Development and Validation of an RP-HPLC Analytical Method for Determination of Lisinopril in Full and Split Tablets

 

Khairi M. S. Fahelelbom¹*, Moawia M. M. Al-Tabakha², Nermin A. M. Eissa¹, Dana Emad Eddin Obaid¹, Sadik Sayed¹

¹Department of Pharmaceutical Sciences, College of Pharmacy, Al Ain University of Science and Technology, P.O. Box 64141 Al Ain, UAE

²Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Ajman University,

P.O. Box 346 Ajman, UAE

 

ABSTRACT:

Objective: The aim of this study is to develop and optimize a new an RP-HPLC method for the analysis of lisinopril from pure samples, full and split tablet dosage forms by investigating all relevant factors in order to obtain a simple, reproducible and sensitive technique for the quantitative determination of lisinopril. Method: A number of chromatographic factors have been tested such as the mobile phase composition, retention rate, column temperature and the effect of excipients using  C18 column as the stationary phase for the determination of lisinopril. While the tested mobile phase was a mixture of methanol, acetonitrile and phosphate buffer solution. Results: The retention time of lisinopril was found to be 2.6 min at the flow rate of 1.0 ml/min. Excellent linearity was found for the drug concentrations in the range 40-200 μg/mL (r²= 0.9993). The small RSD value (< 4%) obtained indicate that the method is quite precise. The high percentage of recovery was 100.174% (range: 98.7 to 102.4%) which complies with all of pharmacopeias with SD of 1.1. The absence of additional peaks in the chromatogram indicates non-interference of the commonly used excipients in the tablets and hence the method is specific. The small values of LOD (0.210) and LOQ (0.636 µg/mL) obtained by this method indicate the sensitivity of the method. Conclusion: The proposed RP-HPLC method is rapid, sensitive, precise and accurate for the determination of lisinopril. It can be reliably adopted for routine quality control analysis of lisinopril bulk and in its full and splitted tablet dosage forms

 

 

INTRODUCTION:

Lisinopril dihydrate an important oral antihypertensive agent belongs to the angiotensin converting enzyme inhibitors class (ACE), it has introduced into therapy in the early 1990s with daily doses of 10-80 mg.^[1]  It is chemically defined as 1-[N-[(s)-1-carboxy-3-phenylpropyl]-L-lysyl]-L-proline dihydrate, with the IUPAC name: (2S)-1-[(2S)-6- amino-2-[[(1S)-1-carboxy-3-phenylpropyl]amino] hexanoyl]pyrrolidine-2-carboxylic acid (Fig. 1).^[2]

 

Figure 1. Chemical structure of lisinopril dihydrate.

 

Several analytical techniques have been reported for the analysis of lisinopril including spectrophotometric methods^[3-5], spectrofluorometric^[6-7] and HPLC analytical techniques and are considered as the methods of choice by many researchers^[8-13]. The development and validation of RP-HPLC method has been reported for the analysis of many pharmaceutical formulations^[14-18].

The analysis of lisinopril by many Pharmacopoeias^[19-21] recommended the use of hexanesulfonate and phosphate buffer (pH 2.0 to 5.0) in the mobile phase and a column temperature of 40°C up to 60°C, to avoid lisinopril peak splitting and tailing. Different HPLC methods that make use of the above-mentioned critical parameters: mobile phases in a wide pH region, from 2 to 7.5, with or without the use of ion-pair reagents and column temperatures from ambient up to 60°C have been published. The use of extreme pH in the range 2.5 – 8.0 and elevated temperature can decrease the HPLC column lifetime expectancy, while the use of ion-pair reagents such as hexanesulfonate limit the subsequent use of the column^[22].

 

Splitting of tablets has been applied to many drugs including lisinopril tablets to adjust the dose and reduce the cost of the dosage form; in our previous study we investigated the uniformity of the lisinopril split tablets by applying two different methods for tablet splitting, the drug contents were also determined using spectrophotometric analysis.^[23]

 

The main objective of this work was to investigate the development and validation of a simple, sensitive, precise, and accurate reverse phase HPLC method for the analysis of lisinopril in pure form and in full and split tablet dosage forms by obtaining an efficient separation and quantitative analysis of lisinopril while avoiding the problem that can affect the performance of the HPLC column.

 

EXPERIMENTAL:

Chemicals and Reagents:

Lisinopril standard was obtained as a gift sample from Neopharma Pharmaceutical co. UAE respectively. Water, acetonitrile, methanol, phosphate buffer pH 7 and orthophosphoric acid of HPLC grade were obtained from Carlo Erba Reagent (Val de Reuil, France).

 

Instrumentation:

A tablet splitter has been used for splitting of lisinopril tablets was purchased from a local pharmacy (Fig. 2). The pH meter (Metteler Toledo, Switzerland) was used for buffer pH measurements.

