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\begin{document}


\def\RunningAuthor{Srinivas L and Neelu Jain}
\firstPage{786}
\articleType{Original Article}
\receivedDate{20 Dec 2020}
\acceptedDate{25 Jan 2021}
\revisedDate{23 Jan 2021}
\journalVolume{2021, 12}
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\journalDoi{ijrps.v12i1.4182}
\copyrightYear{2021}
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\title{Stability Indicative and Cost Effective Creation and Validation of Analytical Method of Lopinavir and Rilpivirine by High Performance Liquid Chromatography}
\author{Srinivas~L\textsuperscript{*}, 
            Neelu~Jain~\\[5pt]{Department of Chemistry, Sri Satya Sai University of Technology and Medical Sciences, Sehore- 466001, Madhya Pradesh, India}}

\begin{abstract}
We have developed a completely unique and reliable HPLC technique for simultaneous quantification of Lopinavir and Rilpivirine. Chromatographic detachment was attained on a X-bridge phenyl column (150x4.6mm, 3.5 \ensuremath{\mu}) using isocratic elution with a buffer containing buffer and acetonitrile with the proportion of 70:30 as movable phase with a flow of 1 ml/min at room temperature and UV detection was carried out at 250 nm. Dissolve 1ml of tri ethylamine in 1 lt of HPLC grade water and filter through 0.45 \ensuremath{\mu} filter paper, this solution was used as buffer. 8 min. run time was used to separate Lopinavir and Rilpivirine. Analysis was achieved within 15 min over an honest linearity within the concentration range from 20-300 \ensuremath{\mu}g/ml of Lopinavir and 2.5-37.5 \ensuremath{\mu}g/ml of Rilpivirine. By injecting the standard six times, system suitability parameters were studied and the outcomes were under the acceptable limit. Precision and recovery study results were found to be within the suitable limit. By using the above technique, assay of Lopinavir and Rilpivirine was performed and found to be within the limit. Degradation studies were carried out on Lopinavir and Rilpivirine, with a purity threshold greater than purity angle in all conditions and within the allowable range. The above mentioned technique was validated according to ICH guidelines. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}HPLC,\newline Lopinavir,\newline Rilpivirine,\newline Development,\newline Validation
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Srinivas~L~\\ Phone:\ 8008958967~\\  Email:\ sri\_bpa@yahoo.in
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
        \textsc{DOI:}\ \href{https://doi.org/10.26452/\@journalDoi}{\textcolor{blue}{\underline{\smash{https://doi.org/10.26452/\@journalDoi}}}}\par%
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        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
        \vspace*{-11pt}\rule{\linewidth}{1.2pt}
        \makeatother
        
    
\section{Introduction}
Lopinavir is an anti retroviral product \unskip~\citep{995473:21337913}  in the class of protease inhibitors. It is used as a fixed dose formulation against HIV infection \unskip~\citep{995473:21337911}  with another protease inhibitor, ritonavir (lopinavir/ritonavir). Lopinavir is 98-99 percent strongly bound to plasma proteins. There are contradictory reports concerning the penetration of Lopinavir into the cerebrospinal fluid (CSF) \unskip~\citep{995473:21337910,995473:21337908}. According to anecdotal reports \unskip~\citep{995473:21337909}, Lopinavir cannot be recognized in the CSF, however a review of paired CSF- plasma samples from 26 patients receiving Lopinavir/Ritonavir found Lopinavir CSF levels above the IC50 in 77\% of samples \unskip~\citep{995473:21337916}. Only the medication combination Lopinavir/Ritonavir was side effects, interactions and contraindications assessed. 

Rilpivirine (TMC 278, trade name Edurant) is a prescription medication, for the treatment of HIV infection \unskip~\citep{995473:21337917}. It is a non-nucleoside reverse transcriptase (NNRTI) \unskip~\citep{995473:21337907,995473:21337915}  second generation inhibitor with higher potency, extended half life and decreased side effect profile contrast with older NNRTIs, such as Efavirenz \unskip~\citep{995473:21337912}. Like Etravirine a second generation NNRTI accepted in 2008, Rilpivirine is a diarylpyrimidine (DAPY) \unskip~\citep{995473:21337914}. In patients with baseline HIV viral loads greater than 100,000 copies/mm\ensuremath{^{3}}, Rilpivirine in union with Emtricitabine and Tenofovir has been shown to have higher virologic failure rates than Atripla \unskip~\citep{995473:21337906}. Figure~\ref{f-13afda1f34c7}  shows the chemical structures of Lopinavir and Rilpivirine. 


