Abstract
The present study describes development and subsequent validation of stability indicating HPLC and HPTLC methods for simultaneous estimation of Telmisartan (TLM) and Atorvastatin (ATV) in their combined formulation. The proposed RP-HPLC method utilizes a Phenomenex Luna C18 column using acetonitrile: 0.025 M ammonium acetate (38 : 52%, v/v) as mobile phase (pH 3.8), flow rate of 1.0 mL/min. Quantification was achieved with UV detection at 281 nm over concentration range of 12 to 72 μg/mL for TLM and 3 to 18 μg/mL for ATV respectively. In HPTLC, separations were performed on silica gel 60 F254 using toluene-methanol-ethyl acetate-acetic acid (5 : 1 : 1 : 0.3, v/v) as mobile phase. The compact bands of TLM and ATV at 0.37 ± 0.02 and 0.63 ± 0.01 respectively were scanned at 279 nm. Linear regression analysis revealed linearity in the range of 40 to 240 ng/band for TLM and 10 to 60 ng/band for ATV respectively. For both the methods, dosage form was exposed to thermal, photolytic, acid, alkali and oxidative stress. The methods distinctly separated the drugs and degradation products even in actual samples. In conclusion, the proposed HPLC and HPTLC methods were appropriate for routine quantification of TLM and ATV in tablet formulation.
1. Introduction
Telmisartan, chemically 4′-[(1,4′-Dimethyl-2′-propyl-[2,6′-bi-[1H]-benzimidazol]-1′-yl) methyl]-[1,1′-biphenyl]-2-carboxylic acid, is a nonpeptide angiotensin-II receptor antagonist which selectively and insurmountably inhibits angiotensin-II AT1 receptor subtype without affecting other systems involved in cardiovascular regulation (Figure 1(a)). Atorvastatin chemically [R-(R*,R*)]-2-(4-Fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)-carboxyl]-1H-pyrrol-1-heptanoic acid calcium salt is a second generation synthetic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor which decreases de novo cholesterol synthesis (Figure 1(b)). ATV decreases the amount of LDL-cholesterol in blood, reduces blood levels of triglycerides, and slightly increases levels of HDL-cholesterol [1–3]. The literature survey reveals several methods for determination of TLM and ATV individually in biological fluids and formulation like HPLC and TLC-densitometric and derivative spectrophotometry [4–16]. HPLC and HPTLC methods were reported for the determination of TLM and ATV in combination [17, 18].
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None of the reported analytical procedures described a stability indicating method for simultaneous determination of TLM and ATV in presence of their degradation products. Hence, the present study was aimed to establish inherent stability of TLM and ATV through stress induced studies under a variety of ICH recommended test conditions [19] and to develop stability indicating HPLC and HPTLC methods. Validation of the developed method was carried out as per ICH guidelines [20]. The developed methods were applied to two marketed tablet dosage forms.
2. Experimental
2.1. Materials and Chemicals
Telmisartan and Atorvastatin were provided by Atoz Laboratories, Chennai, India. Tablets, Telsartan-ATR (Dr. Reddy’s Laboratories Ltd., Hyderabad, India) and Arbitel-AV (Micro Labs Ltd., Bangalore, India) both labelled to contain 40 mg TLM and 10 mg ATV, were purchased from local market. Analytical grade methanol, toluene, ethyl acetate, and acetic acid (97 to 98%, v/v) were obtained from M/S. Merck, Mumbai, India. HPLC grade acetonitrile, ammonium acetate, and acetic acid were obtained from M/S. Merck, Mumbai, India.
2.2. Instrumentation
The HPLC system consisted of a pump (Shimadzu LC 10 ATVP) with 20 μL Rheodyne injector, Phenomenex Luna C18 (5 μm × 25 cm × 4.6 mm id) column, and SPD-10 AVP photodiode array (PDA) UV-visible detector set at 281 nm and equipped with CLASS-VP software (Shimadzu, Kyoto, Japan). In HPTLC, chromatographic separation of drug was performed with silica gel 60 F254 (10.0 × 10.0 cm with 250 mm layer thickness) from E. Merck, Germany. Samples were applied as 8 mm bands by means of Camag 100 μL, sample syringe (Hamilton, Switzerland) with Linomat 5 applicator (Camag, Switzerland). Densitometric scanning was performed in the absorbance/reflectance mode at 279 nm using Camag TLC scanner 3 with deuterium source, slit dimension settings of length 2 mm, width 0.1 mm, monochromator band width 30 mm, and scan rate of 4 mm s−1. Win CATS software (V 1.4.2, Camag, Switzerland) was used for scanner control and data processing.
