INTRODUCTION:

Azelnidipine (AZE) is a new dihydropyridine CCB that is selective for L-type calcium channels and is used to treat hypertension patients. From the extensive literature search, it can be inferred that UV spectroscopy¹, High-performance liquid chromatography^2-4 and LC–ESI-MS⁵ are the only techniques used for estimating AZE concentration in various matrix.

Telmisartan (TEL) is an active, long-acting nonpeptide AT1 receptor antagonist used to treat essential hypertension. TEL is not a prodrug, and its terminal removal half-life (24 hr) is longer than that of other commercially available sartans, rendering it ideal for once-daily dosing. According to the literature review, the concentration of TEL in a sample can be measured using various methods such as UV spectroscopy⁶, HPLC^7-10. and LC-MS/MS¹¹.

Under the brand name UNIAZ T 40, an FDC containing 8 mg of AZE and 40 mg of TEL is available in the market. The rationale for the fixed combination of AZE and TEL is additive BP-lowering effects, lower incidences of adverse events, and improving adherence to therapy by providing combination antihypertensive therapy for administration as a single, once-daily tablet ¹².

The fixed-dose combination of AZE and TEL can be considered a convenient and well-tolerated treatment choice for hypertension based on the above results^13-14.

According to the literature review, there is a lot of study, literature, and scientific papers on Analytical method validation, stability-indicating methods, and formulation development studies on individual drugs like AZE and TEL, but there isn't a single research paper, literature work, or scientific paper on in-vitro dissolution method development and validation for a combination of drugs like AZE and TEL.

The present investigation reports a successful attempt to develop a short, simple, precise, yet repeatable method for simultaneous estimation of AZE and TEL by HPLC using Acetonitrile and Buffer in gradient mode which is capable of estimating the value of each of these drugs in tablet dosage form. Thus developed method was successfully validated for specificity, precision (system precision, method precision, intermediate precision), linearity, recovery, stability in aqueous solution (SIAS), and robustness tests were all used to verify the system.

MATERIAL AND METHODS:

Chemicals and reagents:

The working standards of Azelnidipine and Telmisartan were received as gift samples from M/s. Synokem Pharmaceutical Limited, Haridwar, Uttrakhand. A fixed-dose combination product of AZE and TEL was prepared with a label claim of 8mg and 40mg respectively. Reagents like Ammonium dihydrogen orthophosphate, Orthophosphoric acid were AR grade, solvent like Acetonitrile and Methanol were HPLC grade and Water (Milli-Q-grade) was obtained from M/s. Kusum Healthcare Pvt. Ltd., Bhiwadi, and Rajasthan.

METHOD DEVELOPMENT USING HPLC:

Instrument details and Chromatographic Conditions:

The HPLC instrument (Shimadzu make, model-LC-2010 CHT, Empower software) with Photodiode-Array detector (PDA) was employed. Inertsil ODS 3V, C18 column was used with dimensions of 150 x 4.6mm and 5µm particle size with flow rate 1.5mL/min. The autosampler temperature was maintained at 10⁰C with a run time of 11 minutes. The ƛ max selected was 254 nm. The injection volume was kept at 10µL. Different combinations of mobile phases were used in gradient mode as shown in Table 1. Other types of instruments used were sonicator, pH meter, analytical balance.

Table 1: Gradient mode of mobile phases at a different time interval.

Gradient Time	Pump A % (Buffer)	Pump B % (Acetonitrile)
0.01	55	45
3.00	55	45
5.00	70	30
7.00	70	30
8.00	55	45
11.00	55	45

Dissolution Conditions:

About 900mL of 1% w/v solution of sodium lauryl sulphate (SLS) was maintained in USP type II i.e. paddle apparatus (Lab India, model DS8000+). The rotation speed of the paddle was kept at 75rpm at the temperature of 37.0±0.5°C. The time set for a single point was 45 minutes.

Preparation of Dissolution media:

Weigh accurately about 60gm of Sodium Lauryl Sulphate (SLS) was added into 1000mL of purified water and stirred continuously until the content dissolves. The final volume was made up to 6000mL and mixed well.

Preparation of Buffer:

Weigh accurately 2gm of Ammonium dihydrogen orthophosphate in a 1000mL volumetric flask add 900 mL of milli-Q water. The pH was adjusted to 3.0±0.05 using dilute Orthophosphoric acid. 0.45µm PVDF filter was used to conduct filtration.

