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\begin{document}


\def\RunningAuthor{Shanmugasundaram P and Haritha G}
\firstPage{2417}
\articleType{Original Article}
\receivedDate{05 Oct 2020}
\acceptedDate{10 Nov 2020}
\revisedDate{07 Nov 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4488}
\copyrightYear{2020}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{\textbf{Stability indicating RP-UPLC method development and validation for the simultaneous determination of ertugliflozin and metformin in plasma}}
\author{Haritha~G\textsuperscript{1}, 
            Shanmugasundaram~P\textsuperscript{*}\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Research Scholar, Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai, Tamil Nadu, India}~\\{\textsuperscript{2}Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai, Tamil Nadu, India}}

\begin{abstract}
New stability-indicating RP-UPLC technique was developed for the quantification of ertugliflozin and metformin in human plasma and validated as per the regulatory guidelines. Both the drug components and internal standard were spiked to blank plasma and subjected to liquid-liquid extraction with the mobile phase. The resultant solution was infused into Acquity BEH-C18 (1.7 \ensuremath{\mu }, 100\ensuremath{\times}2.1mm) non-polar column comprising NaH\ensuremath{_{2}}PO\ensuremath{_{4\ }} buffer (pH-3.5), methanol and acetonitrile in the ratio of 50:10:40\% \textit{v/v/v } as mobile phase. The detector response and flow of the mobile phase were monitored at 240nm and 0.5ml/min, respectively. The linearity plot was made in the concentration range of 0.1-3.0 \ensuremath{\mu}g/ml for metformin and 0.05-1.5 \ensuremath{\mu}g/ml for ertugliflozin with correlation coefficient value of more than 0.999. The developed method was subjected for bench-top, freeze and thaw, long-term and short-term stability studies and the drug components were stable over the respective conditions. The Lower limit of quantification (LLOQ) for ertugliflozin and metformin were 0.05 and 0.1 \ensuremath{\mu}g/ml, respectively. The findings of precision and accuracy were present in between 2.6 to 4.2 \%RSD and -2 to 3.99 \%RE, respectively. The findings of the stability data were presented below. The \%stability of ertugliflozin and metformin were varying from 96\% to 104\% for ertugliflozin and 96\% to 105\% for metformin. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Ertugliflozin,\newline Metformin: RP-UPLC,\newline US-FDA guidelines,\newline Accuracy,\newline Human Plasma
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Shanmugasundaram~P~\\ Phone:\ 9840126575~\\  Email:\ director.sps@velsuniv.ac.in
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Ertugliflozin (ERGZ) marketed as Steglatro trade name, is utilized in the management of type-2 diabetes. This drug was approved by US-FDA for administration as a monotherapy and also combined with either metformin or sitagliptin \unskip~\citep{935386:20787252}. ERGZ combination with metformin is marketed as Segluromet. In the normal physiological conditions, the blood glucose is filtered for elimination and in glomerulus again reabsorbed, so less than 1\% of this glucose is eliminated in the urination \unskip~\citep{935386:20787273}. This reabsorption process is facilitated by the Na-dependent glucose co-transporter (SGLT), mainly the type-2 which is accountable for reabsorption of glucose up to 90\%. ERGZ is a small inhibitor of the SGLT-2, and its action improves glucose elimination, decreasing hyperglycemia without the necessity of excessive insulin secretion \unskip~\citep{935386:20787272,935386:20787270}. It is chemically designated as (1\textit{S},2\textit{S},3\textit{S},4\textit{R},5\textit{S}) -5- [4- Chloro -3- (4-ethoxybenzyl) phenyl] -1-(hydroxymethyl) -6, 8-dioxabicyclo [3.2.1] octane- 2,3,4-triol with molecular formula and weight of C\ensuremath{_{22}}H\ensuremath{_{25}}ClO\ensuremath{_{7\ }} and 436.89 g\ensuremath{\cdot}mol\ensuremath{^{\ensuremath{-}1 }} respectively (Figure~\ref{f-b65b614144bb}a). 

