A New Simultaneous HPLC Analytical method for Quantification of Benazepril Hydrochloride and its related Impurities in Bulk Drug Product

 

Tentu NageswaraRao¹*, Karri Apparao¹, N. Krishnarao¹, A. Vijayalakshmi²

¹Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India.

²Department of Marine Living Resources, Andhra University, Visakhapatnam, Andhra Pradesh, India.

 

ABSTRACT:

A simple and inexpensive method was developed with high performance liquid chromatography with PDA detection for determination of benazepril hydrochloride and its related impurities. The chromatographic separations were achieved on (250×4.6 mm), 5.0 µm make: Symmetry Shield column employing 0.02M tetrabuthylammonium  hydroxide + 0.05 % v/v acetic acid : methanol in the ratio of 50:50 (v/v) as mobile phase with isocratic at flow rate 1.0mL/min was chosen. All impurities were eluted within 30 minutes. The column temperature was maintained at 25^oC and a detector wavelength of 240 nm was employed.   The method was successfully validated by establishing System Suitability, Specificity, Linearity, Precision, Accuracy, Limit of detection and Limit of quantification.

 

 

INTRODUCTION:

Benazepril hydrochloride is chemically 3S-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3S)-benzazepine-1-acetic acid monohydrochloride. The empirical formula of BEN is C24H28N2O5·HCl with a molecular weight of 460.96 g/mole¹. Benazepril hydrochloride is obtained as a white to off-white crystalline powder. Benazepril hydrochloride, an angiotensin-converting enzyme (ACE) inhibitor, is used in the treatment of hypertension and heart failure². benazepril is converted into its active form benazeprilat, a non-sulfhydryl angiotensin-converting enzyme (ACE) inhibitor³. It acts on the renin-angiotensin-aldosterone system by inhibition of the conversion of the inactive angiotensin I to the highly potent vasoconstrictor angiotensin II.

 

It also reduces the degradation of bradykinin. benazepril HCl is applied in pharmacotherapy as a first-choice drug for treatment of arterial hypertension, ischemic heart disease, hypertrophy of the left heart ventricle and post infarction heart dysfunction^4,5. Elementary osmotic tablets of benazepril Hydrochloride were developed using Sodium chloride as a key ingredient which gives osmogent property which provides driving force inside the core tablet and which leads to release of drug⁶. In the present research article benazepril hydrochloride and its related impurities were successfully validated by using HPLC. As on date, there were no research articles for method validation of related substances of Benazepril hydrochloride.

 

Benazepril hydrochloride: 3-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3S)-benzazepine-1-acetic acid monohydrochloride.

Impurity A: ((3R)-3-[[(1R)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-2,3, 4,5-tetrahydro-2-oxo -1H-1-benzazepine- 1-acetic acid, monohydrochloride)

Impurity B: ((3S)-3-[[(1R)-1-(eth oxycarbonyl)-3-phenylpropyl]amino]-2,3, 4,5-tetrahydro-2-oxo -1H-1-benzazepine- 1-acetic acid, monohydrochloride).

Impurity C: ((3S)-3-[[(1S)-1-carb oxy-3-phenylpropyl] amino-2,3,4,5-tetrah ydro-2-oxo-1H-1-be nzazepine]-1-acetic acid)

Impurity D: (3-(1-Ethoxycarbony l-3-cyclohexyl-(1S)-propyl)amino-2,3,4,5-tetrahydro-2-oxo-1H- 1-(3S)-benzazepine- 1-acetic acid monohydrochloride)

Impurity E: (3-amino-2,3,4,5-tetr ahydro-2-oxo-1H-1-( 3S)-benzazepine-1- acetic acid, monohydrochloride)

Impurity F: (3-Amino-2,3,4,5-tetr ahydro-2-oxo-1H-1-( 3S)-benzazepine-1- acetic acid, tert-butyl ester)

Impurity G: ((3-(1-Ethoxycarbon yl-3-phenyl-(1S)-propyl) amino-2,3,4,5-tet rahydro-2-oxo-1H-1- (3S)-benzazepine)-1 -acetic acid, ethyl ester)

 

MATERIAL AND METHODS:

Materials:

Standard gift samples of  benazepril  and impurities were provided by Dr Benarji Patrudu, Associate Professor, Gitam University, and Hyderabad. All the chemicals and reagents used were of analytical grade.