 

Figure 2. Tablet splitting device.

 

 

Shimadzu HPLC systems LC 20 series with UV/DAD detector (SPD-20A, Shimadzu, Kyoto, Japan) and isocratic/gradient pump, injector with a 20μl loop was used for the injection of the samples. The column C18 (Restek, Bellefonte, PA, USA) with particle size 5µm, length of 250mm and internal diameter of 4.6mm was the stationary phase employed in this study. The applied column temperature was 45°C.

 

The chromatographic data were analyzed using Lab Solutions software, Version 5.51 (Shimadzu Corporation, Kyoto, Japan) running on Windows 7 Professional (Microsoft Corporation, Redmond, WA, USA).

 

Chromatographic Condition:

The mobile phase was prepared by mixing two phase as follows; mobile phase A contains methanol: phosphate buffer at pH 3 with a ratio of 70:30, v/v and mobile phase B containing methanol: acetonitrile: phosphate buffer pH 3 in ratio of 70:10:20 v/v. Ortho-phosphoric acid was applied for pH adjustment of the buffer. The final mobile phase then filtered by using 0.22µm membrane filters and vacuum pump (Rocker 300) and then degassed for 5 min. The selected flow rate was 1.0 ml/min. The drug showed good absorbance at 215nm, which was used as wavelength for further analysis throughout the study.

 

Mobile phase:

Methanol: acetonitrile: phosphate buffer of pH 3 were prepared in 70:20:10 v/v ratios for preparing mobile phase then was filtered using 0.22 mm membrane filters and was degassed for 5 min before use.

 

Diluent:

A mixture of 800 volumes of water and 200 volumes of methanol was prepared.

 

Standard preparation:

An accurately weighed amount of 20.0mg of lisinopril was transferred into 100mL volumetric flask followed by the addition of. 75ml of the diluent to the lisinopril solution and sonicated by using (Bransonic CPX3800) for 5 min, the resulted dissolved solution was completed to the mark using the mentioned diluent.

 

Calibration curve Preparation:

To prepare the calibration plot, several serial volumes were taken from the standard stock solution and pipetted into a number of 10ml volumetric flasks. The volume was completed to the mark with the mobile phase in order to obtain a set of solutions having the concentration, ranging from 40-200µg/ml of Lisinopril. The solutions were injected, and the curve was generated to represent the concentration against the peak area.

 

Assay Preparation:

20 tablets were accurately weighed, crushed by using mortar and pestle, a powdered tablet containing an equivalent to 20mg of lisinopril was a transferred into 100ml volumetric flask. Diluting vehicle (75ml) was initially added and sonication was carried out for 10 min. The volume was then adjusted up to 100ml and filtered through 0.45μ polypropylene membrane filter. The first 3-4ml of the filtrate was discarded. The prepared sample and standard solution were transferred into separate vials for chromatographic run.

 

Method Validation:

The proposed RP-HPLC method was validated using the ICH guidelines for the validation of analytical procedures Q2 (R1)^[22], the following parameters were considered during the performance of the validation process: linearity, specificity, system suitability, precision, accuracy and ruggedness.

 

Limit of Detection (LOD) and Limit of quantitation (QL):

The limit of detection (LOD) represents usually the smallest concentration of the analyte which can be detected, while the quantification limit (LOQ) is considered as the lowest amount of analyte in the sample that can be quantitatively determined with proper accuracy and precision. The following equations were used for the calculation of the LOD and LOQ values from the calibration curve:

 

LOD = 3.3 × σ / S   and   LOQ = 10 × σ / S

 

Where σ is the standard deviation obtained from the linear line while S represents the slope of the curve.

 

System suitability:

In order to ensure the validity of the proposed RP-HPLC method for lisinopril determination, it is important to evaluate the system suitability factors for the proposed RP-HPLC method. The following factors were tested and evaluated; the retention time, talking factor, number of theoretical plates and the resolution.

 

Precision and intermediate precision:

The proposed method precision was evaluated repeatability as follows; samples of six solutions containing 40µg/mL of lisinopril were injected and the corresponding area for each solution were recorded and analyzed in one day (for the intra-day precision) and in two consecutive days (for the inter-day precision).

 

Accuracy:

Determination of the accuracy for the proposed RP-HPLC is considered as one of the important parameters for the validation process; the standard addition method was applied for the determination of the method accuracy. A fixed amount of standard drug was added to the placebo. The percent recovery was calculated by adding three levels of concentrations: 50%, 100% and 150%. The recovery results were obtained from the average of three reading, the percent recovery and the mean % recovery were calculated.

 

Ruggedness:

In order to study the proposed method ruggedness a slight change in certain parameters of the proposed method was performed. Robustness testing for the analysis of lisinopril was conducted by making deliberate changes in the: temperature of the column oven (±5°C), mobile phase flow rate (±0.2ml/min) and percentage of methanol in the mobile phase (±5%).

