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\begin{document}


\def\RunningAuthor{Subbarao M et al.}
\firstPage{1961}
\articleType{Original Article}
\receivedDate{22 Dec 2020}
\acceptedDate{03 Feb 2021}
\revisedDate{28 Jan 2021}
\journalVolume{2021, 12}
\journalIssue{3}
\journalDoi{ijrps.v12i3.4800}
\copyrightYear{2021}
\def\authorCount{4}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{\textbf{Validated stability indicating RP-HPLC method for the determination of dolutegravir and rilpivirine in bulk and pharmaceutical dosage forms}}
\author{Gopinath~K\textsuperscript{1},  
            Padmavathi~K~V\textsuperscript{2},  
            Murali~Krishna~N\textsuperscript{3}, 
            Subbarao~M\textsuperscript{*}\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur - 522510, Andhra Pradesh, India}~\\{\textsuperscript{2}D. S. Govt. Degree College for Women, Ongole - 523001, Prakasam District, Andhra Pradesh, India}~\\{\textsuperscript{3}Department of Chemistry, V R Siddhartha Engineering College, Chalasani Nagar, Kanuru, Vijayawada - 520007, Andhra Pradesh, India}}

\begin{abstract}
For simultaneous analysis of Dolutegravir and Rilpivirine utilizing RP-HPLC, an accurate, rapid, economical, quick and reliable assay technique was developed and tested. Successful chromatographic detachment using acetonitrile and 0.1 percent tri ethyl amine in the water of pH-2.5 adjusted with 0.1\% orthophosphoric acid in 40:60 v/v as a movable phase with a flow of 1 ml/min and UV observation at 230 nm. Chromatography at ambient temperature was performed isocratically, and the run time was 10 min. By injecting the norm six times, device suitability parameters were studied and the findings were far below the acceptance criteria. The linearity analysis was performed at levels ranging from 10\% to 150\% and the R\ensuremath{^{2}} value was found to be 0.999. Precision has been found to be 0.8 for repeatability and 1.2 for intermediate precision. Assay of the commercialized formulation was performed by using the above method, and we get 100.01 percent was present. For routine analysis in drug testing, this chromatographic approach can be effectively implemented. By using the above technique, an assay of the marketed formulation was performed and found to be within the limit. Degradation studies were carried out on Dolutegravir and Rilpivirine, with a purity threshold greater than purity angle in all conditions and within the acceptable range. The above-mentioned technique was validated according to ICH guidelines.  
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Development,\newline Dolutegravir,\newline Rilpivirine,\newline RP-HPLC,\newline Validation
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Subbarao~M~\\ Phone:\  9989523339~\\  Email:\ msubbarao282@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
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        \makeatother
        
    
\section{Introduction}
Dolutegravir (DTG) and Rilpivirine (RLP) are used to monitor HIV infection \unskip~\citep{1144392:22585566,1144392:22585556,1144392:22585549}. This helps to reduce the amount of HIV in your body so that your immune system \unskip~\citep{1144392:22585559} can function preferable. This decreases your risk of having complications of HIV (such as HIV infection, cancer) and enhances your quality of life. DTG is an antiretroviral medication \unskip~\citep{1144392:22585560} used, together with other medication, to treat HIV/AIDS \unskip~\citep{1144392:22585568}. It may also be used as part of post display prophylaxis to prevent HIV infection following potential exposure \unskip~\citep{1144392:22585570}. It is taken by mouth. Usual side effects involve trouble sleeping, feeling tired, diarrhea \unskip~\citep{1144392:22585548}, high blood sugar \unskip~\citep{1144392:22585563} and headache. Critical consequences may include allergic reactions \unskip~\citep{1144392:22585551} and liver problems \unskip~\citep{1144392:22585565}. It is unclear if used through pregnancy \unskip~\citep{1144392:22585569} or breastfeeding \unskip~\citep{1144392:22585552} is safe. DTG is an HIV integrates strand shift inhibitor which blocks the work of HIV integrase \unskip~\citep{1144392:22585543}, which is needed for viral replication \unskip~\citep{1144392:22585564}. 