2.3. Preparation of Standard Solution
For HPLC, individual stock solutions of TLM (100 μg/mL) and ATV (100 μg/mL) were prepared by transferring 10 mg each of TLM and ATV standard in separate 100 mL volumetric flasks, dissolved in 50 mL of acetonitrile and made up to volume using the same. From this, suitable dilutions was made using acetonitrile to obtain mixture of solution containing 12 to 72 μg/mL of TLM and 3 to 18 μg/mL of ATV, respectively. Aliquots (20 μL) of each solutions were injected and chromatographed in replicate. For HPTLC, standard stock solution was prepared separately by dissolving accurately weighed 10 mg each of TLM and ATV in 100 mL methanol. From this stock solutions, suitable dilution was made using methanol to obtain a combination solution containing TLM (40 to 240 μg/mL) and ATV (10 to 60 μg/mL) taking into consideration their ratio (4 : 1) present in combined tablet formulation.
2.4. Preparation of Sample Solution
Twenty tablets from each brand of one batch were accurately weighed; their mean weight was determined and powdered using glass mortar. For HPLC, an amount of powder equivalent to 50 mg TLM was transferred into 50 mL volumetric flask. 25 mL of acetonitrile was added, followed by sonication for 10 min. The contents were restored to room temperature and diluted to volume with acetonitrile to furnish sample stock solution. The solution was filtered using 0.45 μm nylon filter paper (Millipore, Milford, USA), and dilutions were made with acetonitrile to obtain a solution of TLM (40 μg/mL) and ATV (10 μg/mL). Suitable dilutions were made to achieve optimum concentration for analysis. For HPTLC, an amount equivalent to label claim of each active ingredient was accurately weighed and transferred into a suitable volumetric flask. The volume was adjusted with methanol, and the resultant solution was sonicated for 15 min and filtered through 0.45 μm nylon filter. From the resulting solution, suitable aliquots were transferred into 100 mL volumetric flask and completed to volume with methanol to have a final concentration of 120 μg/mL of TLM and 30 μg/mL of ATV, respectively.
2.5. Forced Degradation Studies
To evaluate the stability indicating properties of the developed methods, forced degradation studies were carried out in accordance with ICH guidelines.
For HPLC, 10 mL of TLM and 25 mL of ATV from previously mentioned stock solutions of standard (100 μg/mL each) were transferred separately into 25 mL standard flask containing 5 mL each of 0.1 M hydrochloric acid, 0.1 M sodium hydroxide and 3% hydrogen peroxide (v/v), respectively. The mixture was refluxed at 60°C for 1 hour and completed to volume with acetonitrile. The resulting solutions were run under optimized chromatographic conditions.
For HPTLC, 5 mL each of 0.1 M hydrochloric acid, 0.1 M sodium hydroxide, and 3% v/v hydrogen peroxide solutions was added to 25 mL volumetric flask containing 3.0 mL and 0.75 mL of methanolic stock solutions of TLM and ATV, respectively. The mixture was refluxed at 60°C for 30 min and completed to volume with same solvent. One microlitre of resulting solutions (120 μg/mL TLM and 30 μg/mL ATV) was spotted as bands to furnish concentration of 120 ng/band TLM and 30 ng/band ATV, respectively. In both HPLC and HPTLC, forced degradation studies using 0.1 M hydrochloric acid, 0.1 M sodium hydroxide, and 3% v/v hydrogen peroxide were performed in dark and exclude possible degradation effect of light. Thermal degradation study using HPLC and HPTLC methods was performed by refluxing appropriate dilutions of standards and samples at 60°C for 1 hour. The resulting solutions were run under optimized chromatographic conditions. For photodegradation study, appropriate dilutions were exposed to UV light (365 nm) for 6 hours in UV light chamber. The resulting solutions were analyzed under optimized chromatographic conditions.