Preparation of stock and standard solutions:

To prepare a stock solution of AZE and TEL about 8.98 mg of AZE and 43.85mg of TEL were transferred into a 100mL volumetric flask. To this solution, 70mL of diluent i.e. (Buffer: Acetonitrile 25:75% v/v), this solution was sonicated and diluent was added to make up the final volume. Thus the prepared solution is considered a stock solution.

From this solution, about 5mL of solution was taken and transferred into a 50mL volumetric flask. The content of the solution was mixed well to make the final concentration of 8.94µg/mL of AZE and 43.70µg/mL of TEL. This solution was filtered through a 0.45µm PVDF membrane filter and used as the standard solution.

Preparation of placebo solution:

Weigh accurately 206mg of placebo powder (as 1 tablet weight was 254mg) and add to one of the dissolution vessel of dissolution apparatus containing 900mL of dissolution medium which was equilibrate^. to the temperature of 37°C±0.5°C. Operate the apparatus at 75 RPM for 45 minutes. Withdraw 20mL of the sample from each vessel after 30 minutes. Centrifuge the test solution for 10 minutes at 3000rpm and use the clear liquid.

Preparation of sample solution:

One tablet was put in each vessel of dissolution apparatus encompassing about 900 mL of dissolution media. The temperature was maintained to 37^°C±0.5^°C at 75rpm. After a pre-determined time interval, about 20 mL aliquot was taken. The sample should be taken such that from a zone midway between the surface of the dissolution medium and the top of the blade, not less than 1-cm from the vessel wall. Filter the aliquot through Whatman filter paper no.1 discard the initial few mL of the filtrate and collect the clear filtrate. Filter again through a 0.45µm PVDF filter.

Method validation parameters:

System suitability:

The percent relative standard deviation (%RSD) of peak area, tailing factor, resolution factor, retention time (Rt) and the theoretical plate were selected as criteria to conduct the test. The aforementioned parameters confirm the reproducible nature of the protocol to be validated. To record the chromatogram about 6 replicates of standard solution were considered. To pass through the criteria of the system suitability, The %RSD for peak area and Rt of AZE and TEL peak obtained from six replicate standard injections should not be more than 2.0. Tailing factor and resolution Factor should not be more than 2.0 of AZE and TEL peak in standard solution and Theoretical Plates should not be less than 2000 of AZE and TEL peak in standard solution.

Specificity:

It was assessed by single injection of blank, placebo standard and sample solution. No interference was observed at the Rt of AZE and TEL was recorded in the blank and placeo chromatograms. The purity angle is less than the purity threshold in standard and sample solution.

Precision:

The samples were tested for different precision intervals like inter-day, intra-day. To determine system precision Blank was injected as single followed by six injections of standard solution prepared independently. For method precision six injections of sample solution were injected. The %RSD of peak area observed should be NMT 2%. Intermediate precision was carried out at doing purposeful changes in a day, instrument, column and analyst. About six sample from the same batch were injected. The %RSD for dissolution release values for samples must be NMT 2% and overall % RSD must not exceed 2%.

Accuracy/Recovery:

From the label claim of AZE 8 mg and TEL 40mg strength, different test samples were prepared at the level of 50%, 100 %, and 150%), covering the specified range (i.e. three concentrations and three replicates of each). The outcomes were evaluated for the respective standard solutions as per their strength. The % recovery was noted and criteria must be applied that “The percent standard deviation at all recovery level NMT 2.0%.”

Linearity and Range:

In a separate volumetric flask the aliquot of samples of AZE and TEL was taken and diluted such that to achieve a final concentration in the range of 1.788-10.731µg/mL and 8.740-52.441µg/mL of AZE and TEL respectively. The solutions of concentration in the range 20%-120% were injected. The regression coefficient value NLT 0.995.

Robustness:

The ability of the system to remain non deviated after introducing intentional changes is calculated using the robustness. The few parameters tested here are the (flow rate with a deviation of ±10%, the temperature of column ±5°C, pH±0.2, gradient changes ±2%, wavelength ±5nm). When system suitability was established as mentioned in protocol, inject sample solution in the deliberately changed method to performed robustness study.

SIAS:

To validate the stability of the sample in analytical solution was done by injecting the sample prepared initially and observing the change in concentration of analyte with time.