Rilpivirine is a pharmaceutical medicine \unskip~\citep{1144392:22585554} evolved by Tibotec \unskip~\citep{1144392:22585555} for the nursing of HIV infection \unskip~\citep{1144392:22585567}. It is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) \unskip~\citep{1144392:22585561} with big potency, longer half-life \unskip~\citep{1144392:22585557} and reduces side effect profile \unskip~\citep{1144392:22585550} compared with older NNRTIs such as Efavirenz \unskip~\citep{1144392:22585562,1144392:22585545}. Rilpivirine entered phase III clinical trials in April 2008 \unskip~\citep{1144392:22585553,1144392:22585544} and was approved for use in the United States in May 2011 under the brand name Edurant. The chemical representation of Dolutegravir and Rilpivirine was shown in Figure~\ref{f-172562a70af2}.

The literature survey revealed that there is only a few methods reported HPLC \unskip~\citep{1144392:22585547,1144392:22585571} and in UV spectrophotometric method \unskip~\citep{1144392:22585542}, two reports for only DTG \unskip~\citep{1144392:22585546,1144392:22585558}. Most of the methods are cost-effective, and run time is high. The author has therefore tried to develop a more sensitive HPLC approach for the testing of the selected drugs in pharmaceutical formulations. In terms of accuracy, precision, linearity, Limit of detection, Limit of quantification, robustness and forced degradation, the established technique was validated. 
    
\section{Materials and Methods}
\textbf{Reagents and Chemicals}

Acetonitrile, Tri ethyl amine (TEA), Ortho phosphoric acid (OPA) and water (HPLC) were purchased from Merck (India) Ltd. Worli, Mumbai, India. All APIs of DTG and RLP (purity-99.9\%) as reference standards were procured from Glenmark pharmaceutical private Ltd, Andheri (E), Mumbai, India.

\textbf{Instrumentation and chromatographic conditions}

The Waters Alliance e-2695 chromatographic system consisting of the quaternary pump, the PDA detector 2996 with a chromatographic software Empower-2.0 was used. Chromatographic separation was achieved in isocratic mode at room temperature using a waters X-bridge C\ensuremath{_{8}} (150x4.6mm, 3.5\ensuremath{\mu}) column. The mixture of 0.1\% TEA in water change pH-2.5 with OPA: acetonitrile (60:40 v/v) at a flow rate 1.0 ml/min was used as a mobile process. The amount of injection was 10\ensuremath{\mu}l and the eluents were monitored at 230 nm using a PDA detector. 7 minutes of run time was used in the separation of Dolutegravir and Rilpivirine. The peaks were recognized by retention time. A typical chromatogram was shown in Figure~\ref{f-a1f49614afac}.

\textbf{Preparation of standard}

\textbf{\space }Carefully weigh and transfer 100 mg of DTG and 50 mg of RLP working standards in a volumetric flask of capacity 100 ml, app. add 70 ml of diluents and sonicate it for 15 min. to dissolve the components, and made up to the mark with diluents. Further, dilute 5 ml of the above solution to a 50 ml volumetric flask and diluted to volume with diluents. 

\textbf{Preparation of sample solution }

Carefully weigh 20 tablets and take the average of one tablet weight. Powder the tablets using mortar and pistil, and take 250 mg of sample into a flask of capacity 100 ml, app. add 70 ml of diluents and sonicated for 30 min to dissolve the components and then diluted up to the mark with diluents. Further, dilute 5 ml of the above solution to 50 ml diluents and it was filtered through a 0.45 \ensuremath{\mu} nylon syringe filter.
    
\section{Results and Discussion}
Different trials were conducted to achieve good resolution between dolutegravir and rilpivirine. Several types of buffers and movable phases were used for developing the method. None of this movable phase was able to achieve a good resolution between dolutegravir and rilpivirine. After a selected mobile phase slightly raised the resolution and gave better resolution between dolutegravir and rilpivirine. 

The selected mobile phase was 0.1\% TEA and acetonitrile in 60:40 v/v. Development trials were performed with C\ensuremath{_{8}}, C\ensuremath{_{18}}, cyano, phenyl columns, but the separation of the selected drugs were achieved with an X-bridge C\ensuremath{_{8}} column (150x4.6 mm, 3.5 \ensuremath{\mu}) connected to a PDA detector. 