 

Results and Discussions:

Many factors were investigated in order to develop the proposed HPLC method, including the choice of suitable mobile phase for the RP-HPLC method, the appropriate wavelength for maximum absorption in lisinopril determination and the effective column as stationary phase; in this regard and in order to get sharp symmetrical peaks with minimum tailing and ideal base line separations of the lisinopril, several experiments were carried out to find the suitable mobile phase, by changing  the composition of different solvents and altering their flow rates. For this purpose many solvents were used as water, methanol and acetonitrile in the presence or absence of buffer solutions with different composition ratios have been tested to choice the suitable mobile phases. Methanol: acetonitrile: phosphate buffer of pH 3 were prepared in 70:20:10 v/v ratios, was found to be the most appropriate mobile phase, since the obtained peaks of chromatographic analysis were  well defined, better resolved and free from tailing. To select the optimum mobile phase media, pH range of 2.5 to 3.5 were tested; perfect performance was achieved at pH 3.0. Information about the calculated lisinopril pKa indicate four values (two acidic and two basic): 1.62, 3.66, 6.07 and 10.33.^[24]  It was suggested to avoid the pH close to the analyses pKa,^[25]  this explains the better performance of our method of analysis at pH 3.0 compared to pH 3.5.

 

Short time for the analysis <10min is recommended for the routine analysis and this was evident with all lisinopril analyzed samples which were totally achieved within 5 min.

 

Varying the column temperature by ±5°C does not significantly change the system suitability parameters which indicates that the method used is robust. While retention time decreased with the increased temperature, the peak symmetry was best with the prescribed column temperature of 45°C, similar with the findings of other researchers.^[25] Splitting peak for lisinopril can be observed when lisinopril is routinely analyzed with a column at room temperature.^[26] As shown in figures 4A-4D.

These achieved satisfactory results were obtained using 1mL/min as flow rate and 215nm as the maximum wavelength of absorption.

 

Figure 3A. Chromatograms of linearity for the calibration curve

 

Figure 3B. Calibration Curve of Lisinopril using HPLC

 

Table 1. Data analysis of Lisinopril calibration curve

 

Linearity of the method was confirmed by preparing lisinopril standard curve for the range of 40-200 g/mL. Table 1 Summarizes of the linearity results which demonstrate an excellent correlation between the peak area and the drug concentrations as represented by the value of the coefficient of determination r²= 0.9993, the linearity were clearly indicated as shown in Fig.3a and 3b.

 

The characterization of calibration plot values is shown in Table 1. LOD and LOQ values for lisinopril were found to be 0.210 and 0.636 µg/mL respectively, which demonstrates high sensitivity of the proposed method.

 

The obtained chromatograms show no interference from the excipients and additives present in the tablet formulation, good correlations were obtained as well between the retention times of sample and standard solutions. Figure 3C shows no peaks at the retention time of Lisinopril for blank and placebo.

 

The tested critical parameters including the retention time, talking factor, number of theoretical plates and the resolution were analyzed and found be within the acceptance criteria; in all of the tested samples the tailing factors were less than 2.00 and the number of theoretical plates were higher than 2000 plates, Table 2 shows the summary of the system suitability parameter.

 

Table 2. Precision by repeatability of the proposed HPLC method

 

 

Figure 4A. Lisinopril Reference Standard HPLC Chromatogram

 

Figure 4B. Lisinopril Full Tablet HPLC Chromatogram

 

Figure 4C. Lisinopril First Half Tablet HPLC Chromatogram

 

Figure 4D. Lisinopril Second Half Tablet HPLC Chromatogram

 

Table 3. Percent recovery of Lisinopril

 

Summary of the obtained results for Intra-day and inter-day precision for the proposed HPLC method are given in Table 3, the obtained results indicate that the method is quite precise.

 

The results of method accuracy were shown in Table 4. The percent recovery of Lisinopril was 99.4 to 100.579 and the mean recovery was 100.417 which demonstrate the high accuracy of the proposed method compared to the pharmacopeial methods applied for lisinopril analysis.

 

Therefore, our developed method was validated by following the ICH guidelines for the validation of analytical procedures Q2 (R1). ^[27]

 

Conclusion:

The proposed chromatographic RP-HPLC method is considered as sensitive, accurate and precise for the lisinopril determination and can be easily applied for the quantitative determination and quality control analysis of lisinopril samples in its pure form and in full and splitted tablet dosage forms

 

ACKNOWLEDGMENTS:

The authors are thankful to the Neopharma Pharmaceutical Co., Abu Dhabi, UAE for providing the lisinopril reference standard.

 

CONFLICTS OF INTEREST:

The authors of this article declare no conflict of interest

 

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Accepted on 26.11.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6):2647-2652.