 

HPLC Chromatographic Parameters:

Chromatographic separation was performed on The HPLC-UV system used, consisted shimadzu high performance liquid chromatography with LC- 20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed phase C18 analytical column of 250 mm x 4.6 mm and particle size 5 µm (Symmetry Shield -C18) Column oven temperature was maintained at 25°C and flow rate 1.0mL/min An HPLC method was developed for benazepril hydrochloride and related impurities by using photo diode array detector. benazepril hydrochloride and all related impurities were injected into HPLC system by changing the different composition of 0.02M tetrabuthylammonium  hydroxide + 0.05 % v/v acetic acid: methanol in the ratio of 50:50 (v/v).

 

The absorption maxima for benazepril hydrochloride, Impurity –A to Impurity – G were found to be at 240 nm and the compound was scanned form 200 – 400 nm. Column temperature was set up at 25°C and injection volume as set to 25µL. By follow this analytical method conditions, benazepril hydrochloride and related impurities were separated. Hence, it was concluded that HPLC method was suitable for method validation.

 

Method Validation:

Specificity:

The specificity will be confirmed comparing the chromatogram of the blank run to the chromatogram of the single impurity run. Then a solution containing a mix of impurities and Benazepril Hydrochloride will be injected. The Benazepril peak has to be separated and has to meet the suitability parameters.

 

Preparation of Specificity Solution:

Impurity A: Weigh 15.0 mg of the Impurity-A in a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Impurity B: Weigh 15.0 mg of the Impurity-B in a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Impurity C: Weigh 15.0 mg of the Impurity-C in a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Impurity D: Weigh 15.0 mg of the Impurity-D in a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Impurity E: Weigh 15.0 mg of the Impurity-E in a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Impurity F: Weigh 15.0 mg of the Impurity-F in a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Impurity G: Weigh 15.0 mg of the Impurity-G in a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Benazepril hydrochloride: Weigh 15.0 mg of the benazepril hydrochloride in a 100mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

From the above stock solution, 1 ml taken into a 100ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

Selectivity Solution:

From the solutions A prepared above take 1ml and dilute to 100ml and inject six times.

 

Linearity:

Standard stock solution of benazepril hydrochloride and impurities having concentrations of 100µg/mL was taken for linearity test. 1.0, 2.0, 5.0, 10 and 20.0mL of standard stock solution of benazepril hydrochloride and impurities was transferred into separate 100mL volumetric flask and diluted up to the mark with diluent to get the final concentration of 1.0, 2.0, 5.0, 10 and 20.0µg/mL respectively. The linearity was in the range of 10 – 200 % for A.I and impurities. The resulting solutions were injected into HPLC in three replications. Correlation coefficient was calculated for A.I and impurities by plotting the graph between concentrations versus peak Area.

 

Precision at 0.15% level:

The repeatability will be determined in agreement with ICH guidelines; injecting six different test solutions obtained weighing six times the homogeneous sample of impurities and Benazepril Hydrochloride.

 

 

Precision Solution:

Weigh 15.0 mg of A, B, C, D, E, F, G and Benazepril in a100mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 150µg/mL).

 

From the above stock solution, 1 ml taken into a 100 ml volumetric flask and diluted to volume with the diluents (Sol B-1.5µg/ml) and injected into HPLC.

 

LOD and LOQ:

LOD and LOQ were assessed in accordance with ICH guidelines. The method chosen was based on the visual inspection of the linearity graphs for impurities at 0.1% level of a,b,c,d,e,f and benazepril and the signal to noise ratio, using the following formulas:

 

LOD=	3.3 x s		LOQ=	10 x s
	S			S

 

RESULTS AND DISCUSSIONS:

Specificity and Selectivity:

The specificity was confirmed comparing the chromatogram of the blank run to the chromatogram of the single impurity run. Then a solution containing a mix of impurities and Benazepril Hydrochloride will be injected.

 

The Benazepril peak has to be separated and has to meet the suitability parameters.

 

Linearity:

The linearity regression curve for benazepril Hydrochloride and their impurities were drawn between concentrations and peak areas. The correlation coefficient is above 0.99 at wavelength of 240 nm for benazepril hydrochloride and their impurities. The results are mentioned in Table 1. A calibration curves were showed in Figure 1.

