A Novel RP-HPLC Method for Determination of Lisinopril (LP) in Pure and Pharmaceutical Formulation

 

Manju Latha. Y.B¹*, Gowri Sankar. D².

¹Sri Vasavi Institute of Pharmaceutical Sciences, Tadepalligudem-534 101. (A.P), India

²University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, (A.P), India

*Corresponding Author E-mail: blessythall@gamil.com

A simple, rapid and accurate and stability indicating RP-HPLC method was developed for the determination of LP in pure and tablet forms. The method showed a linear response for concentrations in the range of 20-100 μg/mL using methanol: Phosphate Buffer solution in the ratio (50:5 0) as the mobile phase with detection at 215 nm and a flow rate of 1.0 mL/min and retention time 2.611 min. The value of correlation coefficient, slope and intercept were, 0.999, 67074.3and18126.355, respectively. The method was validated for precision, recovery, ruggedness and robustness. The drug undergoes degradation under acidic, basic, peroxide and thermal degradation conditions. All the peaks of degraded product were resolved from the active pharmaceutical ingredient with significantly different retention time. As the method could effectively separate the drug from its degradation product, it can be employed as a novel stability indicating one.

 

 

 

INTRODUCTION:

Lisinopril is a drug of the angiotensin converting enzyme (ACE) inhibitor class primarily used in treatment of hypertension, congestive heart failure, and heart attacks and also in preventing renal and retinal complications of diabetes. Its indications, contraindications and side effects are as those for all ACE inhibitors. Lisinopril is chemically described as (S)-1-[N2-(1-carboxy-3-phenylpropyl)-L-lysyl]-L-proline dihydrate¹ (Figure 1).

 

A few spectroscopic^2,3, HPLC^4-6 and LC-MS⁷ methods were reported earlier for the determination of lisinopril in bulk and pharmaceutical dosage forms. In the present study the authors report a rapid, sensitive, accurate and precise HPLC method for the estimation of lisinopril in bulk and in tablet dosage forms.

 

Figure 1. Chemical structure of lisinopril

 

MATERIALS AND METHODS:

Chromatographic Conditions:

The analysis of the drug was carried out on a Waters HPLC system equipped with a reverse phase ODS column (150 mmx4.6mm,5μm), a 2695 binary pump, a 20 μl injection loop and a 2487 dual absorbance detector and running on Waters Empower software.

 

Chemicals and Solvents:

The reference sample Lisinopril of was supplied by Sun Pharmaceutical Industries Ltd., Baroda. HPLC grade water and methanol were purchased from E. Merck (India) Ltd., Mumbai. Potassium dihydrogen phosphate and orthophosphoric acid of AR Grade were obtained from S.D. Fine Chemicals Ltd., Mumbai.

 

Preparation of Phosphate buffer:

Seven grams of KH₂PO₄ was weighed into a 1000 ml beaker, dissolved and diluted to 1000 ml with HPLC water. 2 ml of Triethylamine was added and pH adjusted to 3.0 with orthophosporic acid.

 

Preparation of Mobile phase and Diluents:

300 ml of the phosphate buffer was mixed with 700 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45μ filter under vacuum

 

Chromatographic Conditions:

Mobile phase consist Conditions of methanol-Phosphate buffer in the ratio of 50:50 at a flow rate of1 ml/min and pH of buffer was adjusted to 3.5. UV detection was performed at 215nm. The mobile phase was degassed by an ultrasonic water bath for 5 min. Filter through 0.45µ filter under vacuum filtration. The column was equilibrated for at least 30 min with the mobile phase flowing through the system. chromatogram showing the separation of the drug is given in Figure 2.

 

Preparation of the Lisinopril Standard and Sample Solution:

Standard Solution Preparation:

Accurately weigh and transfer 10mg of LP Working standard into a 10 mL volumetric flask add about 7 mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

 

Further pipette 0.4 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45µm filter.

 

Sample Solution Preparation:

Weigh 5 LP Tablets and calculate the average weight. Accurately weigh and transfer the sample equivalent to 10 mg of LP into a 10 ml volumetric flask. Add about 7 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45µm filter.

 

Further pipette 0.4 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45µm filter. 

 

Preparation of Calibration graph:

The linearity of response for LP assay method was determined by preparing and injecting solutions with concentrations of about 20, 40, 60, 80, 100 μg/ml of  LP. The relevant data are furnished in Table 1.The regression equation of this curve was computed. This regression equation was later used to estimate the amount of lisinopril in tablet dosage form.

