INTRODUCTION:

Benazepril Hydrochloride^1,2:

It is chemically 2-[(3S)-3-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl] amino} - 2-oxo-2, 3, 4, 5-tetrahydro-1H-1-benzazepin-1-yl] acetic acid (Fig.1). It is an ACE inhibitor, used to inhibit blood clots in coronary artery disease, peripheral vascular disease and cerebro vascular disease. It acts on the rennin 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.

.HCl

Fig.1 Chemical structure of Benazepril Hydrochloride

Benazepril hydrochloride appears as a white or almost white crystalline powder having 461.0 gm. it is practically insoluble in cyclohexane, freely soluble in anhydrous ethanol and slightly soluble in water and very slightly soluble in ethyl acetate. A simple and precise method was developed and validated by using solvents like methanol and water respectively^[3,4].

Instrumentation:

Double beam UV- visible spectrophotometer, Make: Jasco spectrophotometer, model Jasco V630 connected to a computer loaded with spectra manager software was used for all the spectrophotometric measurements in all proposed spectrophotometric methods. It consists of pair of 10 mm matched quartz cells was used for experiments. The absorption spectra of reference and test solution were carried out in a 1 cm quartz cell over the range 200-400 nm.

MATERIALS AND METHODS:⁵

Preparation of stock solution of Benazepril Hydrochloride:

Standard Benazepril Hydrochloride of 10 mg was accurately weighed and transferred into a 100 ml volumetric flask. Add small quantity of methanol in volumetric flask and dissolve the drug into it and then adjust the volume with water upto 100 ml this solution form 100 ppm of concentration and then prepared further dilutions.

Selection of analytical concentration ranges:

From the standard stock solutions of Benazepril Hydrochloride (100 µg/ml), appropriate aliquots of 2 ppm, 4 ppm, 6 ppm upto 24ppm.

Determination of λ max:

10ug/ml solution of Benazepril Hydrochloride was prepared and scanned in UV range of 200-400nm and spectrum was obtained. The λmax was found to be at 237nm wavelength where absorbance was maximum at this wavelength.

Validation parameters for Benazepril Hydrochloride^6,7:

1.1 Accuracy:

Accuracy is the closeness of the results obtained by the method to the true value. This study can be done with the help of preparing three concentrations of 11, 21 and 31 ppm each respectively.

Precision:

The precision of an analytical method is the degree of agreement among individual test results, when the method is applied repeatedly to multiple sampling of homogeneous samples. It provides an indication of random error results and is expressed as relative standard deviations [% RSD].

Robustness:

The solution were prepared and analyzed with change in the analytical conditions like change in wavelength, change in solvent etc. This can be studied with the help of using minimum six concentrations. Absorbance can be calculated using UV spectrophotometer.

Limit of detection [LOD] and limit of Quantitation [LOQ]:

The sensitivity of the proposed method for the measurement of Benazepril Hydrochloride was estimated in terms of limit of detection [LOD] and limit of Quantitation [LOQ]. The LOD and LOQ were calculated by using the slope and SD of response [intercept]. The mean slope value and the SD of response were obtained from the calibration curve. The LOD and LOQ calculations were done and reported.

Linearity, Range and Calibration:

The method can be validated by using ICH guidelines. The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in the sample within the range. The range of the analytical method is the interval between the upper and the lower levels that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity.

EXPERIMENTATION:

Table1: Calibration of Benazepril Hydrochloride-

Sr. no.	Concentration (ppm)	Absorbance
1	2	0.1118
2	4	0.1534
3	6	0.1760
4	8	0.2346
5	10	0.2641
6	12	0.2945
7	14	0.3221
8	16	0.3511
9	18	0.4363
10	20	0.5061
11	22	0.6143

Table2: Accuracy-

Sample

Amount of Standard

Drug Added ( μg/ml)

Absorbance

Absorptivity

[x-x*]²

Standard Deviation

% Relative Standard Deviation

0.2842

0.2711

0.2791

2.583

2.464

2.537

0.0030

0.0040

0.000081

0.038

0.015

100

0.4712

0.4704

0.4723

2.243

2.24

2.249

0.000001

0.000016

0.000025

0.0045

0.002

120

0.6552

0.6531

0.6496

2.113

2.106

2.095

0.000081

0.000004

0.000081

0.0091

0.0043

Table3: Precision-

Concentration %	Absorbance Mean	Absorptivity	*[x-x]²**	Standard Deviation	% Relative Standard Deviation
Intraday Precision (n=6)
25	0.5811	2.324	0.000027	0.0384	0.01658
25	0.5623	2.249	0.0049
25	0.5763	2.305	0.00019
25	0.5891	2.356	0.00138
25	0.5834	2.333	0.000207
25	0.5866	2.346	0.00739
Inter-day Precision(n=6)
25	0.5952	2.38	0.3958	0.03065	1.26
25	0.5887	2.35	0.3992
25	0.5903	2.36	0.3960
25	0.5893	2.35	0.3952
25	0.5876	2.35	0.3960
25	0.5861	2.34	0.3919

