Rapid Simultaneous Determination of Telmisartan, Amlodipine Besylate and Hydrochlorothiazide in a Combined Poly Pill Dosage Form by Stability-Indicating Ultra Performance Liquid Chromatography

A simple, precise and rapid stability-indicating ultra-performance liquid chromatography (UPLC) method is developed for the simultaneous quantitative determination of Telmisartan, Amlodipine besylate and Hydrochlorothiazide from their innovative poly pill combination drug product in the presence of degradation products. It involves a 100 mm x 2.1 mm, 1.7 μm C-18 column. The separation is achieved on a simple gradient method. The mobile phase A contains a mixture of sodium perchlorate buffer pH 3.2 (0.053M): acetonitrile in the ratio 90:10, v/v, and mobile B contains a mixture of sodium perchlorate buffer pH 3.2 (0.053M): acetonitrile in the ratio 20:80, v/v. The flow rate is 0.6 mL min−1 and the column temperature is maintained at 55°C.The gradient program (T/%B) is set as 0/5, 1.2/5, 1.6/40, 4/40, 4.1/5 and 4.5/5. The detector wavelength is 271 nm for Hydrochlorothiazide and Telmisartan and 237 nm for Amlodipine. The retention times of Telmisartan, Amlodipine, and Hydrochlorothiazide are 3.6 minutes, 3.2 minutes and 0.9 minutes; respectively. The total runtime for the separation of the three active compounds and their degradation products is 4.5 minutes. The described method is validated with respect to system suitability, specificity, linearity, precision and accuracy. The precision of the assay method is evaluated by carrying out six independent assays of T, A and H (0.032 mg mL−1 of T, 0.004 mg mL−1 of A, 0.01 mg mL−1 of H). The accuracy of the method is evaluated in triplicate at three concentration levels, i.e. 50%, 100% and 150% of target test concentration (0.64 mg mL−1 of T, 0.08 mg mL−1 of A, 0.2 mg mL−1 of H). The described method is linear over the range, 16 to 48 μg mL−1 for T, 2 to 6 μg mL−1A and 5 to 15 μg mL−1 for H. The method is fast and suitable for high-throughput analysis allowing the analysis of about 250 samples per working day.


Introduction
Cardiovascular diseases (CVDs) are the disorders of heart and blood vessels and primarily include coronary heart disease, hypertension, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease and heart failure. CVDs are the major cause of death in developed countries and also are rapidly emerging as a main cause of death in the developing world. An estimated 17.5 million people died from CVDs till 2005, representing almost 30% of all the global deaths. It is projected that almost 20 million people will die from CVDs by 2015. The major risk factors involved in CVDs are high low density lipoprotein (LDL) cholesterol, raised blood pressure, increased serum homocysteine level and platelet aggregation, which are primarily caused by unhealthy diet, physical inactivity and tobacco use. A novel polypill formulation is developed using drugs Telmisartan, Amlodipine besylate and Hydrochlorothiazide for CVDs.
Hydrochlorothiazide (H) is a thiazide diuretic (water pill) that decreases the amount of fluid in the body by increasing the amount of salt and water lost in the urine. Hydrochlorothiazide is used to lower blood pressure and to decrease edema (swelling), it is chemically described as 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide ( Fig. 1).
The ever-increasing need for speed and efficient use of time in the pharmaceutical and other fields, there is demand for the development of fast and high throughput analytical procedures. The rapid quantitative determination of combination drugs with big difference in label claims (40 mg for T and 5 mg for A) with shorter run times is a challenge. For UPLC based assays, the processes of reducing analysis time while adequately resolving analytes from degradation products is often accomplished with column with small particles. The theoretical advantages for small particles are to get well resolved peaks with high theoretical plates over small concentration.
Present drug stability test guidance Q1A (R2) issued by international conference on harmonization (ICH) [1] suggest that stress studies should be carried out on a drug product to establish its inherent stability characteristics, leading to identification of degradation products and hence supporting the suitability of the proposed analytical procedures. It also requires that analytical test procedures for stability samples should be stability indicating and they should be fully validated.
Accordingly, the aim of the present study was to establish inherent stability of Telmisartan, Amlodipine besylate, and Hydrochlorothiazide through stress studies under a variety of ICH recommended test conditions [1][2][3] and to develop a rapid stability-indicating reverse phase assay method [4][5][6].
Literature survey reveals that a variety of spectrophotometric and chromatographic methods including UV, colorimetric determination, ratio derivative, and a stabilityindicating HPLC methods have been reported for determination T A and H either single or in combination with other drugs [7][8][9][10][11][12]. Whereas no liquid chromatography method has been reported for simultaneous quantitative determination of T, A and H in the combined dosage form.
Hence a rapid simple reproducible Ultra performance liquid chromatography method was developed for simultaneous quantitative determination of T, A and H in polypill pharmaceutical dosage forms in the presence of degradation products.