RESULT AND DISCUSSION:

System Suitability:

Retention time (Rt) of TEL and AZE was 2.302 and 4.466 respectively. Tailing factor of TEL and AZE was 1.3 and 1.2 respectively. Theoretical plate count of TEL and AZE was 2739 and 3506 respectively and Resolution factor was 9.06. The %RSD (relative standard deviation) for replicas of six injections area in standard solution was NMT 2.0% .

Specificity:

No interference from blank and placebo at the retention time of TEL and AZE peak confirmed the specific nature of the method developed as per the values observed in (Figure 1 A, B and C).

Figure 1 (A): The chromatogram of Blank under optimized chromatographic conditions.

Figure 1(B): The chromatogram of Placebo under optimized chromatographic conditions.

Figure 1(C): The chromatogram of the standard solution under optimized chromatographic conditions.

Precision:

After injecting six replicate injection of standard solution %RSD was observed as 0.1% and 0.1% for AZE and TEL respectively in system precision. For Method precision %RSD of peak area of six sample solution were observed as 0.5% and 0.4% for AZE and TEL respectively.T he % dissolution release of the formulation was calculated by considering the area of injections while the injection of six independent samples in a single injection was taken for calculation of method precision. The % RSD of six intermediate precision sample solutions was 0.5% and 0.5% for AZE and TEL respectively.The overall % RSD for 12 dissolution release injection, six method precision samples, and six intermediate samples was found to be was 0.7% and 1.1% for AZE and TEL respectively. All the parameters and outcomes of the precision study can be observed in Table 2.

Accuracy (Recovery):

This method is accurate from 50% to 150% of the largest stock concentration as shown in Table 3.

Wt. taken x potency of Working standard

Corrected amount added = -----------------------------------------------------

100

Mean area counts of sample solution x standard dilution x standard Potecy

Recovery = -----------------------------------------------

Mean area counts of standard solution x sample dilution x 100

Mean area counts of sample sol. x std. dilution x std. Potency x Av. wt

% Recovery = ----------------------------------------- x 100

Mean area counts of standard solution x sample dilution x claim x 100

Table 2: Summary of validation parameters for AZE and TEL.

Sr.No	Parameters	AZE	TEL
1	Specificity	No interference	No interference
2	Peak purity	1.00	1.00
3	Linearity and range (μg/ml)	1.788-10.731	8.740-52.441
4	Slope	16015.40	16129.84
5	Intercept	128.86	1636.96
6	Regression coefficient	0.99618	0.99698
7	System Precision (%RSD)	0.1%	0.1%
8	Method precision	0.5%	0.4%
9	Intermediate precision	0.5%	0.5%
10	Ruggedness (n=6*2)	0.7%	1.1%
11	Theoretical plate	3506	2739
12	Accuracy %	99.1‑101.5	96.4‑97.5
13	Robustness (% RSD)	0.9	0.5
14	% Dissolution	101	98

Linearity and Range:

The HPLC method was found linear in the range of 1.788-10.731 µg/ml and 8.740-52.441 µg/ml for AZE and TEL respectively after observing the calibration curve as shown in Figure 2 (A and B). Whereas regression coefficient (R²) was calculated as 0.99618 and 0.99698 for AZE and TEL (Table 4).

Table 3: % Recovery of AZE and TEL

Sample Name	Corrected amount added (mg)	Area counts	Amount Recovered(mg)	% Recovery	Mean	Corrected amount added(mg)	Area counts	Amount Recovered(mg)	% Recovery	Mean
% Recovery of AZE						% Recovery of TEL
50% -1	4.26	76209	4.34	101.8	101.5	20.05	347209	19.56	97.6	97.5
50% -2	4.33	75695	4.37	100.8		20.08	345068	19.44	96.8
50% -3	4.22	75520	4.30	101.8		20.01	348756	19.65	98.2
100% -1	8.02	140381	7.99	99.7	99.1	40.04	693570	39.08	97.6	97.5
100% -2	8.08	139238	7.92	98.1		40.07	692237	39.00	97.3
100% -3	8.00	139626	7.95	99.4		39.96	691467	38.96	97.5
150% -1	11.99	214005	12.18	101.6	99.9	59.92	1025565	57.79	96.4	96.4
150% -2	12.05	210135	11.96	99.3		60.03	1026542	57.84	96.4
150% -3	12.11	210455	11.98	98.9		60.04	1027852	57.92	96.5
Mean				100.2	Mean					97.1
SD				1.38	SD					0.64
%RSD				1.4	%RSD					0.7

Table 4: Data obtained showing the linearity for both drugs AZE and TEL.

