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\def\citeauthor{~Zahid~Zaheer,
              ~Sarfaraz~Khan,
              ~Mohammad~Sadeque,
              ~Hundekari~G.~I.,
              ~Rana~Zainuddin}
\def\citetitle{\textbf{Development and validation for determination of lisinopril dihydrate in bulk drug and formulation using RP-HPLC method}}
\def\copyrightstmt{This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial- ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.}
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\begin{document}


\def\RunningAuthor{Sarfaraz Khan et al.}
\firstPage{14}
\articleType{Original Article}
\receivedDate{04 May 2018}
\acceptedDate{17 Jun 2018}
\revisedDate{09 Jun 2018}
\journalVolume{3}
\journalIssue{2}
\journalDoi{jibps.v3i2.1426}
\copyrightYear{2018}
\def\authorCount{5}
\def\affCount{1}

\def\journalTitle{Journal of Inventions in Biomedical and Pharmaceutical Sciences}

\title{\textbf{Development and validation for determination of lisinopril dihydrate in bulk drug and formulation using RP-HPLC method}}
\author{Zahid~Zaheer,  
            Sarfaraz~Khan\textsuperscript{*},  
            Mohammad~Sadeque,  
            Hundekari~G.~I., 
            Rana~Zainuddin~\\[5pt]{Department of Quality Assurance, Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad {\textendash} 431001. Maharashtra, India}}

\begin{abstract}
A simple, reproducible and efficient reverse phase high performance liquid chromatographic method was developed for Lisinopril in bulk drug and formulation. A column having 150 \ensuremath{\times} 4.6 mm in isocratic mode with mobile phase containing acetonitrile: phosphate buffer (70:30; adjusted to pH 3.0) was used. The flow rate was 0.8 ml/min and effluent was monitored at 216 nm. The retention time (min) and linearity range (\ensuremath{\mu }g/ml) for Lisinopril was (1.510) and (10-35). The developed method was found to be accurate, precise and selective for determination of Lisinopril in bulk and formulation.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Lisinopril,\newline RP-HPLC,\newline Validation
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright{\Large$^\ast$}Corresponding Author\par Name:\ Sarfaraz~Khan~\\ Phone:\ 
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        \makeatother
        
    
\section{Introduction}
Lisinopril (Figure~\ref{f-4815fb52c8a6}) is an orally bioavailable, long-acting angiotensin-converting enzyme (ACE) inhibitor with antihypertensive activity. Lisinopril, a synthetic peptide derivative, specifically and competitively inhibits ACE, which results in a decrease in the production of the potent vasoconstrictor angiotensin II and, so, diminished vasopressor activity. In addition, angiotensin II-stimulated aldosterone secretion by the adrenal cortex is decreased which results in a decrease in sodium and water retention and an increase in serum potassium \unskip~\cite{1281324:23775456,1281324:23775467,1281324:23775464}. Literature survey reveals the availability of several methods by using various Mobile phases but no method was available on this Mobile phase that is acetonitrile: phosphate buffer (70:30; adjusted to pH 3.0) which was a unique method with better results \unskip~\cite{1281324:23775466,1281324:23775463,1281324:23775459}.
    
\section{Materials and Methods}
\textbf{Chemicals and reagents}

The reference sample of Lisinopril was supplied by wockhardt Pharmaceutical Industries Ltd., Aurangabad. HPLC grade water and acetonitrile were purchased from Merck (India) Ltd., Mumbai. Potassium dihydrogen phosphate and orthophosphoric acid of AR Grade were obtained from, Research Lab (India) Ltd \unskip~\cite{1281324:23775455,1281324:23775462}.