 

S.No	Linearity Level	Concentration	Area
1	I	20µg/ml	285883
2	II	40µg/ml	659445
3	III	60µg/ml	1045527
4	IV	80µg/ml	1366878
5	V	100µg/ml	1744802
Correlation Coefficient			0.999

 

Validation of the Proposed Method:

After chromatographic method development and optimization it was validated. The validation of an analytical method verifies that the characteristics of the method satisfy the requirements of the application domain. The proposed method was validated according to ICH guidelines for linearity, precision, sensitivity, and recovery. For linearity studies, working standard solutions equivalent to 20 to 100 mg/ml of LP were prepared with the mobile phase.

 

Precision and Accuracy:

According to ICH, precision is the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions and may be considered at three levels: repeatability, intermediate precision and reproducibility. The intra-day and inter-day variations of the method were determined by using five replicate injections of 60 mg/ml and analyzed on the same day and different days, and the results are furnished in Table 2. The accuracy of the HPLC method was assessed by analyzing solutions of lisinopril at 50, 100 and 150% concentrated levels by the proposed method. The results are furnished in Table 3. The system suitability parameters are given in Table 4.

 

Table 2. Precision of the proposed HPLC method

Injection	Area
Injection-1	1033593
Injection-2	1037996
Injection-3	1039429
Injection-4	1040714
Injection-5	1040878
Average	1038522
Standard Deviation	2989.5
%RSD	0.29

Table 3. Accuracy studies

%Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	1403084	5.0	5.03	100.6%	99.6%
100%	2052260	10.0	9.89	98.9%
150%	3087076	15.0	14.8	99.2%

Table 4. System suitability parameters

Parameter	Result
Linearity, μg/mL	20-100
Correlation coefficient	0.99
Theoretical plates (N)	2423.0
Tailing factor	2423.0
LOD, μg/mL	0.099
LOQ, μg/mL	0.33

 

Estimation of lisinopril in tablet dosage form:

Two commercial brands of tablets were chosen for testing the suitability of the proposed method to estimate lisinopril in tablet formulation. 5 lisinopril tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 10 mg of lisinopril was transferred into a 10 mL volumetric flask and dissolved in 7mL of a 50:50 v/v mixture of phosphate buffer and methanol. The contents of the flask were sonicated for 15 min. The volume was made up with the diluent and the solution was filtered through a 0.45 μ membrane filter. This solution containing 60 μg/mL of lisinopril was injected into the column six times. The average peak area of the drug was computed from the chromatograms and the amount of the drug present in the tablet dosage form was calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table:5

 

Table 5. Assay and recovery studies:

Formulation	Label claim mg	Amount found mg	% Amount found
Formulation 1	5	4.995	99.9
Formulation 2	5	4.994	99.8

 

RESULTS AND DISCUSSION:

In the proposed method, the retention time of lisinopril was found to be 2.611 min. Quantification was linear in the concentration range of 20-100 μg/mL. The regression equation of the linearity plot of concentration of lisinopril over its peak area was found to be Y=18126.355+670743X (r2=0.999), where X is the concentration of lisinopril (μg/mL) and Y is the corresponding peak area. The number of theoretical plates calculated was 2423, which indicates efficient performance of the column. The limit of detection and limit of quantification were found to be 0.099 μg/mL and 0.33 μg/mL respectively, which indicate the sensitivity of the method. The use of phosphate buffer and methanol in the ratio of 50:50v/v resulted in peak with good shape and resolution. The high percentage of recovery indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulation did not interfere with the estimation of the drug by the proposed HPLC method.

 

CONCLUSION:

The proposed HPLC method is rapid, sensitive, precise and accurate for the determination of lisinopril and can be reliably adopted for routine quality control analysis of lisinopril in its tablet dosage form.

 

ACKNOWLEDGMENT:

The authors are thankful to M/s Sun Pharmaceutical Industries Ltd., Baroda for providing a reference sample of lisinopril.

 

REFERENCES:

1      www.drugs.com/lisinopril.html

2      Jamakhandi C M, Javali C, Disouza J I, Chougule U S and Mullani A K, Int J Pharm Pharm Sci., 2011, 3(2), 185-187.

3      Permender R, Sushila R, Shyama T and Vikash K, Int J Pharm Tech Res., 2010, 2(1), 556-562.

4      El-Emam A A, Hansen S H, Mohamed A M, El-Ashry S M and El-Sherbiny D T, J Pharm Biomed Anal., 2004, 34(1), 35-44.

5      Beasley C A, Shaw J, Zhao Z and Reed R A, J Pharm Biomed Anal., 2005, 37(3), 559-567.

6      Tzvetkova D, Obreshkova D and Pencheva I V, Acta Pharm Turc., 2005, 47, 179-187.

7      Zhou N, Liang Y, Chen B, Wang P, Chen X and Lu F, J Chromatogr Sci., 2008, 46, 848-853.

 

 

 

Received on 05.11.2014         Modified on 17.11.2014

Accepted on 01.12.2014         © AJRC All right reserved