The movable phase flow rate was maintained at 1 ml/min and the PDA detector was observed at 230 nm. The final chromatographic conditions provide better resolution between dolutegravir and rilpivirine. The chromatographic, standard, method and intermediate precision results were represented in Table~\ref{tw-5d9e01234297}.


\begin{table*}[!htbp]
\caption{\boldmath {Outcomes of System Suitability studies and validation} }
\label{tw-5d9e01234297}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5539\linewidth-2\tabcolsep}p{\dimexpr.22229999999999997\linewidth-2\tabcolsep}p{\dimexpr.22380000000000003\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Method characteristics & \cAlignHack Dolutegravir & \cAlignHack Rilpivirine\\
\tblmidrule 
\cAlignHack Theoretical plates &
  \cAlignHack 6368 &
  \cAlignHack 10858\\
\cAlignHack Resolution &
  \cAlignHack - &
  \cAlignHack 5.82\\
\cAlignHack Tailing factor &
  \cAlignHack 1.02 &
  \cAlignHack 1.11\\
\cAlignHack Standard \% RSD &
  \cAlignHack 0.71 &
  \cAlignHack 0.68\\
\cAlignHack Accuracy \% RSD* &
  \cAlignHack 0.36 &
  \cAlignHack 0.30\\
\cAlignHack Method precision (\% RSD)** &
  \cAlignHack 1.26 &
  \cAlignHack 0.87\\
\cAlignHack Intermediate precision (\% RSD)** &
  \cAlignHack 0.44 &
  \cAlignHack 1.12\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*Results are average of three sample preparations with three different drug concentration levels}
    
\item{**Results are average of six sample preparations}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Linearity study results} }
\label{tw-1f3dbfdd05de}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.113\linewidth-2\tabcolsep}p{\dimexpr.23349999999999994\linewidth-2\tabcolsep}p{\dimexpr.362\linewidth-2\tabcolsep}p{\dimexpr.29150000000000002\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Analyte & \cAlignHack Linearity Range & \cAlignHack Equation of calibration curve & \cAlignHack Correlation coefficient\\
\tblmidrule 
\cAlignHack DTG &
  \cAlignHack 10.0-150.0 \ensuremath{\mu}g/ml &
  \cAlignHack Y=28760x+121.8 &
  \cAlignHack 0.999\\
\cAlignHack RLP &
  \cAlignHack 5.0-75.0 \ensuremath{\mu}g/ml &
  \cAlignHack Y=10064x+10930 &
  \cAlignHack 0.999\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/5c8bad71-93eb-4932-852f-0648907bef9a-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/5c8bad71-93eb-4932-852f-0648907bef9a-upicture1.png}{\includegraphics{images/5c8bad71-93eb-4932-852f-0648907bef9a-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Chemical representation of (A) Dolutegravir and (B) Rilpivirine}}
\label{f-172562a70af2}
\end{figure*}
\egroup
\textbf{Validation of the method}

The method was confirmed by using ICH guidelines for precision, accuracy, linearity, specificity, ruggedness, robustness and forced degradation. 

\textbf{System precision}

Six replicate injections of the standard solution containing 100 \ensuremath{\mu}g/ml of DTG and 50 \ensuremath{\mu}g/ml of RLP were assessed to check the system suitability. The values obtained for tailing factor, resolution, plate count were well under the acceptance criteria. 

\textbf{Linearity}

A standard solution containing 100 \ensuremath{\mu}g/ml of DTG and 50 \ensuremath{\mu}g/ml of RLP was established to assess the linearity of the process. (100\% of the amount of the assay targeted concentration). Sequential dilutions were carried out at 10, 25, 50, 100, 125 and 150\% of the target concentration to plot calibration curves. This was injected and the peak areas were used against the concentration. The correlation coefficient values of these analytes were 0.9997 for DTG and 0.9992 for RLP, slope values of DTG and RLP were 28760, 10064 and the intercept values are 121.8, 10930 respectively. Linearity results were shown in Table~\ref{tw-1f3dbfdd05de}, and linearity plots of DTG and RLP were shown in Figure~\ref{f-56eebeb23fc5}, Figure~\ref{f-47f610a56b3b} respectively.


