1. INTRODUCTION:

Hypertension is a serious public health concern because of its enormous prevalence across the world^1,2. Chronic heart disease, stroke, and coronary heart disease are all linked to high blood pressure^3-5. Every year, the FDA approves various medication combinations to treat hypertension, one of which is Benidipine (BEN) with Telmisartan (TEL)^6,7. A combination of these medications is used to successfully control blood pressure and reduce the mortality rate among middle-aged persons. BEN is a calcium channel antagonist who blocks all the three L, N and T calcium channel and also is a reno-cardioprotective^8-10.

TEL is an angiotensin-converting enzyme inhibitor that is used to treat hypertension^11,12. TEL binds to the angiotensin II type 1 (AT1) receptor with high affinity, inhibiting angiotensin II's effect on vascular smooth muscle and, as a result, lowering arterial blood pressure^13,14 The structure of the mentioned drugs have been shown in the given figure 1.

BEN and TEL are two medications that are used to treat hypertension. For the quantification of BEN and TEL alone or in combination with other antihypertensive agents, methods such as UV spectroscopic have been published¹⁵. Individually and in conjunction with other anti-hypertensive or anti-diuretic medications, the RP-HPLC approach has been extensively described^16-18. Following a review of the literature, it was discovered that no RP-HPLC technique has been described for the indicated combination till date. TEL has been made official in BP and IP¹⁹, but BEN has yet to be added in IP and BP but it has been officially included in JP²⁰. The presented work has meant to develop new, validated, fast and sensitive RP-HPLC for the simultaneous estimation of BEN and TEL in the combination. The primary goal of method development is to adapt these methods for bioanalytical applications.

A B

Figure 1. Chemical structure of A. Benidipine; B. Telmisartan

2. MATERIAL AND METHOD:

2.1. Reagents and Chemicals:

The working standards of Benidipine and Telmisartan were obtained as a gift sample from ISFAL and Macleods. Acetonitrile (HPLC grade) and Methanol (HPLC, AR grade) were obtained from SD fine chemicals (Ambala, India). The Glacial acetic acid were procured from SD fine- chem limited (Mumbai, India) and Triethylamine (TEA), (HPLC grade) was bought from Sigma Aldrich, Mumbai, India. Highly pure water was obtained from a MilliQ system Waters purification system ELIX 03 (MILLIPORE, USA. The marketed formulation used for the assay determination was obtained from market as Binastar-TL tablets from Jabs Biotech private limited (Patiala, India).

2.2 Method development:

2.2.1. Chromatographic method:

2.2.2.1 Instrumentation and chromatographic conditions:

Waters HPLC with 515 pumps and 2489 UV-VIS detector, as well as a manual rheodyne 7125i injector with a 20 loop, comprised the reverse phase chromatography system. The chromatographic separations were done on an Xbridge C18 column (4.6mm x 250mm) with a particle size of 5µm (Waters, USA). The mobile phase was an 80:20 v/v combination of Methanol and ACN with 0.1 percent v/v TEA, adjusted to pH 5.2 using glacial acetic acid, with an isocratic flow rate of 1.0mL/min with total run duration of 10minutes. At dual wavelenght 237 and 297nm, the absorbance of all chemicals of interest (BEN and TEL) was measured. The Empower^TM 2 software was used to process the data.

2.2.2.2. Preparation of stock and standard solutions:

The stock solutions were made by gently shaking 10mg of BEN and TEL properly weighed in a 10mL volumetric flask containing 8mL Methanol. To achieve a concentration of 1mg/mL (1000g/mL), the volume was adjusted to the desired level and the flasks were sonicated for 5 minutes.

BEN standard solutions in the 2-10µg/mL concentration range were made in triplicate by obtaining an appropriate aliquot from a secondary stock solution containing 100 µg/mL. In the same way, a standard solution containing TEL 5-25µg/mL was made. The standard laboratory binary mixture was created by diluting a 100µg/mL secondary stock solution into 2, 4, 6, 8, and 10µg/mL of BEN and 5, 10, 15, 20, and 25µg/mL of TEL.