Chemicals and reagents
Standards and tablets (40 mg of Telmisartan, 5 mg of Amlodipine besylate, 12.5 mg of Hydrochlorothiazide) were supplied by Dr. Reddy's laboratories limited, Hyderabad, India. The HPLC grade acetonitrile and methanol, analytical grade sodium per chlorate and disodium hydrogen phosphate were purchased from Merck, Darmstadt, Germany. Water was prepared by using Millipore MilliQ Plus water purification system Instrumentation Acquity UPLC TM system (Waters, Milford, USA) used consisting of a binary solvent manager, a sample manager and a UV detector. The out put signal was monitored and processed using empower software, water bath equipped with MV controller (Julabo, Seelbach, Germany) was used for hydrolysis studies. Photo stability studies were carried out in a photo stability chamber (Sanyo, Leicestershire, UK). Thermal stability studies were performed in a dry air oven (MACK Pharmatech, Hyderabad, India).

Chromatographic conditions
The chromatographic column used was Acquity UPLC BEH shield RP-18, 100 mm x 2.1 mm i.d with 1.7 µm particles. The mobile phase A contains a mixture of sodium perchlorate buffer pH 3.2(0.053M): acetonitrile (90:10, v/v) and mobile phase B contains a mixture of sodium perchlorate buffer pH 3.2(0.053M): acetonitrile (20:80, v/v). The flow rate was 0.6 mL min −1 and column temperature was maintained at 55°C. The detection wavelength was 271 nm for Hydrochlorothiazide and Telmisartan, and 237 nm for Amlodipine besylate. The diluent contains a mixture of sodium perchlorate buffer pH 3.2(0.053M): methanol in the ratio 90:10, v/v.

Preparation of standard solutions
Stock standard solutions of T, A and H (0.54 mg mL −1 of T, 0.2 mg mL −1 of A, 0.5 mg mL −1 of H) were prepared by dissolving an appropriate amounts of the compounds in methanol. Working solutions 0.032 mg mL −1 of T, 0.004 mg mL −1 of A, 0.01 mg mL −1 of H were prepared from above stock solution in mobile phase for assay determination.

Chromatographic Method Development
The method was optimized to separate major degradation products formed under different stress conditions. The main target of the chromatographic method is to get the separation for closely eluting degradation products, mainly the degradation product at 1.26 RRT (with respect to A) that is eluting between A and T. The degradation samples were run using different stationary phases like C18, C8, and Mobile phases containing buffers like phosphate and perchlorate with different pH (2.5-7) and using organic modifiers like acetonitrile and methanol in the mobile phase. The isocratic method was not working since telmisartan and amlodipine peaks were not separated with a proper resolution and also the degradents of hydrochlorothiazide were not separating from three actives. Hence the method is optimized with a gradient program. As the concentration of amlodipine is very less as compared to telmisartan, so that to improve response, resolution and peak shapes the method was tried at different column temperatures. But the separation, response of peaks and peak shapes were satisfactory in the adopted chromatographic conditions only. It indicated that the gradient method with 10% acetonitrile as organic modifier in mobile phase A and 80% in mobile phase B was successful in separating drugs and all chromatographic degradation products. Some of the trials were summarized as below.

Selection of Diluent (Extracting solvent):
As the tablets are film coated,and all the active drugs are stable in acidic conditions, the 0.1% orthophosphoric acid is added in order to open the film coating and to use the same assay method for quantification of uniformity of dosage units by UPLC.The trials were 0.1 N HCl also but the peak shapes are not good in 0.1N HCl. The chromatograph shown in Fig.2a. As all the three actives T, A, & H are freely soluble in methanol, so it is used as the extracting solvent.

Selection of Stationary Phase:
The stationary phases like C8 and C18 were tried but on C8 the peak shape for T and the peak symmetry factor for hydrochlorothiazide is more than 1.7, the separation between A and T is not adequate as shown in Fig.2b. On the other hand the peak shapes for all the three components are good and all the three peaks are well separated from each other and their degradents. So that the column with C18 stationary phase is selected.

Selection of mobile phase buffer:
The buffers like Phosphate buffer With Buffer with pH 3.2, pH 7.5 and the perchlorate buffer with pH 2.5 and 3.2 were tried as mobile phase buffer. But with pH 2.5, pH 7.5 phosphate and per chlorate buffer with pH 2.5 the results were not enough good in terms of peak separation and peak shapes, shown in Fig. 2c. On the other hand the perchlorate buffer with pH 3.2, the results were found satisfactory so that the mobile phase with pH 3.2 perchlorate buffer is finalized.

Optimization of gradient program:
Before going to gradient program, an isocratic program with organic modifier methanol and acetonitrile were tried but all the three components are eluted at the same retention time with both methanol and acetonitrile. As we are using the column temperature 55°C the acetonitrile is preferred as an organic modifier rather than methanol. The different gradient programs tried are shown in the Fig. 2d.

Finalization of Chromatographic conditions
By considering all the experiments the chromatographic conditions are finalized. The chromatograph with finalized chromatographic condition was shown in the Fig.3.

System suitability
In order to find the adequate peak separation (resolution) and repeatability of the proposed method, suitability parameters including retention factor, selectivity and asymmetry factor were investigated and the results were abridged in Table 1.

