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\begin{document}


\def\RunningAuthor{Amitkumar J. Vyas et al.}
\firstPage{2435}
\articleType{Original Article}
\receivedDate{19 Oct 2020}
\acceptedDate{23 Nov 2020}
\revisedDate{21 Nov 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4495}
\copyrightYear{2020}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{\textbf{Validated stability-indicating RP-HPLC method for simultaneous estimation of cilnidipine and chlorthalidone in tablet dosage form}}
\author{Ashok~B.~Patel\textsuperscript{1},  
            Amitkumar~J.~Vyas\textsuperscript{*}\textsuperscript{1},  
            Shital~Faldu\textsuperscript{2},  
            Arvind~N~Lumbhani\textsuperscript{3},  
            Nikunj~J.~Patel\textsuperscript{1},  
            Nilesh~K.~Patel\textsuperscript{1},  
            Ajay~I.~Patel\textsuperscript{1}, 
            Jayant~R~Chavda\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Quality Assurance, B.K. Mody Government Pharmacy College, Rajkot, Gujarat, India}~\\{\textsuperscript{2}Smt. R. D. Gardi B. Pharmacy College, Nyara, Rajkot, Gujarat, India}~\\{\textsuperscript{3}Gyanmanjari Pharmacy College, near ISCON Eleven, Bhavnagar, Gujarat, India}}

\begin{abstract}
A novel, simple, specific, accurate \& precise stability-indicating Gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous estimation of Cilnidipine \& Chlorthalidone in tablet dosage form, validated as per ICH guideline. The separation was achieved on Inertsil ODS column (250 mm x 4.6 mm, 5 \ensuremath{\mu }m) in a gradient mode.  The mobile phase consisted of Methanol, 0.025 M Potassium dihydrogen phosphate Buffer pH 5.5 adjusted by 10\% v/v Ortho Phosphoric Acid (50:50 v/v) (Solution A) and Acetonitrile, 0.025 M Potassium dihydrogen phosphate Buffer pH 5.5 adjusted by 10\%v/v Ortho Phosphoric Acid (75:25 v/v) (Solution B)\textbf{, }gradient programming for 20 min at 1 ml/min rate of flow and response was detected at 225 nm. The retention time was found to be 3.580 min and 12.606 mins for Chlorthalidone and Cilnidipine, respectively. The method is validated according to ICH guideline, which includes linearity, specificity, accuracy, precision and robustness. Linearity was obtained over the concentration range of 10-60 \ensuremath{\mu }g/ml for Cilnidipine and 6.25-37.5 \ensuremath{\mu }g/ml for Chlorthalidone, had a regression coefficient (r\ensuremath{^{2}}) almost 0.9966. The \% Recovery was found to be 99.63-100.59 \% and 100.24-100.51 \% for Cilnidipine and Chlorthalidone, respectively. The method was found to be specific enough to separate all degradation products from the active compound. Drug samples were exposed to various stress conditions like photolysis, oxidation, heat conditions, and hydrolysis (acidic and alkaline), there was no interference of any degradants and excipient in the determination of drugs so that methods can be successfully applied for routine QC analysis. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Cilnidipine,\newline Chlorthalidone,\newline Stability indicating method,\newline RP-HPLC,\newline Gradient,\newline ICH guideline
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Amitkumar~J.~Vyas~\\ Phone:\ 9879114519~\\  Email:\ amitvyas77@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Chlorthalidone (CHL) chemically 2-chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1- yl) benzene-1-sulfonamide (Figure~\ref{f-d8c6374bc91a}) \unskip~\citep{942440:20858041}. It avoids the reabsorption of Cl\ensuremath{^{-}} \& Na\ensuremath{^{+}}. CHL is a diuretic drug indicated in edema (associated with Congestive Heart Failure), fluid retention (associated with kidney disease and hypertension) \unskip~\citep{942440:20858047}.  Cilnidipine (CIL) chemically O3-(2-methoxyethyl) O5-[(E)-3-phenylprop-2-enyl] 2,6- dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine- 3,5- dicarboxylate (Figure~\ref{f-639bcb2944b6}) \unskip~\citep{942440:20858041}. It is a Ca\ensuremath{^{+}} channel blocker. CHL and CIL both are commonly prescribed drugs for hypertension \unskip~\citep{942440:20858047}. The addition of these two drugs shows a synergic effect. So, the combination of these two drugs concluded beneficial in the treatment of hypertension. 