		AZE			TEL
Vol. Linearity stock taken (ml)	Dilution (mL)	Conc. (ppm)	Area	Standard Area	Conc. (ppm)	Area	Standard Area
2.0	100	1.788	28016	141365	8.740	137556	696571
5.0	100	4.471	70205	141518	21.850	347204	698854
3.5	50	6.260	105752	141418	30.591	519908	698978
4.5	50	8.048	128990	141384	39.331	638786	698485
5.0	50	8.942	139063	141446	43.701	689626	697372
6.0	50	10.731	173207	141273	52.441	848740	697868

Table 5:Compiled Robustness Data for Dissolution of AZE and TEL.

% Release of AZE
S. No.	FP (1.65 mL/min)	FM(1.35 mL/min)	CTP (45ºC)	CTM (35ºC)	WP (259 nm)	WM (249 nm)	pH-P (3.2)	pH-PM (2.8)	GP-P (+2%)	GP-M (-2%)
1	103	103	103	103	102	103	103	103	103	103
2	103	103	103	103	102	103	104	103	104	104
3	104	104	104	104	102	105	104	103	104	104
Overall Mean	101.6	101.6	101.6	101.6	101.1	101.7	101.7	101.4	101.7	101.7
Overall SD	1.4	1.4	1.4	1.4	0.8	0.8	0.5	0.4	0.5	1.0
Overall % RSD	1.4	1.4	1.4	1.4	0.8	0.8	0.5	0.4	0.5	1.0
Mean of Overall % RSD	0.9
% Release of TEL
S. No.	FP (1.65 mL/min)	FM(1.35 mL/min)	CTP (45ºC)	CTM (35ºC)	WP (259 nm)	WM (249 nm)	pH-P (3.2)	pH-PM (2.8)	GP-P (+2%)	GP-M (-2%)
1	99	98	99	98	98	98	98	99	98	99
2	98	98	98	98	99	98	99	98	99	98
3	98	98	99	98	98	98	98	98	98	98
Overall Mean	98.0	97.9	98.1	97.9	98.0	97.9	98.0	98.0	98.0	98.0
Overall SD	0.5	0.4	0.5	0.5	0.4	0.4	0.4	0.5	0.5	0.5
Overall % RSD	0.5	0.4	0.5	0.5	0.4	0.4	0.4	0.5	0.5	0.5
Mean of Overall % RSD	0.5

Where, FP=Flow Plus, FM=Flow Minus, CTP=Column Temperature Plus, CTM=Column Temperature Minus, WP= Wavelength Plus, WM=Wavelenght Minus, pH-P=pH Plus, pH-M=pH-Minus, GP-P=Gradient Plus, and GP-M=Gradient Minus.

Robustness:

The observance of % RSD as low as 2% confirms the robust nature of the method developed. The values for the same can be observed in Table 5.

Stability in analytical solution (SIAS):

The stability of the analytical solution was assessed by examining the sample solution at 0 hr and 3, 10, 14, 18, 23 and 26 hours time interval and changes were monitored in response with time. The results were presented in terms of the initial (nominal) concentration at time zero. Stability was defined as less than 10% loss of the initial concentration.

CONCLUSION:

The developed method was successfully validated for various ICH parameters like system suitability, specificity, precision (system, method precision, intermediate precision), accuracy, range, robustness, linearity and stability of the solution and all the parameters comply with predetermined criteria. The validated method was specific, linear, precise, accurate, robust, and rugged for the dissolution of AZE and TEL Tablets 8/40mg. Therefore the results obtained showed that the method can be reliably used for laboratory analysis of In-vitro dissolution testing of drugs AZE and TEL.

CONFLICT OF INTEREST:

The author declares no conflict of interest for the present manuscript.

ACKNOWLEDGMENTS:

The authors are thankful to M/s. Synokem Pharmaceutical Limited, Haridwar, Uttrakhand, M/s. Kusum Healthcare Pvt. Ltd., Bhiwadi, Rajasthan, and School of Medical and Allied Sciences, K.R. Mangalam University, Gurugram, for their support towards completion of the present analytical investigation.

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Received on 24.04.2021 Modified on 27.05.2021

Research J. Pharm. and Tech. 2022; 15(5):1967-1972.

DOI: 10.52711/0974-360X.2022.00327