\bgroup
\fixFloatSize{images/ef53ed66-a901-4f95-9e52-0815ba27b928-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/ef53ed66-a901-4f95-9e52-0815ba27b928-upicture2.png}{\includegraphics{images/ef53ed66-a901-4f95-9e52-0815ba27b928-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatogram of standard}}
\label{f-a1f49614afac}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/d365e336-06e8-4bbb-9a61-cbb2817f8f44-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/d365e336-06e8-4bbb-9a61-cbb2817f8f44-upicture3.png}{\includegraphics{images/d365e336-06e8-4bbb-9a61-cbb2817f8f44-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Linearity plot for DTG}}
\label{f-56eebeb23fc5}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/44e0bb4c-742b-458b-8285-ec0281f60db5-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/44e0bb4c-742b-458b-8285-ec0281f60db5-upicture4.png}{\includegraphics{images/44e0bb4c-742b-458b-8285-ec0281f60db5-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Linearity plot for RLP}}
\label{f-47f610a56b3b}
\end{figure}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Outcomes of Accuracy} }
\label{tw-30b5547fd3ac}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.06169999999999999\linewidth-2\tabcolsep}p{\dimexpr.15909999999999997\linewidth-2\tabcolsep}p{\dimexpr.1518\linewidth-2\tabcolsep}p{\dimexpr.1732\linewidth-2\tabcolsep}p{\dimexpr.16130000000000003\linewidth-2\tabcolsep}p{\dimexpr.1554\linewidth-2\tabcolsep}p{\dimexpr.1375\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack \% & \multicolumn{3}{p{\dimexpr(.4841\linewidth-2\tabcolsep)}}{\cAlignHack Dolutegravir} & \multicolumn{3}{p{\dimexpr(.4542\linewidth-2\tabcolsep)}}{\cAlignHack Rilpivirine}\\
\rowcolor{kwdboxcolor}\cAlignHack level & \cAlignHack \ensuremath{\mu}g/ml added & \cAlignHack \ensuremath{\mu}g/ml found & \cAlignHack \% Accuracy & \cAlignHack \ensuremath{\mu}g/ml added & \cAlignHack \ensuremath{\mu}g/ml found & \cAlignHack \% Accuracy\\
\tblmidrule 
\cAlignHack 50 &
  \cAlignHack 50 &
  \cAlignHack 50.1 &
  \cAlignHack 100.28 &
  \cAlignHack 25 &
  \cAlignHack 25.3 &
  \cAlignHack 100.38\\
\cAlignHack 100 &
  \cAlignHack 100 &
  \cAlignHack 100.2 &
  \cAlignHack 100.36 &
  \cAlignHack 50 &
  \cAlignHack 25.1 &
  \cAlignHack 100.69\\
\cAlignHack 150 &
  \cAlignHack 150 &
  \cAlignHack 150.2 &
  \cAlignHack 100.15 &
  \cAlignHack 75 &
  \cAlignHack 25.4 &
  \cAlignHack 100.47\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Outcomes of Robustness} }
\label{tw-d4a0503fafff}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.11510000000000002\linewidth-2\tabcolsep}p{\dimexpr.1661\linewidth-2\tabcolsep}p{\dimexpr.1684\linewidth-2\tabcolsep}p{\dimexpr.1104\linewidth-2\tabcolsep}p{\dimexpr.1578\linewidth-2\tabcolsep}p{\dimexpr.1388\linewidth-2\tabcolsep}p{\dimexpr.1434\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Drug name & \cAlignHack Flow plus  \mbox{}\protect\newline (1.2 ml/min) & \cAlignHack Flow minus  \mbox{}\protect\newline (0.8 ml/min) & \cAlignHack Org Plus (45:55) & \cAlignHack Org minus (35:65) & \cAlignHack pH plus (3.0) & \cAlignHack pH minus (2.0)\\
\rowcolor{kwdboxcolor}\cAlignHack  & \multicolumn{6}{p{\dimexpr(.8849000000000001\linewidth-2\tabcolsep)}}{\cAlignHack \% RSD}\\
\tblmidrule 
\cAlignHack Dolutegravir &
  \cAlignHack 0.22 &
  \cAlignHack 0.16 &
  \cAlignHack 0.28 &
  \cAlignHack 0.35 &
  \cAlignHack 0.48 &
  \cAlignHack 0.65\\
\cAlignHack Rilpivirine &
  \cAlignHack 1.01 &
  \cAlignHack 0.28 &
  \cAlignHack 0.39 &
  \cAlignHack 1.21 &
  \cAlignHack 0.68 &
  \cAlignHack 0.36\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Outcomes of Forced Degradation} }
\label{tw-bfd9e2b27376}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.205\linewidth-2\tabcolsep}p{\dimexpr.1983\linewidth-2\tabcolsep}p{\dimexpr.1967\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Degradation & \cAlignHack DTG (\% Assay) & \cAlignHack \% Degradation & \cAlignHack RLP (\% Assay) & \cAlignHack \% Degradation\\
\tblmidrule 
\cAlignHack Control &
  \cAlignHack 100.58 &
  \cAlignHack 0.00 &
  \cAlignHack 100.36 &
  \cAlignHack 0.00\\
\cAlignHack Acid &
  \cAlignHack 92.36 &
  \cAlignHack 8.22 &
  \cAlignHack 95.18 &
  \cAlignHack 5.18\\
\cAlignHack Alkali &
  \cAlignHack 88.78 &
  \cAlignHack 11.8 &
  \cAlignHack 92.36 &
  \cAlignHack 8.00\\
\cAlignHack Peroxide &
  \cAlignHack 90.36 &
  \cAlignHack 10.22 &
  \cAlignHack 96.45 &
  \cAlignHack 3.91\\
\cAlignHack Reduction &
  \cAlignHack 98.68 &
  \cAlignHack 1.9 &
  \cAlignHack 97.25 &
  \cAlignHack 3.11\\
\cAlignHack Thermal &
  \cAlignHack 95.36 &
  \cAlignHack 5.22 &
  \cAlignHack 93.68 &
  \cAlignHack 6.68\\
\cAlignHack Photolytic &
  \cAlignHack 93.36 &
  \cAlignHack 7.22 &
  \cAlignHack 92.14 &
  \cAlignHack 8.22\\
\cAlignHack Hydrolysis &
  \cAlignHack 91.36 &
  \cAlignHack 9.22 &
  \cAlignHack 90.87 &
  \cAlignHack 9.49\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Limit of detection and quantification}