The literature reveals that CHL and CIL FDC is not official in any pharmacopeia, however individual drug, as well as a combination with other drugs, have been determined by pharmacopoeial method \unskip~\citep{942440:20858048} including UV spectroscopy \unskip~\citep{942440:20858037,942440:20858036}, Derivative Spectrophotometric Method \unskip~\citep{942440:20858049,942440:20858039}, HPTLC \unskip~\citep{942440:20858031,942440:20858035}, HPTLC for Cilnidipine And Metoprolol \unskip~\citep{942440:20858045}, RP-HPLC for four drugs simultaneously \unskip~\citep{942440:20858034,942440:20858042}, for two drugs \unskip~\citep{942440:20858050,942440:20858046}, for Telmisartan and Chlorthalidone \unskip~\citep{942440:20858038}, in Biological Fluids \unskip~\citep{942440:20858040}\ensuremath{^{,}} for CIL and CHL \unskip~\citep{942440:20858043,942440:20858030} and Chlorthalidone, Cilnidipine and Telmisartan \unskip~\citep{942440:20858032}. RP HPLC (Stability indicating) of drugs contains CIL, CHL with telmisartan also reported \unskip~\citep{942440:20858033,942440:20858044}, in which \% degradation is insufficient as it should be in between 5-20\%,\unskip~\citep{942440:20858051}. Also, degrading data \& chromatograms for degradation study are not given; the only method for how to perform study is given. Thus, it is worthwhile to develop a stability-indicating RP-HPLC method, which can be used in routine by Pharmaceutical industry \& analytical laboratories.
    
\section{Materials and Methods}
\textbf{Chemicals and reagents}

CIL \& CHL working standards were gifted from Pure chem. Pvt Ltd., Marketed formulation of CIL and CHL (Label claim: CIL 10 mg and CHL 6.25 mg), NEXOVAS-CH6.25 tablets (Macleod Pharmaceuticals Ltd.) was purchased from the market. Acetonitrile, methanol, water all chemicals HPLC grade and HCL, Orthophosphoric acid, H2O2, triethylamine, KH\ensuremath{_{2}}PO4, NaOH, are of AR Grade, were purchased from Merck.