2.6. Method Validation
2.6.1. Linearity
In case of HPLC, linearity was studied by injecting six concentrations of standard TLM (12, 24, 36, 48, 60, and 72 μg/mL) and ATV (3, 6, 9, 12, 15, and 18 μg/mL) in triplicate. In HPTLC, a series of combination dilutions and standard curves were prepared over a concentration range from 60 to 240 ng/band of TLM and 10 to 60 ng/band of ATV from stock solution. In both methods, peak area versus concentration data was performed by least square linear regression analysis, whereby slope, intercept, and correlation coefficient were determined.
2.6.2. Sensitivity
The sensitivity of the methods was determined with respect to LOD and LOQ. The LOD and LOQ parameters were determined from regression equations of TLM and ATV: LOD = 3.3 × SD/s, LOQ = 10 × SD/s, where “SD” is the standard deviation of response and “s” slope of calibration curve.
2.6.3. Precision and Accuracy
Intra- and interday precisions of the methods were determined by performing replicate () analyses of standards and samples. This procedure was replicated on different days (). Recovery studies by standard addition method were performed in view of justifying accuracy of the proposed methods. Previously analysed samples containing TLM and ATV were spiked with standard TLM and ATV, and the mixtures were analysed in triplicate () by proposed methods. Precision was calculated from percentage relative standard deviation (RSD %) for repeated measurements, whereas accuracy was expressed as % of recovery.
2.6.4. Specificity
For HPLC, the specificity of the method was established through study of resolution factor of drug peaks from the nearest resolving peak and also among all other peaks. In HPTLC, the bands for TLM and ATV in sample were identified by comparing values and spectrum of band with those of the bands from standard. The peak purity of TLM and ATV was assured by comparing spectra acquired at three different positions on peaks (peak start, apex, and end).
2.6.5. Robustness
Robustness of HPLC method was determined by deliberately varying certain parameters like detection wavelength, temperature, flow rate, and pH of mobile phase. For HPTLC, the conditions altered were mobile phase composition, development distance, time of spotting, temperature, and detection wavelength. For all changes in conditions, the samples were analysed in triplicate. When the effect altering one set of conditions was tested, the other conditions were held constant at optimum values.
2.6.6. System Suitability
The system suitability test was performed to confirm that the LC system to be used was suitable for intended application. A standard solution containing 40 μg/mL of TLM and 10 μg/mL of ATV was injected six times. The parameters retention time, resolution, capacity factor, theoretical plates, tailing factor, and % RSD were determined.
3. Results and Discussion
3.1. Reversed Phase High Performance Liquid Chromatography
A satisfactory separation was obtained when using acetonitrile 0.025 M ammonium acetate (38 : 52, v/v), pH 3.8 using acetic acid under isocratic conditions, and a flow rate of 1.0 mL/min. From the overlain spectra (Figure 2), it was observed that the drugs exhibited strong absorbance at 281 nm, which is selected for detection. Peaks were well defined, resolved, and almost free from tailing. Retention times of TLM and ATV were observed at and , respectively (Figure 3), and the optimum wavelength was determined to be 281 nm.
