Analytical Method Development and Validation for the Simultaneous Estimation of Azelnidipine and Telmisartan by RP-HPLC in Bulk and Tablet Dosage Forms

 

Sufiyan Ahmad*, Nikhil Sonawane, A. U. Tatiya

Department of Quality Assurance, Gangamai College of Pharmacy, Nagaon, Dist. Dhule (M.S.), India.

 

ABSTRACT:

An accurate, precise and reproducible RP-HPLC method was developed for the simultaneous quantitative determination of Azelnidipine and Telmisartan in tablet dosage forms. Agilent (S.K) Gradient System UV Detector and C₁₈ column with 250mm x 4.6mm i.d and 5μm particle size Acetonitrile: ph Buffer (80:20v/v) pH 3 was used as the mobile phase for the method. The detection wavelength was 237nm and flow rate was 0.9 ml/min. In the developed method, the retention time of Azelnidipine and Telmisartan were found to be 4.77 min and 11.97min. The developed method was validated according to the ICH guidelines. The linearity, precision, range, robustness was within the limits as specified by the ICH guidelines. Hence the method was found to be simple, accurate, precise, economic and reproducible. The developed method was validated according to the ICH guidelines. In this methods linearity, precision, range, robustness were observed. The method was found to be simple, accurate, precise, economic and reproducible. So the proposed methods can be used for the routine quality control analysis of AZN and TMZ in bulk drug as well as in formulations.

 

 

INTRODUCTION:

High blood pressure, also called hypertension, is a common condition that is characterized by having a higher amount of pressure in your blood vessels than normal. Hypertension (HT) is a very common disorder, particularly past middle age. It is not a disease in itself, but is an important risk factor for cardiovascular mortality and morbidity. For improvement activity of hypertension, Azelnidipine and Telmisatan newer combination in market, which is effective in Hypertension. This combination was developed to improve medication for Stage II Hypertension.^1-2

 

Azelnidipine is dihydropyridine derivative and chemically 3-[1-(Benzyldrylazetidin-3-yl] 5-isopropyl- 2- amino6methyl-4-(3-nitrophenyl)-1,4- dihydropyridine -3,5dicarboxylate (figure 1).

 

Azelnidipine category is Dihydropyridine calcium channel blocker. Azelnidipine calcium channel blocker. Azelnidipine inhibits trans-membrane Ca2+ influx through the voltage dependent channels of smooth muscles in vascular walls. It is a vasodilator that induces a gradual decrease in blood pressure in hypertension. When calcium channels are blocked, the vascular smooth muscle does not contract, resulting in relaxation of vascular smooth muscle walls and decreased blood pressure. It is Used in Treatment of Hypertension which lower the Blood Pressure due to Block calcium Channel and Decreases Blood Pressure. and oral dose is 8 – 16 mg once daily. It metabolized in hepatic cytochrome P450 (CYP) 3A4.^3-5

 

Telmisartan is benzimidazole derivative and chemically 2-{4-[[4-methyl-6-(1-methylbenzimidazol-2yl)-2 –propylbenzimidazol-1yl]methyl]biphenyl)-benzoic acid (figure 1). Telmisartan category is Angiotensin II receptor blocker. Telmisartan interferes with the binding of to the angiotensin Ⅱ AT1-receptor by binding reversibly and selectively to the receptors in vascular smooth muscle and the adrenal gland. As angiotensin II is a vasoconstrictor, which also stimulates the synthesis and release of aldosterone, blockage of its effects results in decreases in systemic vascular resistance. Telmisartan does not inhibit the angiotensin converting enzyme, other hormone receptors, or ion channels. It is Used in Hypertension. Lowering high blood pressure helps prevent strokes, heart attacks, and kidney problems. Telmisartan is metabolized by conjugation to form a pharmacologically inactive acyl glucuronide; the glucuronide of the parent compound is the only metabolite that has been identified in human plasma and urine.^6-7

 

 

Figure 1. Structure of Azelnidipine and Telmisartan

 

Literature review reveals that, only one HPLC ⁸ methods have been published, during the preparation of the present work for publishing. Methods have been reported for the individual drugs as well as in combination with others drugs in formulation. The present study aimed to develop a simple, sensitive, short retention time and accurate RP-HPLC method for the simultaneous determination of both Azelnidipine and Telmisartan together in pure and tablet dosage forms with high sensitivity, selectivity that can be used for the routine analysis of production samples. Validation of the developed method done in accordance with ICH guidelines.⁹

 

MATERIALS AND METHODS:

Materials and Reagents:

The analysis of the drug was carried out on Agilent (S.K.) gradient system UV detector. Equipped with reverse phase (Agilent) c18 column (4.6mm x 100mm; 2.5µm), a SP930d pump, a 20µl injection loop and UV 730d (dad) absorbance detector and running Chemstation software.

 

Azelnidipine and Telmisartan were procured from R.S.I.T.C Jalgaon. Orthophopsphoric acid (OPA) (Avantor Performance material India Ltd. Thane, Maharashtra) and methanol, acetonitrile, (HPLC grade Merck Specialties Pvt. Ltd. Shiv Sager Estate ‘A’ Worli, Mumbai.), water, 0.45µm filter (Millipore, Bangalore). A combination of Azelnidipine (46mg) and Telmisartan (40mg) in tablet formulation was procured from Merck and Co. (Zepatier 16 : 40mg brand).