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\caption{\boldmath {Chemical structure of CHL}}
\label{f-d8c6374bc91a}
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\caption{\boldmath {Chemical structure of CIL}}
\label{f-639bcb2944b6}
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\caption{\boldmath {Optimized Chromatogram of Cilnidipine and Chlorthalidone}}
\label{f-70895444eb73}
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\caption{\boldmath {(A) Acidic (B) Basic (C) Oxidative (D) Thermal degradation Chromatograms}}
\label{f-237e7f9d4046}
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\caption{\boldmath {Calibration curve of Cilnidipine}}
\label{f-fb80be64d85b}
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\caption{Calibration curve of Chlorthalidone}
\label{f-d11318da5ee8}
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\label{tw-b7ee81611af5}
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  \cAlignHack 2 hrs  &
  \cAlignHack 60 \text{\textordmasculine}C \\
\cAlignHack 3\% H2O2  &
  \cAlignHack 4 hrs  &
  \cAlignHack 60 \text{\textordmasculine}C \\
\cAlignHack Thermal   &
  \cAlignHack  24 hrs &
  \cAlignHack 105\ensuremath{^\circ}C \\
\cAlignHack Photolytic  &
  \cAlignHack one ICH cycle  &
  \cAlignHack ----\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {System Suitability parameter} }
\label{tw-ee0d5f217a74}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.42\linewidth-2\tabcolsep}p{\dimexpr.27360000000000007\linewidth-2\tabcolsep}p{\dimexpr.30639999999999993\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack System suitability parameter & \cAlignHack CHL (n=6) & \cAlignHack CIL (n=6)\\
\tblmidrule 
\cAlignHack Retention times (tR) &
  \cAlignHack 3.580 \ensuremath{\pm} 0.049 &
  \cAlignHack 12.606 \ensuremath{\pm} 0.295\\
\cAlignHack Theoretical plates (N) &
  \cAlignHack 2229.4 \ensuremath{\pm} 17.47 &
  \cAlignHack 20865.2 \ensuremath{\pm} 374.13\\
\cAlignHack Tailing &
  \cAlignHack 0.9906 \ensuremath{\pm} 0.046 &
  \cAlignHack 1.4642 \ensuremath{\pm} 0.029\\
\cAlignHack Resolution (RS) &
  \multicolumn{2}{p{\dimexpr(.58\linewidth-2\tabcolsep)}}{\cAlignHack 26.409}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Forced Degradation Summary} }
\label{tw-86eb278be74d}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.40339999999999996\linewidth-2\tabcolsep}p{\dimexpr.12079999999999998\linewidth-2\tabcolsep}p{\dimexpr.16040000000000003\linewidth-2\tabcolsep}p{\dimexpr.1514\linewidth-2\tabcolsep}p{\dimexpr.164\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Stress Condition & \multicolumn{2}{p{\dimexpr(.2812\linewidth-2\tabcolsep)}}{\cAlignHack  \% of Degradation} & \multicolumn{2}{p{\dimexpr(.3154\linewidth-2\tabcolsep)}}{\cAlignHack Peak Purity Index}\\
\rowcolor{kwdboxcolor} & \cAlignHack CHL & \cAlignHack CIL & \cAlignHack CHL & \cAlignHack CIL\\
\tblmidrule 
Acid Degradation 0.1N HCl / 40\ensuremath{^\circ}C &
  \cAlignHack 26.62 &
  \cAlignHack 13.39 &
  \cAlignHack 1.000 &
  \cAlignHack 0.999\\
Base Degradation 0.1N NaOH / 60\ensuremath{^\circ}C &
  \cAlignHack 17.77 &
  \cAlignHack 25.41 &
  \cAlignHack 1.000 &
  \cAlignHack 0.999\\
Peroxide Degradation 3\% H2O2 / 60\ensuremath{^\circ}C &
  \cAlignHack 16.19 &
  \cAlignHack 18.97 &
  \cAlignHack 1.000 &
  \cAlignHack 1.000\\
Thermal Degradation Heated at 60\ensuremath{^\circ}C &
  \cAlignHack 7.60 &
  \cAlignHack 6.56 &
  \cAlignHack 1.000 &
  \cAlignHack 1.000\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{\space }