Metformin (MTFN)\textbf{\space }is the first-line medicine for the management of type-2 diabetes, mostly in weighty people.\ensuremath{^{}} It is also utilized in the management of polycystic ovary syndrome \unskip~\citep{935386:20787260,935386:20787275}. It is not accompanying with weight gaining. The oral route administers it. MTFN reduces the blood glucose concentration by reducing hepatic glucose production (gluconeogenesis), reducing the intestinal glucose absorption and increases insulin sensitivity by an increase in peripheral glucose uptake and utilization \unskip~\citep{935386:20787255,935386:20787276}. It is well recognized that MTFN prevents mitochondrial complex-I action, and it has since been generally assumed that its powerful antidiabetic activities arise through this mechanism \unskip~\citep{935386:20787261,935386:20787274}. The above progressions lead to reductions in blood glucose levels, managing type-2 diabetes and employing positive activities on glycemic control \unskip~\citep{935386:20787267,935386:20787262}. It is chemically designated as \textit{N},\textit{ N}-Dimethylimidodicarbonimidic diamide with molecular formula and weight of C\ensuremath{_{4}}H\ensuremath{_{11}}N\ensuremath{_{5\ }} and 129.167 g\ensuremath{\cdot}mol\ensuremath{^{\ensuremath{-}1, }} respectively (Figure~\ref{f-b65b614144bb}b).


\bgroup
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\centering \makeatletter\IfFileExists{images/a7fe09a4-739e-43e0-975e-373e917ada44-upicture1.png}{\includegraphics{images/a7fe09a4-739e-43e0-975e-373e917ada44-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Chemical structure of a) Ertugliflozin and b) Metformin}}
\label{f-b65b614144bb}
\end{figure}
\egroup
Thorough literature on ERGZ and MTFN reveals that no single method was reported on the stability-indicating bio-analytical method by RP-UPLC in a plasma sample. All the reported methods were on UV-Visible spectrophotometer, RP-HPLC \unskip~\citep{935386:20787254,935386:20787269}  and LC-MS/MS techniques \unskip~\citep{935386:20787250,935386:20787266}  either in single or in combination. Hence, there was the need to develop new stability-indicating RP-UPLC technique for the quantification of ERGZ and MTFN in human plasma.
    
\section{Materials and Methods}
\textbf{Chemicals and Reagents}

ERGZ, MTFN and dapagliflozin (IS) were acquired from Hetero drugs, Hyderabad, India. Methanol and acetonitrile of HPLC grade purity and NaH\ensuremath{_{2}}PO\ensuremath{_{4}} and orthophosphoric acid of analytical reagent grade were attained from Merck Ltd, Mumbai, India. Milli-Q purification system was utilized for the purification of water to get the HPLC grade water for the entire research work.


\bgroup
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\centering \makeatletter\IfFileExists{images/2af5801a-9399-41e2-b295-6d7b29ef5ac6-upicture2.png}{\includegraphics{images/2af5801a-9399-41e2-b295-6d7b29ef5ac6-upicture2.png}}{}
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\caption{\boldmath {Typical chromatogram of ERGZ and MTFN blank plasma}}
\label{f-02329a058367}
\end{figure}
\egroup

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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/2a7b30b3-4ac4-4fd8-8c51-534d91d15e2a-upicture3.png}{\includegraphics{images/2a7b30b3-4ac4-4fd8-8c51-534d91d15e2a-upicture3.png}}{}
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\caption{\boldmath {Typical chromatogram of ERGZ and MTFN at LLOQ level}}
\label{f-fbfefd9b42b2}
\end{figure}
\egroup

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\centering \makeatletter\IfFileExists{images/2cc8720f-6314-4231-95e4-12d3ee9f0e00-upicture4.png}{\includegraphics{images/2cc8720f-6314-4231-95e4-12d3ee9f0e00-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatogram of ERGZ and MTFN at LQC level}}
\label{f-5d69f0ab49a2}
\end{figure}
\egroup

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\centering \makeatletter\IfFileExists{images/481eb053-2cf6-4848-8bf7-6a405804ff8e-upicture5.png}{\includegraphics{images/481eb053-2cf6-4848-8bf7-6a405804ff8e-upicture5.png}}{}
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\caption{\boldmath {Typical chromatogram of ERGZ and MTFN at MQC level}}
\label{f-34afc332efbb}
\end{figure}
\egroup