\bgroup
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\centering \makeatletter\IfFileExists{images/909eb7cb-3145-4878-9e86-488db268a1d0-upicture1.png}{\includegraphics[width=.65\linewidth]{images/909eb7cb-3145-4878-9e86-488db268a1d0-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Structural representations of (A) Lopinavir and (B) Rilpivirine}}
\label{f-13afda1f34c7}
\end{figure}
\egroup
\textbf{Experiment}

\textbf{Chemicals and Reagents}

Acetonitrile and Ortho phosphoric acid, tri ethyl amine, water (HPLC mark) were acquired from Merck (India) Ltd, Worli, Mumbai, India. The reference standards of Lopinavir and Rilpivirine APIs were took from Spectrum Pharma Research Solutions Pvt. Ltd., Hyderabad.

\textbf{Equipment}

An HPLC system (Waters alliance e2695 model) consisting of quaternary pump, PDA detector-2998 was used. Data processing was performed with Empower 2.0 software. 

\textbf{Chromatographic Conditions}

Chromatographic detachment was achieved in isocratic mode at room temperature using X-bridge phenyl (150x4.6 mm, 3.5 \ensuremath{\mu}). A mixture of acetonitrile and 0.1 percent triethylamine (TEA) in 70:30 v/v at a stream of 1 ml/min was used as movable phase. The injection volume was 10 \ensuremath{\mu}l and the run time was 10.0 min.

\textbf{Preparation of Buffer}

1 mL of tri ethylamine is dissolved in 1 liter of HPLC grade water and filtered through 0.45 \ensuremath{\mu} filter paper. 

\textbf{Diluent}

Movable phase was used as diluent.

\textbf{Standard Preparation}

Carefully weigh and transfer 200 mg of Lopinavir and 25 mg of Rilpivirine in a volumetric flask of capacity 100 ml and add app. 70 ml of diluent, sonicated to melt it for 30 min. and add diluent up to mark. Take 5 ml of the above solution and dilute it to 50 ml and filtered through 0.45 \ensuremath{\mu} nylon syringe filter.

\textbf{Method Validation}

\textbf{System Suitability}

In order to confirm the system performance, system suitability parameters were measured. The parameters including USP plate count, USP tailing and \%RSD were calculated and found to be within the limit. 

\textbf{Specificity}

Specificity is the capacity to evaluate the analyte unambiguously the analyte in the presence of other constituents that may be expected to be combined in the sample and standard solution. It is checked by examining the chromatograms of blank samples and samples spiked with Lopinavir and Rilpivirine.

\textbf{Accuracy}

Accuracy is the closeness of the test findings gained by the technique to the real value. It is assessed by the recovery studies will be evaluated at three different concentration levels. In each level a minimum of three injections were administered at each level and quantity of the drug present, percentage recovery and the associated standard deviations were calculated. 

\textbf{Precision}

Precision of the analytical technique is the degree of agreement among independent test results. It was studied by investigation of multiple sampling of homogeneous sample. The precision of the present technique was assessed in terms of repeatability, intra-day and inter-day variations. It was checked by analyzing the samples at different time intervals of the same day as well as on different days.

\textbf{Linearity}

Linearity of an analytical process is its ability to produce results within a given range directly proportional to the concentration of the analyte in the sample within a definite range. The six series of standard solutions were selected for the evaluation of the linearity range, six series of standard solutions were selected. Using peak area versus concentration of the regular solution, the calibration curve was plotted and the regression equations were calculated. The least squares method was used to calculate the slope, intercept and coefficient of correlation. 

\textbf{Stress Degradation}

Stress Degradation should not have any interference between the peaks of forced degradation preparations obtained for the chromatogram. Stress degradation studies were conducted in accordance with ICH guidelines Q1 (A) R2. The peaks of degradation should be well spaced and the resolution between the peaks should be at least 1.0. The peak purity of the principle peaks will pass only when there is separation. Forced degradation studies were achieved to obtain the degradation of about 20 percent by different types of stress conditions. 