\begin{table*}[!htbp]
\caption{\boldmath {Calibration data of lisinopril} }
\label{tw-8f90f7901046}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5353\linewidth-2\tabcolsep}p{\dimexpr.4647\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Concentration(\ensuremath{\mu }g/ml) & Mean peak area (n=6)\\
\tblmidrule 
10 &
  742315\\
15 &
  1419822\\
20 &
  2121436\\
25 &
  2810895\\
30 &
  3531268\\
35 &
  4265201\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Precision studies for lisinopril} }
\label{tw-52b753ed6251}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5852\linewidth-2\tabcolsep}p{\dimexpr.2035\linewidth-2\tabcolsep}p{\dimexpr.2113\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Concentration of lisinopril (10\ensuremath{\mu }g/ml)  & \multicolumn{2}{p{\dimexpr(.41480000000000004\linewidth-2\tabcolsep)}}{\cAlignHack Peak area }\\
\rowcolor{kwdboxcolor} & Intra-day & Inter-day\\
\tblmidrule 
Injection-1  &
  1021546 &
  1052530\\
Injection-2  &
  1020125 &
  1075203\\
Injection-3  &
   1030560 &
  1061251\\
Injection-4  &
  1019832 &
  1081789\\
Injection-5  &
  1047695 &
  1078415\\
Injection-6  &
  1021346 &
  1084812\\
Average  &
  1026851 &
  1072333\\
Standard Deviation  &
  10965.24 &
  12691.25\\
\% RSD  &
  1.067852 &
  1.183517\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Accuracy studies for lisinopril} }
\label{tw-422c57e11720}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2249\linewidth-2\tabcolsep}p{\dimexpr.1884\linewidth-2\tabcolsep}p{\dimexpr.1867\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\% Concentration & Amount added (mg) & Amount found (mg) & \% Recovery & \% Mean recovery\\
\tblmidrule 
80 &
  18 &
  17.95 &
  99.72 &
  \multicolumn{1}{p{\dimexpr(.20\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{99.64}}\\
100 &
  20 &
  19.80 &
  99.00 &
  \\
120 &
  22 &
  22.05 &
  100.22 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {System suitability parameters} }
\label{tw-45f3add7bc6e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.6307\linewidth-2\tabcolsep}p{\dimexpr.3693\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Parameter & Result of lisinopril\\
\tblmidrule 
Linearity (\ensuremath{\mu }g/ml) &
  10-35\\
Correlation coefficient &
  0.999\\
Tailing factor &
  1.6\\
LOD (\ensuremath{\mu }g /ml) &
  0.010024\\
LOQ (\ensuremath{\mu }g /ml) &
  0.030374\\
Specificity &
  Specific\\
Precision &
  Intra-day 1.067852 \mbox{}\protect\newline Inter-day 1.183517\\
Accuracy &
  99.64\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/790cdd88-9282-4942-8372-b15fb4c6db4d-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/790cdd88-9282-4942-8372-b15fb4c6db4d-upicture1.png}{\includegraphics{images/790cdd88-9282-4942-8372-b15fb4c6db4d-upicture1.png}}{}
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\caption{\boldmath {Structure of Lisinopril dihydrate}}
\label{f-4815fb52c8a6}
\end{figure}
\egroup

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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/5505e14a-e5a5-41a7-b6d9-6c258412dd52-upicture2.png}{\includegraphics{images/5505e14a-e5a5-41a7-b6d9-6c258412dd52-upicture2.png}}{}
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\caption{\boldmath {Linearity peak of lisinopril}}
\label{f-c2ea35e09a77}
\end{figure}
\egroup

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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/78e1f8e3-81f3-4477-ab9a-15e9b088050e-upicture3.png}{\includegraphics{images/78e1f8e3-81f3-4477-ab9a-15e9b088050e-upicture3.png}}{}
\makeatother 
\caption{\boldmath {From the instrument linearity peak of lisinopril}}
\label{f-478c5227338b}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/20175c50-30ab-4fac-96a3-073dfd06aa16-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/20175c50-30ab-4fac-96a3-073dfd06aa16-upicture4.png}{\includegraphics{images/20175c50-30ab-4fac-96a3-073dfd06aa16-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Linearity curve of lisinopril}}
\label{f-c2e5c9374905}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/bf454014-84ab-4caf-85c6-5755e41683d2-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/bf454014-84ab-4caf-85c6-5755e41683d2-upicture5.png}{\includegraphics{images/bf454014-84ab-4caf-85c6-5755e41683d2-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatogram of lisinopril}}
\label{f-c6b376fa7f28}
\end{figure}
\egroup
\textbf{Chromatographic conditions}

The analysis of the drug was carried out on a Waters HPLC system equipped with a reverse phase Xterra C18 column (150mmx4.6mm; 5\ensuremath{\mu }m), a 2695 binary pump, a 20\ensuremath{\mu }l injection loop and a 2487 dual absorbance detector and running on Waters Empower software. The UV spectrum of the drugs was taken using a shimadzu 1800 UV/VIS double beam spectrophotometer.

\textbf{Preparation of phosphate buffer (pH 3.0)}

7 gm of KH\ensuremath{_{2}}PO\ensuremath{_{4}} was weighed into a 1000 ml beaker, dissolved and diluted to 1000 ml with HPLC water and pH adjusted to 3.0 with orthophosporic acid.

\textbf{Preparation of mobile phase and diluents}

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

\textbf{Procedure}

A mixture of buffer and acetonitrile in the ratio of 30:70 v/v was found to be the most suitable mobile phase for lisinopril. The solvent mixture was filtered through a 0.45 \ensuremath{\mu } membrane filter and sonicated before use. It was pumped through the column at a flow rate of 0.8 ml/min. The column was maintained at ambient temperature. The pump pressure was set at 900 psi. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. The detection of the drug was monitored at 216 nm. The run time was set at 9 min. Under these optimized chromatographic conditions the retention time obtained for the drugs lisinopril was 1.510 min \unskip~\cite{1281324:23775460,1281324:23775457}. 