\begin{table*}[!htbp]
\caption{\boldmath {Accuracy and Precision Data of Cilnidipine and Chlorthalidone} }
\label{tw-e79ed3d705cf}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1529\linewidth-2\tabcolsep}p{\dimexpr.1168\linewidth-2\tabcolsep}p{\dimexpr.1477\linewidth-2\tabcolsep}p{\dimexpr.1349\linewidth-2\tabcolsep}p{\dimexpr.1438\linewidth-2\tabcolsep}p{\dimexpr.1516\linewidth-2\tabcolsep}p{\dimexpr.1523\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Level (\%) & \multicolumn{2}{p{\dimexpr(.2645\linewidth-2\tabcolsep)}}{\cAlignHack \% Recovery*} & \multicolumn{2}{p{\dimexpr(.2787\linewidth-2\tabcolsep)}}{\cAlignHack Intraday precision RSD* } & \multicolumn{2}{p{\dimexpr(.3039\linewidth-2\tabcolsep)}}{\cAlignHack Inter day precision RSD*}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack CIL & \cAlignHack CHL & \cAlignHack CIL & \cAlignHack CHL & \cAlignHack CIL & \cAlignHack CHL\\
\tblmidrule 
\cAlignHack 50 &
  \cAlignHack 100.01 &
  \cAlignHack 99.58 &
  \cAlignHack 0.166 &
  \cAlignHack 0.236 &
  \cAlignHack 0.177 &
  \cAlignHack 0.325\\
\cAlignHack 100 &
  \cAlignHack 99.31 &
  \cAlignHack 100.33 &
  \cAlignHack 1.038 &
  \cAlignHack 0.519 &
  \cAlignHack 0.142 &
  \cAlignHack 0.107\\
\cAlignHack 150 &
  \cAlignHack 99.56 &
  \cAlignHack 100.81 &
  \cAlignHack 0.509 &
  \cAlignHack 0.185 &
  \cAlignHack 0.070 &
  \cAlignHack 0.187\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{ * - n = 3}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Analysis of Marketed Pharmaceutical Dosage Form} }
\label{tw-e7d80129975a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.061\linewidth-2\tabcolsep}p{\dimexpr.10909999999999997\linewidth-2\tabcolsep}p{\dimexpr.12299999999999999\linewidth-2\tabcolsep}p{\dimexpr.12090000000000003\linewidth-2\tabcolsep}p{\dimexpr.0981\linewidth-2\tabcolsep}p{\dimexpr.101\linewidth-2\tabcolsep}p{\dimexpr.1509\linewidth-2\tabcolsep}p{\dimexpr.167\linewidth-2\tabcolsep}p{\dimexpr.068999999999999995\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Drug & \cAlignHack Label  & \cAlignHack Amt.  & \multicolumn{3}{p{\dimexpr(.3200000000000001\linewidth-2\tabcolsep)}}{\cAlignHack Area(mAU)} & \cAlignHack Mean \ensuremath{\pm} S.D & \cAlignHack \% Assay \ensuremath{\pm} S.D & \cAlignHack RSD\\
\rowcolor{kwdboxcolor} &
  \cAlignHack claim(mg) &
  \cAlignHack Found(mg) &
  \cAlignHack 1 & \cAlignHack 2 & \cAlignHack 3 & \cAlignHack (mAU) &
  \cAlignHack (n=3) &
  \\
\tblmidrule 
\cAlignHack CHL &
  \cAlignHack 6.25 &
  \cAlignHack 6.22 &
  \cAlignHack 1934337 &
  \cAlignHack 1912651 &
  \cAlignHack 1942136 &
  \cAlignHack 1929708 \ensuremath{\pm} 15277.82 &
  \cAlignHack 99.60 \ensuremath{\pm} 0.862 &
  \cAlignHack 0.865\\
\cAlignHack CIL &
  \cAlignHack 10 &
  \cAlignHack 9.90 &
  \cAlignHack 2579270 &
  \cAlignHack 2574241 &
  \cAlignHack 2586652 &
  \cAlignHack 2580054 \ensuremath{\pm} 6242.56 &
  \cAlignHack 99.06 \ensuremath{\pm} 0.184 &
  \cAlignHack 0.186\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{\textbf{Instrumentation and Chromatographic conditions}}

The desired separation established by using Inertsil ODS (250 \ensuremath{\times} 4.6) mm; 5 \ensuremath{\mu }m i.d. at (35\ensuremath{\pm}1) {\textdegree} C, with 1.0 ml/min flow rate. Mobile phase A: Methanol: 0.025 M KH\ensuremath{_{2}}PO\ensuremath{_{4}} buffer adjusted to pH 5.5 by 10\% OPA (50:50 v/v) and Mobile phase B: ACN: 0.025 M KH\ensuremath{_{2}}PO\ensuremath{_{4}} buffer adjusted to pH 5.5 by 10 \% v/v OPA (75:25 v/v), was used in gradient mode. Photodiode array detector set at 225 nm with a run time of 20 mins. The gradient condition is described in Table~\ref{tw-b7ee81611af5}.

\textbf{Preparation of standard and Sample}

The standard stock solutions 500 \ensuremath{\mu }g/ml of CIL and 125 \ensuremath{\mu }g/ml of CHL were prepared in methanol and later diluted to final concentrations as 20 \ensuremath{\mu}g/ml CIL and 12.5 \ensuremath{\mu}g/ml CHL.

\textbf{Preparation of sample mixture }

About 20 tablets were accurately weighed and crushed. In 50 ml Vol. flask a quantity of powder equivalent to 6.25 mg of CHL or 10 mg of CIL was added. It was sonicated and filtered after the addition of MeOH; appropriately diluted to obtain 20 \ensuremath{\mu}g/ml CIL and 12.5 \ensuremath{\mu}g/ml CHL.

\textbf{Forced degradation study}

CHL and CIL have undergone stress conditions as tabulated in Table~\ref{tw-e9044ab09c93}.