\textbf{Robustness}

An analytical procedure's robustness is a measure of its ability to remain unchanged by small but intentional differences in method parameters and gives sign of its reliability during normal use. Robustness study was carried out by injecting standard solution into the HPLC system and modifying chromatographic settings such as flow rate (\ensuremath{\pm}0.2 ml/min), organic content in the mobile phase (\ensuremath{\pm}10\%). By establishing the effect of the modified parameters the separation factor, retention time and peak asymmetry were estimated.
    
\section{Results and Discussion}
The present study was designed to develop a simple, accurate and rapid analytical RP-HPLC method that can be used for the simultaneous estimation of Lopinavir and Rilpivirine in bulk and pharmaceutical dosage form for the analysis of the assay technique. In order to provide good assay performance the chromatographic conditions were optimized. To optimize mobile phase, various combinations were tried for Lopinavir and Rilpivirine. The final working mobile phase is (0.1\%) triethylamine and acetonitrile in the composition of (30:70 v/v). Mobile phase for each drug was selected on the basis of its polarity. In order to obtain sufficient sensitivity for the two APIs in smaller proportions (Lopinavir and Rilpivirine) detection was carried out in several wavelengths. Finally, the 250 nm wavelength was selected where the two drugs showed good absorption. The flow rate was 1.0 ml/min. For Lopinavir and Rilpivirine the retention time was 6.482 min, 2.925 min respectively. The proposed method is confirmed by all the outcomes within the limits in accordance with the ICH guidelines. The detection was carried out with a total runtime of 8.0 min. Optimized chromatographic conditions were showed in Table~\ref{tw-1e19d801c4db}. 