Limit of detection and quantification minimum concentration levels at which the analyte can be reliably detected, quantified by using the standard formulas (3.3 times \ensuremath{\sigma  }/s and 10 times \ensuremath{\sigma  }/s for LOD and LOQ respectively. LOD values for DTG and RLP were 0.01 \ensuremath{\mu}g/ml, 0.005 \ensuremath{\mu}g/ml and s/n values are 3 and 4 respectively. 

LOQ values of DTG and RLP were 0.1 \ensuremath{\mu}g/ml, 0.05 \ensuremath{\mu}g/ml and s/n values were 23 and 28 respectively. 

\textbf{Precision and Intermediate precision}

For the quantification of DTG and RLP, the method was found to be precise with six sample preparations. The precision was also evaluated on different days. Percent RSD values for DTG and RLP were found to be less than 2.0 percent in precision and intermediate precision.

\textbf{Accuracy}

Accuracy was determined by recovery experiments which were performed at three different levels of concentration (50 percent, 100 percent and 150 percent levels). As per the test method the test solution was injected to three preparations each spike level and the assay was performed. Accuracy outcomes were given in Table~\ref{tw-30b5547fd3ac}.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

\textbf{Robustness}

Robustness of the method was found to be \% RSD should be less than 2.0\%. Slightly variations were done in optimized method parameters like flow rate (\ensuremath{\pm}0.2\%), organic content in the mobile phase (\ensuremath{\pm}5\%), pH of the buffer variations (\ensuremath{\pm}0.5). The results are given in Table~\ref{tw-d4a0503fafff}.

\textbf{Stability}

Stability of standard and sample solutions were prepared from initial to 24 hrs in stored at RT and 2-8\ensuremath{^\circ}C. They are injected at different time intervals and the difference between initial to 24 hrs \% of the assay was not more than 2\%. There is no effect in storage conditions for DTG and RLP drugs. The solutions were stable up to 24 hrs.

\textbf{Forced degradation }

Forced degradation conditions such as acidic, basic, peroxide, reduction, thermal, hydrolysis and photolytic stress were attempted as per IH guidelines. The outcomes of forced degradation were shown in Table~\ref{tw-bfd9e2b27376}.
    
\section{Conclusion}
This method described the quantification of dolutegravir and rilpivirine in bulk and pharmaceutical formulation as per ICH guidelines. The evolved technique was found to be accurate, precise, linear and reliable. The advantage lies in the simplicity of sample preparation and reproducibility data are satisfactory. The evolved chromatographic method can be effectively applied for regular investigation in drug research. 
\section*{Acknowledgement}The authors are thankful to Shree Icon Pharmaceutical Laboratories, Vijayawada, 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 they have conflict of interest for this study.
    

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