume with diluent [25,30,31].
Alkaline degradation: Sodium hydroxide (NaOH) (1 M, 10 ml) was added to 10 ml prepared stock solutions of Amlodipine Besylate, Atorvastatin Calcium and Caduet tablets respectively. These solutions were separately heated at 70°C for 2 hours in the dark (to exclude the possible degradative effect of light). The solutions (2 ml) were then transferred to 10 ml volumetric flasks, neutralized by addition of 1ml of 1 M HCl, and diluted to final volume with diluent [25,30,31].
Oxidation: Hydrogen peroxide (H 2 O 2 ; 10%, v/v, 10 ml) was added to 10 ml prepared stock solutions of Amlodipine Besylate, Atorvastatin Calcium and Caduet tablets respectively. These solutions were separately heated at 70°C for 6 hours in the dark. The solutions (2 ml) obtained were then transferred to 25 ml volumetric flasks and diluted to final volume with diluent [25,30,31].
Neutral degradation (Thermal degradation): Prepared stock solutions of Amlodipine Besylate, Atorvastatin Calcium and Caduet tablets respectively were heated at 70°C for 6 hours in the dark to study the effect of thermal stress. 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 experiment was performed in the dark to exclude the possible degradative effect of light. The solutions (1 ml) obtained were then transferred to 25 ml volumetric flasks and diluted to finalvolume with diluent [25,30,31].
Photo stability: Prepared stock solutions of Amlodipine Besylate, Atorvastatin Calcium and Caduet tablets 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 25 ml volumetric flasks and diluted to final volume with diluent [25,30,31].

Method Validation Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc [32]. A placebo of 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 240 nm. The method were also evaluated by assessing whether impurities like (Amlodipine impurity A, Amlodipine impurity B, Atorvastatin impurity A andAtorvastatin impurity C )and degradation products present in the pharmaceutical formulations-obtained from stress studies involving acid, base, peroxide, and heat stored under ICH stability conditions-interfered with the analysis of Amlodipine Besylate and Atorvastatin Calcium (Figure 2A).

Linearity and range
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. For the establishment of linearity, a minimum of 5 concentrations is recommended [32].

Precision
The precision of the assay was investigated by measurement of both repeatability and Intermediate precision. Repeatability: Repeatability was investigated by injecting a minimum of 9 determinations covering the specified range for the procedure (e.g., 3 concentrations/3 replicates each) and percentage SD were calculated in Table 1.
Intermediate precision: In the inter-day studies, standard and sample solutions prepared as described above, were analyzed in triplicate on three consecutive days at specified range for the procedure (e.g., 3 concentrations/3 replicates each) of the test concentration and percentage SD were calculated in Table 1.

Accuracy
Accuracy was assessed using 9 determinations over 3 concentration levels covering the specified range (80,100 and 120%). Accuracy was reported in Table 1 as percent recovery by the assay of known added amount of analyte in the sample.

Limits of detection and limits of quantization
According to the ICH recommendations, 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 in Table 2, Figure 2B [32].

System suitability tests
System suitability tests were used to verify that the resolution and reproducibility were adequate for the performed analysis. The system suitability tests included number of theoretical plates, resolution, peak tailing, capacity factor and selectivity factor. Results are revealed in Table 3.

Robustness
Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small variations in method parameters and provides an indication of its reliability during normal usage [32]. Robustness was tested by studying the effect of changing mobile phase pH by ± 0.3, the percentage of organic solvent (Acetontrile) in the mobile phase by ± 2%, temperature ± 2°C, wavelengths ± 2 nm and flow rate ± 0.1 ml/min had no significant effect on the chromatographic resolution of the method in Figure 3. Changes in pH degree and Acetonitrile percent in mobile phase have a greater effect on resolution than other factors( Table 4).

Stability of analytical solution
Also as part of evaluation of robustness, solution stability was evaluated by monitoring the peak area response. Standard stock

Conc.% Amlodipine Besylate Atorvastatin Calcium
AV ± SD mg/ml AV ± SD % AV ± SD mg/ml AV ± SD %  solutions in methanol were analyzed right after its preparation 1, 2 and 3 days after at 5°C. The change in standard solution peak area response over 3 days was (0.57 and 0.58%) for Amlodipine Besylate, Atorvastatin Calcium respectively. Their solutions were found to be stable for 3 days at 5°C at least (Figure 3).

Application to Pharmaceutical Preparation
The proposed methods were successfully used to determine Amlodipine Besylate, Atorvastatin Calcium in Caduet 10/10 mg/tablet. Seven replicate determinations were performed. Satisfactory results were obtained for each compound in good agreement with label claims. The results obtained were compared statistically with those from published method [26] by using Student's t-test and the variance ratio F-test. The results 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 (Table 3).