Specificity
The specificity of an analytical method is the ability of the method to determine the analyte response in the presence of additional components such as impurities, degradation products and matrix [4]. The solution of analytical placebo (containing all the excipients except T, A, & H) was prepared according to the sample preparation procedure and injected. To identify the interference by these excipients, a mixture of inactive ingredients, standard solutions, and the commercial pharmaceutical preparations including T, A, and H were analyzed by the developed method.
The specificity of the method was also evaluated to ensure there were no interference products resulting from forced degradation.

Placebo Interference
The placebo (excepients present in the tablet) sample were prepared as per the test method and analyzed in the UPLC. The Fig.4. shows there is no peaks at the retention time of the T, A, & H in the chromatograph indicates that there is no placebo interference.

Fig. 4.
Typical chromatogram of Telmisartan, Amlodipine besylate and Hydrochlorothiazide tablets placebo blend at 271 nm and 237 nm from drug product

Forced degradation studies.
Stress testing of a drug substance can help to identify the likely degradation products, which can help to establish the degradation pathways and the intrinsic stability of the molecule.
All stress decomposition studies were performed at an initial drug concentration 0.64 mg mL −1 of T, 0.08 mg mL −1 of A, 0.2 mg mL −1 of H. The degradation conditions are selected on the basis of literature survey [13][14][15][16][17].

Water induced degradation
Water hydrolysis was performed in purified at 60 °C for 6 hours. Fig. 5a shows the major degradation found at RRT 0.83 with respect to H. and all the major and minor degradation products were well separated from T, A, and H peaks. The peak purity is checked for T, A, and H and the results are summarized in Table 2.

Hydrogen peroxide induced degradation
For hydrogen peroxide-induced degradation, the studies were carried out at room temperature in 1% hydrogen peroxide for 6 hours. The Fig.5a. shows the major degradation found at RRT 0.841 with respect to H and at RRT 1.056 and 1.272 with respect to A. All the major and minor degradation products were well separated from T, A, and H peaks. The peak purity is checked for T, A, and H and the results are summarized in Table 2.

Acid induced degradation
Acid hydrolysis was performed in 1N HCl at 60 °C for 6 hours. Fig. 5a shows the major degradation found at RRT 0.842 with respect to H. and all the major and minor degradation products were well separated from T, A, and H peaks. The peak purity is checked for T, A, and H and the results are summarized in Table 2.

Base induced degradation
The study in basic solution was carried out in 0.1N NaOH at 60 °C for 6 hours. The Fig.  5b. shows all the three T, A, and H were stable in 0.1N NaOH and all the minor degradation products were well separated from T, A, and H peaks. The peak purity is checked for T, A, and H and the results are summarized in Table 2 Fig. 5a. Typical chromatograms of Telmisartan, Amlodipine besylate and Hydrochlorothiazide at 271 nm and 237 nm for force degradation study

Heat induced degradation
The drug product was exposed to dry heat at 105 °C for 24 hours. Samples were withdrawn at appropriate time and subjected to UPLC analysis after suitable dilution (0.032 mg mL −1 of T, 0.004 mg mL −1 of A, 0.01 mg mL −1 of H).The Fig. 5b. shows that the drug product containing T, A, and H is thermally stable. The peak purity is checked for T, A, and H and the results are summarized in Table 2.

Photo-degradation
Photo degradation studies were carried out at according to option 2 of Q1B in ICH guidelines [3]. Samples were exposed to light for an overall illumination of 1.2 million lux hours and an integrated near ultraviolet energy of 200 watt hm 2 . Samples were withdrawn at appropriate time and subjected to UPLC analysis after suitable dilution (0.032 mg mL −1 of T, 0.004 mg mL −1 of A, 0.01 mg mL −1 of H). The drugs T, A and H are stable under photolytic condition. The peak purity is checked for T, A, and H and the results are summarized in Table 2.

Linearity
Linearity solutions were prepared from stock solution at six concentration levels from 50 to 150% of analyte concentrations (16 to 48 µg mL −1 for T, 2 to 6 µg mL −1 A and 5 to 15 µg mL −1 for H). The linear regression analysis of T, A, and H were constructed by plotting the peak area of the analytes (y) versus analytes concentration in (x) axis. The calibration curves were linear in the range of 16 to 48 µg mL −1 for T, 2 to 6 µg mL −1 A and 5 to 15 µg mL −1 for H with a correlation coefficient of more than 0.999 for all the three drugs. The slope, Y-intercept and correlation coefficient were calculated and summarized in Table 3.

Precision
The precision of the assay method was evaluated by carrying out six independent assays of T

Conclusions
The established UPLC method proves to be simple, linear, precise, accurate and specific. The total runtime was 4.5 minutes within which three drugs and their degradation products were separated. The method was validated and shows satisfactory data for all the method validation parameters tested. The Developed method is stability indicating and can be used for simultaneous quantitative determination of the drugs T, A and H in presence of degradation products in stability by the industry. The adopted UPLC method also can be used separately for assay estimation of Telmisartan tablets, simultaneous estimation of Telmisartan and Hydrochlorothiazide tablets and simultaneous estimation of Amlodipine besylate and Hydrochlorothiazide tablets.