RP-HPLC Method Development and Validation for Simultaneous Estimation of Telmisartan, Rosuvastatin Calcium and Amlodipine Besylate in Combination

Introduction: Dyslipidemia-hypertension proves to be a major risk factor for cardiovascular diseases. In order to achieve better adherence and cost-effectiveness than free equivalent combination therapies, a fixed-dose combination therapy with telmisartan (TEL), rosuvastatin calcium (ROS) and amlodipine besylate (AML) is required in this type of patients. Aim: A simple, selective and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for estimation of telmisartan, rosuvastatin calcium, and amlodipine besylate in synthetic mixture. Materials and methods: Chromatographic separation was performed on a reversed-phase Luna C18 100Å column (250 mm × 4.6 mm i.d., particle size 5 μ) using an isocratic elution of mobile phase consisting of methanol and acetonitrile (pH 3.5 adjusted by orthophosphoric acid) (60:40 v/v) at a flow rate of 1.0 ml/min. Results: Ultraviolet (UV) detection was performed at 242 nm and retention time of telmisartan, rosuvastatin calcium and amlodipine besylate was found to be 2.67, 4.70, and 7.44 min, respectively. The calibration curve was linear (correlation coefficient >0.999) in the selected range of analyte. Conclusions: The method was validated for accuracy, precision, linearity, limit of detection, limit of quantitation and ruggedness. The system suitability parameter, such as theoretical plate, asymmetry, and resolution between standard five replicate were well within the limits.


AIM
The present work described a validated reverse phase HPLC method for determination of TEL, ROS, and AML in synthetic mixture used in the management of hypertension with dyslipidemia. 14

Chemicals and materials
The raw materials for telmisartan and Rosuvastatin were received as gift sample from Dano Pharmacham Pvt.Ltd., Ankleswar. Amlodipine Besylate raw material was received as gift sample from Mccoy Drug Pvt. Ltd., Sachin. HPLC Grade Methanol was received from RANKEM and HPLC Grade Acetonitrile was received from E Merck Ltd. Membrane filter: 0.22 µm and Nylon membrane filters were received from RANKEM.

Instrumentation
Chromatographic analysis was carried out on liquid chromatography (UFLC Shimadzu Corporation, Japan) with LC-2010HT series binary pump system using a UV detector with Software CLASS -VP (version 2.31) software to acquire and process the data. HPLC condition is given in Table 1.

Preparation of standard solution
1 ml of the standard stock solutions (1000 µg/ml) of all three drugs (TEL, ROS, and AML) was taken in a common volumetric flask diluted up to 10 ml with mobile phase -acetonitrile : methanol, pH=3.5 adjusted using orthophosphoric acid (60:40) to make the final concentration of 100:100:100 µg/ml.

Preparation of a sample solution (assay procedure)
It was prepared as per the patent (telmisartan: 80 mg, rosuvastatin calcium: 20 mg, and amlodipine besylate: 10 mg) and talc quantity was sufficient. All the excipients were mixed in a 100-ml volumetric flask and sonicated for 15 min. The solution was filtered through Whattman filter paper no. 42. Finally, the solution concentrations were obtained as 800, 200, and 100 µg/ml for each of the drugs, respectively. From that pipette out 1 ml in a 10-ml volumetric flask and volume made up with mobile phase -methanol : acetonitrile (60:40 v/v) to make the final concentration of TEL (80 µg/ml), ROS (20 µg/ml), and AML (10 µg/ml) and recorded peak areas were noted for estimation of TEL, ROS and AML. 15

System suitability studies
The system suitability was evaluated by five replicate analyses of TEL, ROS, and AML mixture at concentrations of 80, 20, and 10 µg/ml of each drug, respectively. The column efficiency, resolution, and peak asymmetry were calculated for the standard solution. The results of system suitability and system precision were presented (Table 2). [16][17][18]

Linearity and range
The linearity response was determined by analysing five independent levels of concentration in the range of 40-200 µg/ml for TEL, 10-50 µg/ml for ROS, and 5-25 µg/ml for AML. [19][20][21] The results are presented in Table 3. A calibration curve was found to be linear with a regression coefficient (>0.99) (Fig. 1).

Accuracy
The difference between theoretical added amount and practically achieved amount is called accuracy of analytical method. Accuracy was determined at three different levels -at 50%, 100%, and 150% of the target concentration in triplicate. The results are presented in Table 4.

Intraday precision and interday precision
The precision of the developed method was assessed by analysing samples of the same batch with three combined solutions of TEL, ROS and AML in the concentration of 80, 120, and 160 μg/ml of TEL, 20, 30, and 40 μg/ml of ROS, and 10, 15, and 20 μg/ml of AML, respectively in three replicates (n=3) each on same day. 22 The percentage of RSD value of the results corresponding to the peak area was expressed for intra-day precision. The precision of the developed method was assessed by analysing samples with three standard solutions of TEL, ROS, and AML similarly like above concentration respectively in three replicates (n=3) each on different day. The results are presented in Table 5.
The results obtained were within 2% RSD.
Limit of detection (LOD) and limit of quantification (LOQ) 23,24 The LOD & LOQ were found to be 1.46 and 4.21 µg/ml for TEL, 0.54 and 1.63 µg/ml for ROS, and 0.78 and 2.01 µg/ml for AML, respectively. The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Robustness
As defined by The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), the robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. Robustness was performed by small variation in the chromatographic conditions and found to be unaffected by ±0.1 ml/min variation in flow rate of mobile phase, and ±0.1 variation in detection wavelength. These results are presented in Table 6.

Assay
The percentage assay of chromatogram analysis for bulk mixture of TEL, ROS, and AML were 99.98%, 100.21%, and 99.24%, respectively The retention time of bulk mixture of TEL, ROS, and AML are 2.67 min, 4.70 min, and 7.44 min, respectively (Fig. 2)

CONCLUSIONS
A rational and valid attempt has been made for the development of telmisartan, rosuvastatin calcium, and amlodipine besylate in synthetic mixture. The accountability of the proposed method has been established by evaluating validation parameters as per ICH guidelines. The developed RP-HPLC methods are simple, economical, precise, and accurate for the simultaneous determination of telmisartan, rosuvastatin calcium, and amlodipine besylate in synthetic mixture.