Optimization of chromatographic condition
Several trials were carried out to obtain optimized chromatographic condition for simultaneous determination of Amlodipine Besylate and Atorvastatin Calcium in their pharmaceutical preparations.
Firstly, maximum absorption wavelengths (240 nm) for Amlodipine Besylate and Atorvastatin Calcium were selected by scanning from 350-200 nm under UV (Figure 4).
Potassium Dihydrogen Phosphate buffer has no effect on absorption at wavelengthmore than 200 nm [33]. Conc. of buffer (0.03M) is adequate for most reversed phase applications. This concentration is also moderate enough to avoid problems with precipitation when significant amounts of organic modifiers are used in the mobile phase [34].
On the basis of pka of Amlodipine Besylate and Atorvastatin Calcium are 8.6 and 4.5 respectively which means that Amlodipine Besylate has more basic properties than Atorvastatin Calcium due to    free and cyclic amino group and vice versa Atorvastatin Calcium has free carboxylic group. Several degrees of pH of phosphate buffer (2.5-6.5) were examined ( Figure 5). At lower (2.5-3.5), it shows a fast eluting of amlodipine (it becomes difficult to separate it from its predictable degradation products, lately one for atorvastatin (long time about 25 min) and co-elution between Amlodipine imp A and Amlodipine imp B. At 4.5, there is interference between Amlodipine imp B and atorvastatin imp A.

Drugs/Parameters Theoretical Plates (N) Resolution (R) Capacity Factor (K) Tailing Factor (T) Selectivity (α)
A good separation had obtained at higher (5.5-6.5), 5.5 is more suitable than 6.5 because resolution between Amlodipine and its impurity A are greater than resolution between atorvastatin and its impurity C at5.5 and the former is more critical than the latter one as it is more affected by increasing in organic solvent percentage.
Ammonium acetate and sodium dihydrogenosate were also tried, no significant difference between sodium dihydrogenosate and potassium dihydrogenosate but Ammonium acetate give less resolution power ( Figure 8).
Methanol exhibits poor separation and peak shape as an organic solvent ( Figure 9). After all of previous had been carried out, our previous mentioned optimized chromatograic conditions were selected.

Forced degradation studies
If the compound is poorly water-soluble, organic co-solvents may be used in combination with acid or base. Organic solvents that have been commonly used for stress-testing studies are DMSO, acetic acid acetonitrile and methanol. As usual, when conducting stress testing, the analyst should be wary of possible side reactions that may affect the drug, for example, methanol should be avoided for compounds containing -CO2H, -CO2R, amide groups. Acetonitrile is the cosolvent of choice for otochemical reaction [30]. Amlodipine Besylate, Atorvastatin calcium authentic standards and Caduet tablets were subjected to various stress condition. In acidic conditions, both drugs exhibited high percentage of degradation about 79% and 62% for Amlodipine Besylate and Atorvastatin calcium respectively ( Figure 10). The pyridine analogue of Amlodipine (impurity D) and Atorvastatin lactone (impurity G) were the main degradation products of Amlodipine Besylate and Atorvastatin calcium respectively (Figure   11a-11c). In alkaline conditions, both drugs degraded about 95% and 14% for Amlodipine Besylate and Atorvastatin calcium respectively. The major degradation products of Amlodipine Besylate eluted early at 0.9 and 1.09 minute ( Figure 12). Amlodipine Besylate and Atorvastatin calcium are more stable under neutral degradation (Thermal degradation); only about 13% degradation of two drugs was observed ( Figure 13). In oxidative conditions, both drugs were found to be highly labile to oxidative hydrolysis in 10% H 2 O 2 at 70% after 6 hours. Approximately 80%, 75% degradation was observed for Amlodipine Besylate and Atorvastatin calcium respectively due to its nitrogenous content. The major degradation product of Amlodipine Besylate was its pyridine analogue ( Figure 14). Both drugs were affected significantly by otolyticdegradation ( Figure 15).

Discussion
New developed method has several merits than other published methods in literature, it didn´t use ion pair buffer in mobile ase like RJ Eranki et al. [28] which shorten column life time and require long time for system stabilization and column washing. Run time is shorter and sensitivity is more than Turabi et al. [29]. Specificity was proven clearly after impurities of both drugs had been separated in addition to degradation products which were identified and its pathways were mentioned, in contrary to BG Chaudhari et al. [27]. Mohammadi et al. [26] didn´t mention how to optimize and develop his method and little information about system suitability test.

Conclusion
A simple, accurate, precise, robust and reliable LC method has been established as stability indicating method for Amlodipine Besylate and Atorvastatin Calcium respectively in bulk and in their pharmaceutical dosage form.