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Original Article
RP-HPLC method development and validation for simultaneous estimation of telmisartan, rosuvastatin calcium, and amlodipine besylate in combination
expand article infoRujuta P. Mistry, Chainesh Shah§, Rakesh Jat|
‡ Shri Jagdishprasad Jhabarmal Tibrewala University, Vidyanagari, India
§ Sigma Institute of Pharmacy, Vadodara, India
| Shree Jagdishprasad Jablamer Tiberwala University, Vidyanagari, India
Open Access

Abstract

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 ortho-phosphoric 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.

accuracy, ICH guideline, mobile phase, precision, retention time.

Introduction

Telmisartan is chemically described as 2-(4- {[4-methyl-6-(1-methyl-1H-1,3-benzodiazol-2-propyl-1H- 1,3benzodiazol-1-yl] methyl}phenyl) benzoic acid. It is used as an angiotensin II receptor antagonist (AT1) in the management of hypertension.[1] It selectively antagonizes angiotensin II binding to the AT1 subtype receptors. It is commonly administered through the oral route.

Rosuvastatin calcium is chemically calcium: (E,3R,5S)-7-[4-(4-fluorophenyl)-2- [methyl(methylsulfonyl)amino]-6-propan-2-ylpyrimidin-5-yl]-3,5-dihydroxyhept-6-enoate. It belongs to a class of drugs called statins, which are employed in lowering hypercholesterolemia, its related conditions and preventing cardiovascular diseases.‌[2]

Amlodipine besylate is chemically described as 3-ethyl-5-methyl (±)-2-[(2-aminoethox) methyl]-4-(2- chlorophenyl)-1, 4- dihydro-6-methyl-3, 5-pyridinedi carboxylate, mono benzene sulphonate. AML is a calcium channel blocker[2], which inhibits the influx of extracellular calcium across the myocardial and vascular smooth muscle cell membranes, thus decreasing the contractile process and hence dilating coronary and systemic arteries[3].

The literature survey revealed that methods available for the determination of telmisartan are such as [UV][4], [HPLC][5], [RP-HPLC][6], and [Tandem mass spectrometry][7]. The methods available for determination of rosuvastatin calcium are [UV][8], [RPHPLC][9], [HPTLC][10], etc. Similarly, for determination of ADB, the methods are [UV][11], [RP-HPLC][12], and [HPTLC][13]. Many methods have been described in the literature for determination of telmisartan, rosuvastatin calcium, and amlodipine besylate individually and in combination with other drugs. No single method was reported for the estimation in the combined form.

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]

Materials and methods

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.

Table 1.

Chromatographic condition

Column Luna C18 100Å column (250 mm * 4.6 mm i.d., particle size 5 μ)
Detector 242 nm
Injection volume 20 µl
Flow rate 0.1 ml/min
Mobile phase Methanol : Acetonitrile : water (60:40 v/v) (PH3.5 Adjusting with orthophosphoric acid)

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]

Results and discussions

Method validation

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).[16–18]

Table 2.

System suitability studies

Parameters Observed values IP specification
TEL* ROS* AML*
Retention time (min) 2.07 4.65 7 6.99 -
Theoretical plates 7506.14 2190.34 11989 Not less than
Asymmetry (10%) 1.57 0.89 0.78 Not greater
Resolution 15.76 - 7.45 >2

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.[19–21] The results are presented in Table 3. A calibration curve was found to be linear with a regression coefficient (>0.99) (Fig. 1).

Table 3.

Calibration curve data

Concentration (μg/ml) TEL (Peak area)* Concentration (μg/ml) ROS (Peak area)* Concentration (μg/ml) AML (Peak area)*
40 10789 10 23450 5 54677
80 157889 20 142311 10 298777
120 246778 30 319008 15 356789
160 334556 40 497665 20 678990
200 410560 50 585261 25 846670
Figure 1.

Calibration curve of telmisartan, rosuvastatin calcium, and amlodipine besylate.

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.

Table 4.

Recovery data (accuracy)

Drug Spiked level (%) Amount taken (μg/ml) Amount found (μg/ml) Total amount Taken(μg/ml) Amount found (μg/ml) % Recovery
TEL 50% 80 40 120 118.14 98.45±0.56
100% 80 80 160 159.13 99.46±0.21
150% 80 120 200 199.48 99.74±0.88
ROS 50% 20 10 30 29.40 98.01±0.77
100% 20 20 40 39.62 99.07±0.89
150% 20 30 50 50.09 100.19±0.76
AML 50% 10 5 15 14.95 99.67±0.98
100% 10 10 20 20.02 100.01±0.13
150% 10 15 25 25.00 100.01±0.87

Precision

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.

Table 5.

Interday and intraday precision studies

INTRADAY PRECISION INTERDAY PRECISION
DRUG Con. taken (μg/ml) Mean Peak Area* %RSD DRUG Con. taken (μg/ml) Mean Peak Area* %RSD
TEL 80 216788 0.67 TEL 80 206789 0.87
120 288780 0.82 120 288685 0.80
160 380998 0.56 160 374566 0.67
ROS 20 145677 0.78 ROS 20 155678 0.80
30 312670 0.67 30 319080 0.69
40 500134 0.21 40 526678 0.24
AML 10 298754 0.37 AML 10 289970 0.39
15 354606 0.80 15 354567 0.81
20 687888 0.88 20 698008 0.89

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.

Table 6.

Robustness studies

Conditions Modification TEL Peak Area TEL %RSD ROS Peak Area ROS %RSD AML Peak Area AML %RSD
Flow rate (ml/min) 0.9 288678 0.56 309901 0.89 347891 0.82
1.1 288689 0.59 309903 0.89 34893 0.83
Change of wavelength 241 276899 0.77 319960 0.70 346800 0.79
243 276897 0.76 319950 0.71 346778 0.78

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).

Figure 2.

Typical chromatogram of TEL, ROS and AML showing the retention times of 2.67, 4.70, and 7.44 min for telmisartan, rosuvastatin calcium, and amlodipine besylate, respectively, in synthetic mixture.

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.

Acknowledgements

The authors are thankful to M/s. Dano Pharmachem Pvt.Ltd. Ankleswar for providing gift sample of TEL and ROS, and to M/s. Mccoy Drug Pvt. Ltd., Sachin for providing gift sample of AML raw material. The authors would also like to thank Laxminarayan Dev College of Pharmacy and Shree Jagdishprashad Jablamer Tiberwala University for providing all facilities.

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