2.3. Method validation:

A full validation of the current method was performed in accordance with the regulatory guidelines [21,22]

2.3.1. Selectivity and Specificity

By comparing the HPLC chromatograms for the blank mobile phase (HPLC) and the drug, the selectivity and specificity of the newly devised technique were determined.

2.3.2. Linearity:

The method's linearity was assessed using linear regression analysis. Least square regression analysis was used to determine the linearity range. Reference standard solutions of 2-10g/mL and 5-25g/mL for BEN and TEL, respectively, were used to create the calibration curve. For each drug sample, five concentration points were tested in triplicate.

2.3.3. Precision and Accuracy:

The method's precision was determined using a repeat analysis (n=6) of the test concentrations. The procedure's precision was achieved by the use of a system, method, and intermediate precision (inter-analyst). The system precision was achieved by analysing the same solution six times, whereas the technique and intermediate precision was achieved by replicating the analysis of six separate test concentration solutions.

Recovery experiments were used to assess the method's accuracy at 80-120 percent of the test concentration in triplicate (n=3). Percent mean recovery was used to determine the quantity of fortified medication recovered, and percent relative standard deviations were used to determine accuracy and precision (percent RSD).

2.3.4. Limit of Detection and Quantification (LOD and LOQ):

The detection and quantitation limits are the smallest amounts that his technology can detect and quantify. The method's LOD and LOQ were derived using equations based on the slope (σ) and residual standard deviation (SD) of the calibration lines.

LOD = 3.3 σ/SD ……………………………………...(1)

LOD = 10 σ/SD ……………………….……………..(2)

2.3.5. Robustness:

It refers to the method's capacity to stay unaffected by tiny purposeful changes. The flow rate, pH of the Mobile Phase, and percent TEA were used to illustrate the HPLC method's robustness. To show the method's robustness, the findings were given as percent RSD.

2.4. Extraction procedure for RP- HPLC Method:

Twenty Binastar-TL tablets were weighed and pulverised precisely. In a 10 mL volumetric flask, the tablet powder corresponding to 10 mg BEN and 10 mg TEL was transferred. Methanol (HPLC) was used to extract the powder, followed by 15 minutes of sonication and filtering through a 0.22 micron syringe filter. Following that, the extracted sample was diluted using dilution solvent to achieve the needed concentration for HPLC analysis.

3. RESULTS AND DISCUSSION:

3.1. Method development:

3.1.1. Chromatographic Method:

In the choosing of Reverse Phase separation, the intrinsic features of the active moieties became visible. As a result, the C18 column was chosen as the best option for their separation since it contains selective and efficient silanol groups. The optimization of the chromatographic condition is a critical step in the development of chromatographic methods. To optimise the chromatographic condition, various mobile phases with varying ratios of aqueous and organic fractions were tested, with the goal of using them in bioanalysis. Water was used as the aqueous fraction, which was evaluated at various pH levels ranging from 4.4 to 5.2, as well as flow rates ranging from 0.5 to 1.2 mL/min. To increase peak sharpness, organic modifiers were also added. Methanol: ACN (80:20, v/v with 0.1 percent v/v TEA), adjusted to pH 5.2 with glacial acetic acid, produced appreciable separation and peak purity. As shown in figure 2, the optimization was based on system appropriateness, with (Rt) 6.18 and 8.55 minutes for BEN and TEL, respectively, and a total run duration of 10 minutes. The method validation was carried out in accordance with regulatory criteria, resulting in a sensitive, selective, accurate, precise, and accurate technique as shown in Tables 1 and 2.

3.2. Method validation:

3.2.1. Selectivity and Specificity:

The capacity of the techniques to assess the analyte in the presence of additional components such as matrix, excipients, solvents, mobile phase, and so on was used to determine specificity and selectivity. At the retention period of BEN and TEL, no unique signal hindrances were observed, indicating that there was no inherent interference.