 

Chromatographic Conditions:

Column C₁₈ (100mm× 4.6mm); particle size packing 2.5 mm; detection wavelength of 237nm; flow rate 0.7 ml/min; temperature ambient; sample size 20µl; mobile phase Methanol : water (75:25) (pH 4.3 adjust with OPA); run time of 15 mins.

 

Preparation of standard stock solution:

The mobile phase was allowed to equilibrate with stationary phase until OPA by baseline was obtained. From the freshly prepared standard stock solution, pipette out 16mg Azelnidipine and 40mgTelmisartan in 25ml of volumetric flask and diluted with mobile phase.

 

Figure 2. Chromatogram of standard combination of Azelnidipine and Telmisartan

From it 0.1, 0.2, 0.3, 0.4 and 0.5 of solution were pipette out in 10ml volumetric flask and volume was made up to 10ml with mobile phase to get final concentration 6.4, 12.8, 19.2, 25.6 and 32µg/ml of Azelnidipine and 16, 32, 48, 64 and 80µg/ml of Telmisartan .sample were injected and peaks were recorded at 237nm as the graph plotted as concentration of drug verses peak area is depicted in (Table 1 and figure 2) respectively.

 

Table 1. Details of chromatogram of standard combination containing AZN and TMZ

 

Method development and validation:

Serial dilutions were done to prepared various concentration stock (Standard solution and diluted to get required concentration for calibration plot and which was injected^10-23.

 

Assay preparation for commercial formulation:

Weigh 20 AZN and TMN combination tablets and calculated the average weight, accurately weigh and transfer the sample equivalent to 5mg Azelnidipine and 10mg Telmisartan into 10ml volumetric flask.

 

 

Figure 3. Chromatogram for marketed formulation

 

Add about 10ml MEOH of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 µm filter. Further pipette 0.1ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluents. (10 µg/ml). The simple chromatogram of test AZN and TMN Shown in (figure 3) the amounts of AZN and TMN per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with tablet formulation. Tablet Assay for %Label claim for %RSD Calculated, Result was shown in (Table 2).

 

Table 2. Analysis of marketed formulation  

 

RESULTS:

Linearity and Range:

The data obtained in the calibration experiments when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range 6.4-32µg/ml for AZN and 16-80 µg/mL for TMN Table 3 and 4 depict the calibration data of AZN and TMN. The respective linear equation for AZN was y = 13.068 x+ 10.07 and TMN equation y = 65.50 x + 43.341 where x is the concentration and y is area of peak. The correlation coefficient was 0.999 and 0.999.

 

Table 3. Linearity data for Azelnidipine

 

Table 4. Linearity data for Telmisartan

 

Accuracy:

Recovery studies were performed to validate the accuracy of developed method. To a pre-analysed tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed. The % recovery was found to be within 98-101%. Statistical validation of recovery studies are shown in (Table 5 and 6).

 

Table 5. Result of Recovery data for Azelnidipine and Telmisartan

*mean of each 3 reading for RP-HPLC method

Table 6: Statistical Validation of Recovery Studies Azelnidipine and Telmisartan

*Denotes average of three determinations for RP-HPLC method

 

System suitability parameters:

To ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of AZN and TMN system suitability parameters were studied. The result shown (Table 7).

 

Table 7. Repeatability studies on RP-HPLC for Azelnidipine and Telmisartan

Precision:

The method was established by analyzing various standards of AZN and TMN. All the solution were analyzed thrice in order to record any intra-day and inter-day variation in the result. The result obtained for interday and intraday variation are shown in the (Table 8).

 

Table 8. Intraday and Inter day Precision studies on RP-HPLC method for AZN and TMN

*Mean of each 3 reading for RP-HPLC method

 

Robustness:

To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of changes in mobile phase composition and flow rate on retention time and tailing factor of drug peak was studied. The results indicate that less variability in retention time and tailing factor were observed Table 9.

 

 

Table 9. Result of Robustness study of AZN and TMN

 

DISCUSSION:

The proposed methods for simultaneous estimation of AZN and TMN in tablet dosage forms were found to be simple, accurate, economical and rapid. The method was validated as per the ICH Q2 (R1) guidelines. Standard calibration yielded correlation coefficient (r²) 0.999 for both AZN and TMN at all the selected wavelengths. The values of % RSD are within the prescribed limit of 2 %, showing high precision of methods and recovery was close to 97%-101% for both drugs. Results of the analysis of pharmaceutical formulations reveal that the proposed method is suitable for their simultaneous determination with virtually no interference of any additive present in pharmaceutical formulations. Hence, the above methods can be applied successfully for simultaneous estimation of AZN and TMN in formulations.

 

CONCLUSION:

The developed HPLC methods in that linearity, precision, range, robustness were found to be more accurate, precise and reproducible. The methods were found to be simple and time saving. All proposed methods could be applied for routine analysis in quality control laboratories.

 

ACKNOWLEDGEMENTS:

The authors are thankful to the Principal, Gangamai College of Pharmacy, Nagaon, Dist. Dhule for providing necessary facilities for research work. They are also grateful to Merck and Co. for giving gift samples of pure drugs.

 

 

 

 

ABBREVIATION USED:

HPLC: High performance liquid chromatography; UV: Ultraviolet; ICH: International Conference on Harmonization; LOQ: Limit of quantitation;
LOD: Limit of detection; RSD: Relative standard deviation; RT: Retention time; OPA: Orthophosphoric acid; AZN: Azelnidipine; TMN: Telmisartan; FDA: Food and Drug Administration; SD: Standard deviation.

 

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Accepted on 06.02.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5736-5740.