\begin{table*}[!htbp]
\caption{\boldmath {Optimized Chromatographic Conditions} }
\label{tw-1e19d801c4db}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.4503000000000001\linewidth-2\tabcolsep}p{\dimexpr.5496999999999999\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Parameter & \cAlignHack Proposed method\\
\tblmidrule 
\cAlignHack Stationary Phase &
  \cAlignHack X-bridge phenyl (150x4.6 mm, 3.5 \ensuremath{\mu})\\
\cAlignHack Mobile Phase &
  \cAlignHack 0.1\% Tri ethyl amine: Acetonitrile (30:70)\\
\cAlignHack Injection Volume &
  \cAlignHack 10 \ensuremath{\mu}l\\
\cAlignHack Flow Rate &
  \cAlignHack 1.0 ml/min\\
\cAlignHack Column Temperature &
  \cAlignHack Ambient\\
\cAlignHack Wave Length &
  \cAlignHack 250 nm\\
\cAlignHack Run Time &
  \cAlignHack 8.0 min\\
\cAlignHack Retention time of Rilpivirine &
  \cAlignHack 2.925 min\\
\cAlignHack Retention time of Lopinavir &
  \cAlignHack 6.482 min\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of System Suitability} }
\label{tw-337f849df2c8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5208\linewidth-2\tabcolsep}p{\dimexpr.25950000000000003\linewidth-2\tabcolsep}p{\dimexpr.2197\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Parameter & \cAlignHack Rilpivirine & \cAlignHack Lopinavir\\
\tblmidrule 
\cAlignHack Theoretical plate count &
  \cAlignHack 2497 &
  \cAlignHack 7815\\
\cAlignHack Tailing factor &
  \cAlignHack 1.52 &
  \cAlignHack 1.23\\
\cAlignHack Resolution &
  \cAlignHack - &
  \cAlignHack 13.23\\
\cAlignHack Retention time &
  \cAlignHack 2.925 &
  \cAlignHack 6.482\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Linearity} }
\label{tw-25b1f22b4078}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No & \multicolumn{2}{p{\dimexpr(.4313999999999999\linewidth-2\tabcolsep)}}{\cAlignHack Rilpivirine} & \multicolumn{2}{p{\dimexpr(.44660000000000004\linewidth-2\tabcolsep)}}{\cAlignHack Lopinavir}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack Concentration (\ensuremath{\mu}g/ml) & \cAlignHack Area & \cAlignHack Concentration (\ensuremath{\mu}g/ml) & \cAlignHack Area\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 2.5 &
  \cAlignHack 104265 &
  \cAlignHack 20 &
  \cAlignHack 178924\\
\cAlignHack 2 &
  \cAlignHack 6.25 &
  \cAlignHack 212452 &
  \cAlignHack 50 &
  \cAlignHack 419862\\
\cAlignHack 3 &
  \cAlignHack 12.5 &
  \cAlignHack 389575 &
  \cAlignHack 100 &
  \cAlignHack 851729\\
\cAlignHack 4 &
  \cAlignHack 25 &
  \cAlignHack 723139 &
  \cAlignHack 200 &
  \cAlignHack 1609432\\
\cAlignHack 5 &
  \cAlignHack 31.25 &
  \cAlignHack 905367 &
  \cAlignHack 250 &
  \cAlignHack 2043458\\
\cAlignHack 6 &
  \cAlignHack 37.5 &
  \cAlignHack 1049622 &
  \cAlignHack 300 &
  \cAlignHack 2473340\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Method Precision} }
\label{tw-cb4f3192866a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1972\linewidth-2\tabcolsep}p{\dimexpr.4917999999999999\linewidth-2\tabcolsep}p{\dimexpr.311\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Area of Rilpivirine & \cAlignHack Area of Lopinavir\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 723139 &
  \cAlignHack 1609432\\
\cAlignHack 2 &
  \cAlignHack 723457 &
  \cAlignHack 1610984\\
\cAlignHack 3 &
  \cAlignHack 723654 &
  \cAlignHack 1610894\\
\cAlignHack 4 &
  \cAlignHack 723689 &
  \cAlignHack 1609538\\
\cAlignHack 5 &
  \cAlignHack 724587 &
  \cAlignHack 1609421\\
\cAlignHack 6 &
  \cAlignHack 724791 &
  \cAlignHack 1609649\\
\cAlignHack Mean &
  \cAlignHack 723886 &
  \cAlignHack 1609986\\
\cAlignHack Std. dev &
  \cAlignHack 654.99 &
  \cAlignHack 743.08\\
\cAlignHack \% RSD &
  \cAlignHack 0.09 &
  \cAlignHack 0.05\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Intermediate Precision} }
\label{tw-e0bc05a80fb6}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.22919999999999998\linewidth-2\tabcolsep}p{\dimexpr.29360000000000003\linewidth-2\tabcolsep}p{\dimexpr.18979999999999997\linewidth-2\tabcolsep}p{\dimexpr.28740000000000002\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Area of Rilpivirine & \cAlignHack Relative standard Deviation & \cAlignHack Area of Lopinavir & \cAlignHack Relative standard Deviation\\
\tblmidrule 
\cAlignHack 723143 &
  \multicolumn{1}{p{\dimexpr(.29360000000000003\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{\cAlignHack 0.05}} &
  \cAlignHack 1609568 &
  \multicolumn{1}{p{\dimexpr(.28740000000000002\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{\cAlignHack 0.07}}\\
\cAlignHack 723471 &
   &
  \cAlignHack 1610984 &
  \\
\cAlignHack 723661 &
   &
  \cAlignHack 1610868 &
  \\
\cAlignHack 723673 &
   &
  \cAlignHack 1609548 &
  \\
\cAlignHack 724576 &
   &
  \cAlignHack 1608421 &
  \\
\cAlignHack 724785 &
   &
  \cAlignHack 1608648 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Accuracy} }
\label{tw-433b0b94e165}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1435\linewidth-2\tabcolsep}p{\dimexpr.26980000000000004\linewidth-2\tabcolsep}p{\dimexpr.15849999999999994\linewidth-2\tabcolsep}p{\dimexpr.26139999999999997\linewidth-2\tabcolsep}p{\dimexpr.1668\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Accuracy & \cAlignHack Amount of Rilpivirine & \cAlignHack \% Recovery & \cAlignHack Amount of Lopinavir & \cAlignHack \% Recovery\\
\tblmidrule 
\cAlignHack 50 &
  \cAlignHack 12.5 &
  \cAlignHack 98.4 &
  \cAlignHack 100 &
  \cAlignHack 100.1\\
\cAlignHack 100 &
  \cAlignHack 25 &
  \cAlignHack 99.3 &
  \cAlignHack 200 &
  \cAlignHack 99.8\\
\cAlignHack 150 &
  \cAlignHack 37.5 &
  \cAlignHack 99.1 &
  \cAlignHack 300 &
  \cAlignHack 99.6\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Robustness} }
\label{tw-a5f9fe3364ca}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.357\linewidth-2\tabcolsep}p{\dimexpr.3258\linewidth-2\tabcolsep}p{\dimexpr.3172\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Parameter & \cAlignHack \% RSD of Rilpivirine & \cAlignHack \% RSD of Lopinavir\\
\tblmidrule 
\cAlignHack Flow (0.8 ml/min) &
  \cAlignHack 0.64 &
  \cAlignHack 0.43\\
\cAlignHack Flow (1.2 ml/min) &
  \cAlignHack 0.71 &
  \cAlignHack 0.77\\
\cAlignHack Organic phase (63:37) &
  \cAlignHack 0.35 &
  \cAlignHack 0.51\\
\cAlignHack Organic phase (77:23) &
  \cAlignHack 0.98 &
  \cAlignHack 0.87\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Forced Degradation} }
\label{tw-454bac026816}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.6288\linewidth-2\tabcolsep}p{\dimexpr.1973\linewidth-2\tabcolsep}p{\dimexpr.1739\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Stress Parameter & \multicolumn{2}{p{\dimexpr(.37120000000000005\linewidth-2\tabcolsep)}}{\cAlignHack \% of Degradation}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack Rilpivirine & \cAlignHack Lopinavir\\
\tblmidrule 
\cAlignHack Acid degradation (1N HCl) &
  \cAlignHack 13.7 &
  \cAlignHack 13.6\\
\cAlignHack Alkali degradation (1N NaOH) &
  \cAlignHack 14.2 &
  \cAlignHack 13.0\\
\cAlignHack Peroxide degradation (30\% Peroxide) &
  \cAlignHack 14.3 &
  \cAlignHack 14.9\\
\cAlignHack Reduction degradation (30\% sodium bisulphate) &
  \cAlignHack 15.3 &
  \cAlignHack 14.7\\
\cAlignHack Thermal (sample, 70\ensuremath{^\circ}C, 6 Hrs) &
  \cAlignHack 14.8 &
  \cAlignHack 12.7\\
\cAlignHack Photo (sample, sunlight, 12 Hrs) &
  \cAlignHack 15.4 &
  \cAlignHack 13.6\\
\cAlignHack Hydrolysis (5ml HPLC water) &
  \cAlignHack 10.9 &
  \cAlignHack 11.2\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{System Suitability}