\textbf{Calibration plot}

About 100 mg of lisinopril was weighed accurately, transferred into a 100 ml volumetric flask and dissolved in 50 ml of a 30:70 v/v mixture of phosphate buffer and acetonitrile. The solution was sonicated for 15 min and the volume made up to the mark with a further quantity of the solvent to get a 1000 \ensuremath{\mu }g/ml solution. From this, a working standard solution of the drugs (10\ensuremath{\mu }g/ml for lisinopril) was prepared by diluting the above solution to 10 ml in a volumetric flask. Further dilutions ranging from 10-35 \ensuremath{\mu }g/ml for lisinopril was prepared from the solution in 10ml volumetric flasks using the above diluents. 20\ensuremath{\mu }l of each dilution was injected six times into the column at a flow rate of 0.8 ml/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The calibration curve constructed by plotting concentration of the drug against peak area was found to be linear in the concentration range of 10-35\ensuremath{\mu }g/ml for lisinopril. The relevant data are furnished in Table~\ref{tw-8f90f7901046} and Typical Chromatogram was shown inFigures~\ref{f-c2ea35e09a77}, \ref{f-478c5227338b} and~\ref{f-c2e5c9374905}. The regression equations of this curves was computed. 

\textbf{Validation of the proposed method}

The specificity, linearity, precision, accuracy, limit of detection, limit of quantification, robustness and system suitability parameters were studied systematically to validate the proposed HPLC method for the determination of lisinopril. Solution containing 10\ensuremath{\mu }g/ml for lisinopril was subjected to the proposed HPLC analysis to check intra-day and inter-day variation of the method and the results are furnished in Table~\ref{tw-52b753ed6251}. The accuracy of the HPLC method was assessed by analyzing solutions of lisinopril at 80\%, 100\% and 120\% concentrated levels by the proposed method. The results are furnished in Table~\ref{tw-422c57e11720}. The system suitability parameters are given in Table~\ref{tw-45f3add7bc6e}.

\textbf{Linearity}

\textbf{LOD and LOQ studies of lisinopril}

The limit of detection and limit of quantification for lisinopril was found to be 0.0101 and 0.0303 respectively, which indicate the sensitivity of the method.

\textbf{Specificity studies of lisinopril}

The specificity of the method was ascertained by analyzing standard drug and sample. The spot for lisinopril in sample was confirmed by comparing the Rf and spectra of the spots with that of standards indicating no interference of any another peak of mobile phase, impurity.

\textbf{Precision studies for lisinopril}

Precision of the method was performed by intra-day and inter-day studies. The \% RSD values obtained from peak area for lisinopril was 1.067852 intra-day and 1.183517 inter-day. The developed method was found to be precise as the RSD values for repeatability and inter-day precision studies were {\textless}2\%, respectively, as recommended by ICH guidelines and Shown in the Table~\ref{tw-52b753ed6251}.

\textbf{Estimation of lisinopril in tablet dosage forms}

Commercial brand of tablets was chosen for testing the suitability of the proposed method to estimate lisinopril in tablet formulations. Twenty tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 100 mg of lisinopril was transferred into a 100 ml volumetric flask and dissolved in 25 ml of a 30:70 v/v mixture of phosphate buffer and acetonitrile. The contents of the flask were sonicated for 15 min and a further 25 ml of the diluent was added, the flask was shaken continuously for 15 min to ensure complete solubility of the drug. The volume was made-up with the diluent and the solution was filtered through a 0.45 \ensuremath{\mu } membrane filter. This solution was further diluted to get the required concentrations. The solution containing 10\ensuremath{\mu }g/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 \unskip~\cite{1281324:23775458,1281324:23775465,1281324:23775461}. 

\textbf{Accuracy studies for lisinopril}

Accuracy of the method was obtained by performing recovery studies by the standard addition method at different levels of standard drug i.e. 80\%, 100\% and 120\% of lisinopril to analyzed tablet powder sample and mixture were reanalyzed by the proposed method. From the amount of drug found percentage recovery was calculated. The relevant results are furnished in Table~\ref{tw-422c57e11720}.
    
\section{Results and discussion}
In the proposed method, the retention time of lisinopril was found to be 1.510 min. Quantification was linear in the concentration range of 10- 35\ensuremath{\mu }g/ml for lisinopril. The regression equation of the linearity plot of concentration of lisinopril over its peak area was found to be y = 14079x - 68594 (r\ensuremath{^{2}}=0.9990) for lisinopril, where X is the concentration of lisinopril (\ensuremath{\mu }g/ml) and Y is the corresponding peak area. The limit of detection and limit of quantification for lisinopril was found to be 0.010024l and 0.030374 respectively, which indicate the sensitivity of the method. The use of phosphate buffer and acetonitrile in the ratio of 30:70 v/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 formulations did not interfere with the estimation of the drug by the proposed HPLC method. Figure~\ref{f-c6b376fa7f28} shows typical chromatogram of lisinopril. All the parameter result of lisinopril was shown in Table~\ref{tw-45f3add7bc6e}.
    
\section{Conclusion}
The proposed HPLC method is rapid, sensitive, precise and accurate for the determination of lisinopril can be reliably adopted for routine quality control analysis of lisinopril bulk and in its tablet dosage forms.

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest for this study.

\textbf{Funding Support}

The authors declare that they have no funding support for this study. 
    
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