\textbf{Method Validation}

\textbf{System Suitability}

Separately injected six replicate injections of Standard preparation of CHL and CIL solution having a concentration of 12.5 \ensuremath{\mu }g/ml and 20 \ensuremath{\mu }g/ml respectively in the HPLC.

\textbf{Linearity}

The linearity of the method was observed in the range of 10-60 \ensuremath{\mu }g/ml for CIL \& 6.25-37.5 \ensuremath{\mu }g/ml for CHL.

\textbf{Accuracy }

The accuracy was ascertained by spiking 3 concentration levels 50, 100, and 150 \% in three replicates of the targeted concentration of the analytes. The accuracy was revealed as percent analytes recovered by the proposed method.

\textbf{Precision}

The intra-day and inter-day precision of the method was carried out by analyzing sample solutions in three sets each of 50, 100, and 150 \% of assay concentration. It is performed within a day (three distinct timings) and 3 consecutive days.

\textbf{Specificity}

The specificity of the method was ascertained by analyzing diluent, placebo, standard, and tablet formulation to examine the \% interference of excipients and their impurities with analytes peak. 

\textbf{Robustness}

The sample solutions were prepared and then analyzed with a change in the typical analytical conditions like Flow rate \ensuremath{\pm} 10\%, Mobile Phase \ensuremath{\pm} 5\%, pH \ensuremath{\pm} 0.1. Observe the area and compare it with normal conditions.
    
\section{Results and Discussion}
\textbf{Optimized chromatographic condition }

The optimized separation was achieved by the chromatographic condition stated earlier, as shown in Figure~\ref{f-70895444eb73}. The System suitability parameters of the RP-HPLC method obtained for CHL and CIL is acceptable, as described in Table~\ref{tw-ee0d5f217a74}.

\textbf{Forced degradation study}

As the peak purity index is almost 0.999, there is no merging of peaks with sufficient degradation, as shown in Table~\ref{tw-86eb278be74d} \& in Figure~\ref{f-237e7f9d4046}.\textbf{\space }

\textbf{Method validation}

The method was validated for various parameters, according to ICH Q2 (R1).

\textbf{Linearity }

The correlation coefficients and regression equations for CHL and CIL were found to be R\ensuremath{^2} = 0.9965, y = 138010x {\textendash} 154200 and R\ensuremath{^2} = 0.9966, y=141724x + 165176 respectively. The calibration curve of CIL and CHL are shown in Figure~\ref{f-fb80be64d85b} and Figure~\ref{f-d11318da5ee8}.

\textbf{Accuracy}

The percentage recovery for CIL was found within 99.63-100.59, and that for CHL was found to be in the range of 100.24-100.51.  Thus, the \% recovery of both drugs is acceptable, as tabulated in Table~\ref{tw-e79ed3d705cf}.

\textbf{Precision}

RSD for Intraday and Inter day precision was found less than 2. Thus, the method is precise. Data for precision is demonstrated in Table~\ref{tw-e79ed3d705cf}.

\textbf{Specificity}

The \% interference was less than 0.5 \%, thus making specificity to be acceptable.

\textbf{Robustness}

RSD was found to be less than 2. Thus, the method is robust. 

\textbf{Assay of tablet dosage form }

The result for CHL and CIL tablets assay was found to be satisfactory, are tabulated in Table~\ref{tw-e7d80129975a}.
    
\section{Conclusion }
A specific, accurate, precise, and robust stability-indicating Gradient RP-HPLC method is developed and validated for the determination of Cilnidipine and Chlorthalidone in a tablet dosage form. Degradation was carried out at oxidation, hydrolysis, photolysis, and heat conditions. The method is good enough to separate the peaks of tablet dosage form from the degradation products (generated during stress condition) and peak purity was also passed. Order of stability for Cilnidipine and Chlorthalidone is Thermal{\textgreater} Oxidative {\textgreater}Alkali {\textgreater}Acidic. All these induce us to assure that stability-indicating assay can be used in a routine.
\section*{Acknowledgement}The authors are thankful to B. K. Mody Govt. Pharmacy College for providing the necessary research facilities and infrastructure to carry out the research work.

\textbf{Funding Support}

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

\textbf{Conflict of interest}

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

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