Table 4: Change in wavelength:

Sr. No.	Concentration [ppm]	Absorbance		Difference	*[d-d]²**	Standard deviation	% Relative Standard Deviation
		237nm	235nm
1.	10	0.2491	0.2351	0.0016	0.0000025
2.	12	0.3258	0.3120	0.0019	0.000036
3.	14	0.4096	0.3826	0.0113	0.0001276	0.00864	0.55
4.	16	0.6345	0.6327	0.0139	0.0001932
5.	18	0.7345	0.7191	0.0003	0.0000009
6.	20	0.8963	0.8738	0.0068	0.0000462

Limit of Detection:

LOD = 3.3 * Standard deviation

Slope

= 5.5

· Limit of Quantitation:

LOQ = 10 * Standard deviation

Slope

= 16.3

RESULT AND DISCUSSION:

The method can be validated using ICH guidelines in order to determine linearity, range, accuracy, precision and robustness of the method.

The standard stock solution of Benazepril Hydrochloride was prepared and this can be scanned from 200-400 nm and absorption spectra were recorded at 237 nm wavelength in UV spectrophotometer using methanol as a solvent.

Linearity:

The absorbance of the solutions of Benazepril Hydrochloride was determined at a wavelength 237 nm. The correlation coefficient was found to be 0.961 and the regression equation was found to be Y= 0.047x-0.017 and this can be shown in table no. 1.

Precision:

Precision can be done for all type of drugs and the limit of RSD value was found to be not more than 2. Here RSD was found to be 0.01658 and 1.26 and this can be shown in table no. 3.

Accuracy:

Accuracy can be determined by performing recovery studies at 3 levels that is taking 3 different concentrations which has replicates and this can be studies using absorbance.

Limit of detection and limit of Quantitation:

The parameters LOD and LOQ were determined on the basis of response and slope the regression equation LOD and LOQ values are 5.5 and 16.6.

CONCLUSION:

According to ICH guidelines slope, intercept and correlation coefficient can be calculated using calibration curve were shown in fig.1.the results show that the methods are reasonable precise. The developed UV spectrophotometric method was found to be simple, economic, easy, accurate, precise, reproducible and highly sensitive and can be used for routine estimation of Benazepril Hydrochloride in bulk.

Table5: Summary of UV spectrophotometric method validation:

PARAMETERS	RESULTS
χ max	237nm
Range	2-22ug/ml
Correlation coefficient	0.961
Regression equation	Y= 0.047x +0.017
Slope [m]	0.047
Intercept [c]	0.017
Precision [%RSD]	0.0165-1.26
LOD	5.5
LOQ	16.6
Accuracy [%RSD]	0.002 for 100%

ACKNOWLEDGEMENTS:

The authors are grateful to R. G. Sapkal College of Pharmacy for providing necessary research facilities to carry out the research work and to Arti Drugs Ltd, India for providing the gift sample of the drug.

REFERENCES:

1. John H. Block, John M. Beale, Wilson and Gisvold’s text book of organic medicinal and pharmaceutical chemistry, 11^th edition, Lippincott Williams and Wilkins, page no. 647.

2. Clarke’s analysis of drugs and poisons, 3^rd edition, Published by the Pharmaceutical Press; volume 2. Page no. 675.

3. Remington the science and practice of pharmacy, 21^st edition volume 2. Lippincott Williams and Wilkins. Page no. 1355.

4. The Indian pharmacopoeia 2014, Government of India, Ministry of Health and Family Welfare, The controller of publications, Ghaziabad; 7 ^th edition, Vol 2, p no-1141.

5. Kavathia A, Mishra M. “Development and validation of RP HPLC and UV spectrophotometric methods for rapid simultaneous estimation of amlodipine and Benazepril in pure and fixed dose combination”, Arabian journal of chemistry, 2013, p. no 1-8.

6. Madhavi L, Shireesha M. “Spectrophotometric estimation of valsartan and benazepril Hydrochloride in pure and pharmaceutical formulations”, International journal of chemtech research, volume- 3, Issue-4, 2011, p no-1630-1834.

7. Stanisz B, Paszun S, Lesniak M. “Validation of UV derivative spectrophotometric method for determination of benazepril Hydrochloride in tablet and evaluation of its stability.” Research Gate, Acta Poloniac-Drug Research, Volume-66, issue-4, 2009, p. no-343-349.

Received on 11.05.2016 Modified on 17.05.2016

Asian J. Research Chem. 2016; 9(8): 369-372.

DOI: 10.5958/0974-4150.2016.00056.0