The System Suitability was performed by injecting standard solution containing 200 \ensuremath{\mu}g/ml of Lopinavir and 25 \ensuremath{\mu}g/ml of Rilpivirine in six replicates. The results indicate that the system suitability parameter is within the limit. Standard chromatogram was showed in Figure~\ref{f-60db17dab7e2}  and system suitability results were represented in Table~\ref{tw-337f849df2c8}.


\bgroup
\fixFloatSize{images/34c3e1f8-2773-45be-b51f-dd27ac8909d3-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/34c3e1f8-2773-45be-b51f-dd27ac8909d3-upicture2.png}{\includegraphics{images/34c3e1f8-2773-45be-b51f-dd27ac8909d3-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of System Suitability}}
\label{f-60db17dab7e2}
\end{figure}
\egroup
\textbf{Specificity}

There was no interference from blank at the retention time of Lopinavir and Rilpivirine. Figure~\ref{f-72c475727730}  shows the blank chromatogram of Lopinavir and Rilpivirine.


\bgroup
\fixFloatSize{images/a5edf394-e7d3-46d0-a998-8028dfd56479-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/a5edf394-e7d3-46d0-a998-8028dfd56479-upicture3.png}{\includegraphics{images/a5edf394-e7d3-46d0-a998-8028dfd56479-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of Blank}}
\label{f-72c475727730}
\end{figure}
\egroup
\textbf{Linearity}

By plotting a calibration curve of the peak area against its respective concentration, linearity was determined. From this calibration curve, it was found that the curve was linear in the range of 20-300 \ensuremath{\mu}g/ml of Lopinavir and 2.5-37.5 \ensuremath{\mu}g/ml of Rilpivirine. The regression equations for calibration curve of Rilpivirine was Y= 27717.07x + 28136.70 (R\ensuremath{^{2}}-0.9992) and Y= 8151.63x + 11034.55 (R\ensuremath{^{2}}-0.9998) for Lopinavir respectively. Linearity results were showed in Table~\ref{tw-25b1f22b4078}  and the calibration plots of Rilpivirine and Lopinavir were represented in Figure~\ref{f-05d9f7ebd4e9}. 