Table 1. Linearity Data of Benazepril hydrochloride and Impurities

Replications		Conc. (µg/mL)	Area in mAU-sec of
			Benazepril ydrochloride	Imp-A	Imp-B	Imp-C	Imp-D	Imp-E	Imp-F	Imp-G
R1	10%	1	32947	61204	25113	29389	33022	22110	31981	30824
R2			32004	61379	26144	28979	33004	21966	31350	30621
R3			31723	60919	24476	30771	33252	21442	31897	29941
R1	20%	2	62884	117551	49573	58193	64838	44604	63797	63128
R2			62313	116306	49746	58281	64760	44562	63698	62749
R3			63846	119658	48030	59380	66371	44418	63326	62654
R1	50%	5	159725	288490	121113	149151	165534	110594	157074	152166
R2			159054	285749	121113	149060	165107	109862	156690	152431
R3			160341	289575	121732	146696	165722	109373	156756	151670
R1	100%	10	327598	587895	246290	309821	338850	226578	321244	311187
R2			326665	588900	245622	307684	338774	226542	321107	313442
R3			328629	583152	254880	306265	339567	225253	320199	310759
Slope			32888	58405	24870	30963	34050	22671	32102	31167
Intercept			-2351	575	-763	-3195	-2491	-1413	-1197	-955
correlation coefficient			0.999	0.999	0.999	0.999	0.999	0.999	0.999	0.999

 

 

Figure 1. Linear regression curve of benazepril  its impurities

 

Precision

The precision test is carried out with six homogenous solution of benazepril hydrochloride test item and the content of Benazepril hydrochloride and their impurities were calculated. The results are mentioned in Table 2.

 

Table 2. Precision of Benazepril hydrochloride and Impurities

 

Accuracy:

Preparation of Test solutions:

Prepare a solution containing all the impurities at a concentration of 100 µg/ml each (10 mg/100 ml; solution from the linearity test can be used). Transfer respectively 0.5 ml, 1 ml and 1.5 ml of this solution to three different 100 ml volumetric flasks, containing 100 mg of benazepril hydrochloride each one. The dilutions have to be carried out for each solution of the linearity test (total: 3x3 test solutions). The representative chromatogram showed in Figure 2 and results were presented in Table 3.

 

Table 3. Recovery results of benazepril and its impurities

 

 

Figure. 2. Representative chromatogram of 100 % fortification level of impurities

 

LOD and LOQ

The LOD and LOQ are established successfully for each impurity in benazepril and its impurities based on Signal-to-noise ratio method^{7, 8, 9}. The results were presented in Table 4.

 

Table 4. Limit of quantification and Limit of detection results of benazepril impurities

 

Calculations:

The Benazepril Hydrochloride impurities assay is determined by comparison of peaks areas with the following formula:

 

where:

At: Peak area of impurity obtained by test solution

Ar: Peak area of Benazepril obtained by Standard solution

C: Benazepril concentration in Standard solution (mg/mL)

D: sample dilution (mL)

W sample: sample weight in test solution (mg)

PS: Purity of reference standard

Rf: response factor of impurity

PS: Purity of reference standard

Fc: Response Factor of Impurity

 

% Recovery	=	Recovered Concentration	×	100
		Fortified Concentration

 

CONCLUSIONS:

The method developed for quantitative determination of benazepril Hydrochloride and its impurities is rapid, precise, accurate and selective. The method was completely validated showing satisfactory data for all method - validated parameters tested. The mobile phase composition water showed good separation and resolution. Satisfactory validation parameters such as linearity, precision, Accuracy, LOD and LOQ were established by following ICH guidelines¹⁰. Therefore, the proposed analytical procedure could be useful for regular monitoring, pharma manufacturing labs and research scholars.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Benerjee patrudu, Gitam University, Hyderabad for providing the gift sample of benazepril and providing necessary facilities to carry out the research work with keen interest and help.

 

REFERENCES:

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10.     International Conference on Harmonisation, Validation of Analytical Procedures. ICH Q2B. 1996.

 

 

 

Received on 19.05.2017          Accepted on 28.07.2017        

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