3.2.2. Linearity:

The evaluation of linearity was done with the use of a calibration curve. Plotting analyte concentration vs peak area was used to evaluate the calibration curve of the analytical technique. For the HPLC procedure, the calibration curves were produced in methanol. The linearity of BEN and TFL was 2-10 g/mL and 5-25 µg/mL, respectively. Figure 3 and 4shows that the regression coefficients (r²) for both samples are > 0.99.

Figure 3: Calibration curve for Benidipine

Figure 4: Calibration curve for Telmisartan

3.2.3. Precision:

The repeatability (system and method) and intermediate precision (intra) of the HPLC technique's test concentration were assessed using repeated analysis (n=6). The table's outcome meets regulatory accuracy criteria, which are determined by percent RSD2 (percent relative standard deviation), which is well within limitations for both organisations (Table 1).

Table 1. HPLC assay validation parameters results.

Validation Parameter	BEN	TEL
λmax	237nm	297nm
Range (µg/mL)	2-10	5-25
Regression equation	y = 69921x + 17250	35209x + 50804
Correlation coefficient (r²)	0.997	0.998
Limit of Detection (µg/mL)	0.108	0.610
Limit of Quantification (µg/mL)	0.330	1.850
Precision (%RSD)
System	0.449	0.741
Method	0.529	0.555
Intermediate
Analyst 1	0.486	0.572
Analyst 2	0.442	0.579

3.2.4. Accuracy:

Recovery experiments at levels 80-120 percent of the test doses given in Table were used to evaluate accuracy. The capacity of a procedure to recover a known amount that has been spiked at varying quantities to the assessed sample is known as accuracy. The recovery investigation yielded a percent recovery of 100%, indicating a high degree of accuracy for the devised approach and compliance with validation standards Table 2.

Table 2. Recovery studies of the HPLC method

Drug	Level (%)	Amount spiked (µg/mL)	Amount recovered (µg/mL)	% Recovery	Mean % recovery	S.D
	80	4.8	4.753	99.03
BEN	100	6	5.941	99.02	99.38	± 0.620
	120	7.2	7.207	100.10
	80	12	11.883	99.03
TEL	100	15	15.162	101.08	100.4	± 1.186
	120	18	18.196	101.09

3.2.5. Limit of Detection and Quantification (LOD and LOQ):

The assay method's detection/quantification limits are the lowest concentrations that can be detected/ quantified. The established LOQ should be 3.3 times the LOD. As shown by the LOQ and LOQ in Tables 1, it was found that the developed method was sensitivity.

3.2.6. Robustness:

The performance of a successful analytical technique is measured by how well it stands up to less-than-ideal implementation. Examined characteristics such as flow rate, pH change, and wavelength have no effect on the HPLC process. The % RSD for the robustness parameter resulted in the Table 3.

Parameters	BEN %RSD	TEL %RSD
Flow rate (±0.1)	0.165	0.374
pH (±0.2)	0.174	0.228
Wavelenght (±2)	0.270	0.625

4. CONCLUSION:

For the estimation of Benidipine and Telmisartan in mixed dose forms, a simple, quick, exact, and reliable approach was established. The findings obtained are within the ICH guidelines' defined limits. The mobile phase and analytical column employed produce superior separation and crisp findings, because both medications had a satisfactory retention time of 6.891 and 8.997 min respectively. The approach was successfully employed for the simultaneous quantitative measurement of BEN and TEL in solid dosage form and was verified in terms of linearity (r2>0.997), robustness, precision, and accuracy. The established HPLC chromatographic technique for concurrently determining BEN and TEL was simple, specific, accurate, precise, sensitive, and cost-effective. The method might be used in quality control laboratories on a regular basis.

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Received on 05.07.2022 Modified on 06.03.2023

Asian J. Pharm. Ana. 2023; 13(4):231-235.

DOI: 10.52711/2231-5675.2023.00037