System suitability tests were also carried out to verify reproducibility, and results are summarized in Table 1. For quantitative applications, linear calibration graphs were obtained with correlation coefficients of 0.9991 and 0.9989 for TLM and ATV, respectively. Calibration plots were linear from 12 to 72 μg/mL for TLM and 3 to 18 μg/mL for ATV. Limits of detection (LOD) were 3.21 μg/mL for TLM and 1.21 μg/mL for ATV; limits of quantification (LOQ) were found to be 8.65 μg/mL for TLM and 2.25 μg/mL for ATV which showed good sensitivity of the proposed method. The low RSD (<2.0%) values of intraday and interday precision for TLM and ATV revealed that the proposed method is precise (Table 2). % RSD of recovery study was found to be 0.58 to 1.71, which indicated that the method is accurate (Table 2). Upon slight variation in the selected parameters, insignificant difference in peak area and retention time was observed. The resolution between TLM and ATV and its major degradation products were found to be ≥2.0 indicating robustness of LC method (Table 3). The assay results for an average of five determinations of two tablets (Telsartan-ATR and Arbitel-AV (40 mg TLM and 10 mg ATV)) were shown in Table 4. The mean assay values were 99.56 and 100.71 for TLM and 98.42 and 97.52 for ATV, respectively. The results of quantitative analysis of tablets indicate that the proposed method can be used for routine quantitative and quality control analysis of TLM and ATV in pharmaceutical dosage forms.
Typical chromatograms obtained following the assay of stressed samples of TLM and ATV show significant degradation in acid (Rt 1.8 and 4.5) and alkali hydrolysis (Rt 1.7 and 7.2). Thermal and photolytic stress conditions show insignificant degradation for TLM and ATV. From the peak purity profile studies, it was confirmed that peak of the degradation product was not interfering with the response of drugs (Table 5). It confirms that, degradation products can be separated from the drugs by this method (Figure 4).
3.2. High Performance Thin Layer Chromatography
Preliminary experiments were carried out to optimise parameters affecting simultaneous estimation of both the drug using HPTLC and detection at 279 nm. Mobile phase consisting of toluene-methanol-ethyl acetate-acetic acid (5 : 1 : 1 : 0.3, v/v/v/v) was found to give best sensitivity, efficiency, and peak shape. It was observed that at 279 nm both drugs could be detected simultaneously with no mobile phase interference, good separation, sensitivity, and consistent baseline (Figure 5). It can also be assumed from peak purity spectra (Figure 6) that the method is specific for these components. Under optimum conditions, the retention factors obtained for TLM and ATV were and , respectively.
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The calibration plots were linear in the concentration range between 40 and 240 ng/band (, ) for TLM and 10 to 60 ng/band (, ) for ATV, respectively. The LOD and LOQ obtained by this method were 8.02 and 26.72 ng/band for TLM and 2.13 and 6.22 ng/band for ATV, respectively. Table 1 shows linearity parameters of calibration curve. The % RSD values were 0.28 to 1.84 and 0.41 to 1.65 for intra- and interday precisions, respectively (Table 6). Recovery study (Table 6) performed at three different concentrations in triplicate shows good recoveries: 99.57 to 100.42% for TLM and 97.86 to 98.81% for ATV, respectively. Results of robustness study are depicted in Table 7. The retention factor () and assay (%) were not significantly affected. RSD (%) value in all robustness parameter was found to be <2%. The validated HPTLC method was applied for simultaneous determination of TLM and ATV in commercial tablets. The results as depicted in Table 4 indicate that each drug in tablet corresponds to requirements of label claim. The low RSD value (<2%) confirmed the suitability of method for routine analysis of TLM and ATV in pharmaceutical dosage form.
Typical chromatograms obtained following the assay of stressed samples of TLM and ATV show significant degradation in acid (12% and 8%) and alkali hydrolysis (15% and 28%). TLM and ATV show extra degradation peaks at values 0.27 and 0.45 for acid; 0.42, 0.58, 0.74, and 0.80 for alkali; and 0.20 for oxidation. Thermal and photolytic stress conditions show insignificant degradation for TLM and ATV. From the peak purity profile studies, it was confirmed that peak of the degradation product was not interfering with the response of drugs (Table 5). It confirms that degradation products can be separated from the drugs by this method (Figure 7).
4. Conclusion
The proposed methods were found to be sensitive, reproducible, and accurate for analysis of Telmisartan and Atorvastatin in tablet dosage form. Both methods envisage stability behaviour of Telmisartan and Atorvastatin individually and in combination as per ICH guidelines. Therefore, the proposed methods could be used as stability indicating methods for simultaneous determination of Telmisartan and Atorvastatin in bulk drug and in pharmaceutical formulations.
Conflict of Interests
The authors declare that they have no conflict of interests.