\bgroup
\fixFloatSize{images/c9fb18c1-fc71-4682-92df-a0d7c7eeb9a5-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c9fb18c1-fc71-4682-92df-a0d7c7eeb9a5-upicture4.png}{\includegraphics{images/c9fb18c1-fc71-4682-92df-a0d7c7eeb9a5-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Calibration plots of (A) Rilpivirine and (B) Lopinavir}}
\label{f-05d9f7ebd4e9}
\end{figure}
\egroup
\textbf{Precision}

In terms of intraday and intermediate precision variations, the accuracy of this method was assessed. Six repeated analyses of the sample solution of Rilpivirine and Lopinavir on the same day under the same experimental conditions were used to determine the intraday studies. In the same laboratory, the intermediate precision of the method was carried out by studying the analysis with different analysts and with different instruments. As percentage RSD values were found to be {\textless}2 percent, the method is highly precise. At each added concentration, good recoveries of the drug were obtained, indicating that the method was exact. Method precision results were showed in Table~\ref{tw-cb4f3192866a}.

\textbf{Intermediate Precision (Ruggedness)}

Intermediate precision results were showed in Table~\ref{tw-e0bc05a80fb6}.

\textbf{Accuracy}

By calculating the recovery experiments at three levels, the accuracy of the method was achieved (50\%, 100\% and 150\%). APIs were prepared with a concentration of 12.5, 25, 37.5 \ensuremath{\mu}g/ml of Rilpivirine and 100, 200, 300 \ensuremath{\mu}g/ml of Lopinavir. For each spike level, the test solution was injected three times and the assay was performed as per test method. The recovery outcomes were close to 100\% and also the RSD values were less than \ensuremath{\pm}2\% as well. The recovery percentage, mean and relative standard deviations have been calculated. Recovery values showed within the desired range, the method was accurate. Table~\ref{tw-433b0b94e165}  shows the accuracy results of Lopinavir and Rilpivirine.

\textbf{Robustness}

Robustness of the chromatographic technique was determined by changing flow rate and movable phase composition. \% RSD was found to be within the acceptable limit. Table~\ref{tw-a5f9fe3364ca}  shows the robustness results of Rilpivirine and Lopinavir.

\textbf{Forced Degradation}

The proposed method can be used for release and stability studies for effective evaluations and can be considered as stability preferable technique. The forced degradation study carried out according to the ICH requirements includes acid, base, oxidation, reduction, thermal, photo and hydrolysis degradation. From the chromatograms, it is evident that the selected drugs were stable under the applied stress conditions though degraded peaks were observed. Results of forced degradation were shown in Table~\ref{tw-454bac026816}. 
    
\section{Conclusion}
In this study, a novel, rapid, economical, sensitive and easily available HPLC method was evolved for the simultaneous estimation of Rilpivirine and Lopinavir in API form. The main advantages of this method are no HPLC methods were reported. Other merits are shorter run time, low price, accessibility, sensitivity, reliability and reproducibility. These properties are important to analyze the sample. The validation of all the parameters like linearity, accuracy, specificity, robustness, method precision were done and found to be within the acceptable limit. The related standard deviation values for all the parameters were found to be less than 2\%, which indicates the validity of the method and the results gained by this technique are in fair agreement. So the proposed method could be easily applied for the routine analysis of Rilpivirine and Lopinavir in quality control laboratories without any preliminary separation.  
    
\section{Acknowledgement}
The authors are thankful to the report guide for supporting in this research work and Shree Icon Pharmaceutical Laboratories for providing laboratory facilities.

\textbf{Funding Support }

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

\textbf{Conflict of Interest}

The authors declare that there is no conflict of interest for this study.
    

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