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\centering \makeatletter\IfFileExists{images/202a081b-6cc0-48ab-97dd-e56083a8c961-upicture6.png}{\includegraphics{images/202a081b-6cc0-48ab-97dd-e56083a8c961-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatogram of ERGZ and MTFN at HQC level}}
\label{f-28fa24e6baca}
\end{figure}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Plasma blank and LLOQ areas of ERGZ and MTFN} }
\label{tw-c6a865a34e0f}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1297\linewidth-2\tabcolsep}p{\dimexpr.22510000000000005\linewidth-2\tabcolsep}p{\dimexpr.30599999999999994\linewidth-2\tabcolsep}p{\dimexpr.3392\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No. & Blank area  & ETGZ LLOQ area & MTFN LLOQ area \\
\tblmidrule 
1  &
  106 &
  27465 &
  35025\\
2  &
  150 &
  27790 &
  35438\\
3  &
  124 &
  33314 &
  42482\\
4  &
  134 &
  25864 &
  32982\\
5  &
  104 &
  30945 &
  39462\\
6  &
  146 &
  22425 &
  28597\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Peak area ratio values of ERGZ and MTFN} }
\label{tw-4486d4802bd3}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2894\linewidth-2\tabcolsep}p{\dimexpr.2002\linewidth-2\tabcolsep}p{\dimexpr.2724\linewidth-2\tabcolsep}p{\dimexpr.238\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.4896\linewidth-2\tabcolsep)}}{\cAlignHack ERGZ} & \multicolumn{2}{p{\dimexpr(.5104\linewidth-2\tabcolsep)}}{\cAlignHack MTFN}\\
\rowcolor{kwdboxcolor}Concentration in \ensuremath{\mu}g/ml  & Area ratio & Concentration in \ensuremath{\mu}g/ml & Area ratio\\
\tblmidrule 
0.05 &
  0.11 &
  0.1 &
  0.14\\
0.075 &
  0.16 &
  0.15 &
  0.22\\
0.125 &
  0.28 &
  0.25 &
  0.35\\
0.375 &
  0.81 &
  0.75 &
  1.05\\
0.675 &
  1.47 &
  1.35 &
  1.89\\
1.00 &
  2.2 &
  2.0 &
  2.72\\
1.25 &
  2.72 &
  2.5 &
  3.51\\
1.50 &
  3.4 &
  3.0 &
  4.21\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Precision and Accuracy of MTFN} }
\label{tw-1e898fb44a30}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.121\linewidth-2\tabcolsep}p{\dimexpr.146\linewidth-2\tabcolsep}p{\dimexpr.12719999999999999\linewidth-2\tabcolsep}p{\dimexpr.1258\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}QC level & Standard concentration (\ensuremath{\mu}g/ml) & \multicolumn{3}{p{\dimexpr(.361\linewidth-2\tabcolsep)}}{\cAlignHack Intra-batch} & \multicolumn{3}{p{\dimexpr(.399\linewidth-2\tabcolsep)}}{\cAlignHack Inter-batch}\\
\rowcolor{kwdboxcolor} &  & Amount found (\ensuremath{\mu}g/ml) & \%RSD & \%RE & Amount found (\ensuremath{\mu}g/ml) & \%RSD & \%RE\\
\tblmidrule 
LLOQ &
  0.1 &
  0.098 &
  2.6 &
  -2 &
  0.097 &
  2.8 &
  -3\\
LQC &
  0.28 &
  0.29 &
  3.1 &
  3.571429 &
  0.287 &
  4.2 &
  2.5\\
MQC &
  2 &
  2.0798 &
  4.2 &
  3.99 &
  2.08 &
  3.7 &
  3.805\\
HQC &
  3 &
  3.11 &
  2.9 &
  3.67 &
  3.107 &
  2.4 &
  3.57\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{RE: Relative error;  RSD: Relative standard deviation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Precision and Accuracy of ERGZ} }
\label{tw-e2a25c3de29d}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1065\linewidth-2\tabcolsep}p{\dimexpr.18849999999999998\linewidth-2\tabcolsep}p{\dimexpr.12830000000000002\linewidth-2\tabcolsep}p{\dimexpr.10229999999999999\linewidth-2\tabcolsep}p{\dimexpr.11280000000000001\linewidth-2\tabcolsep}p{\dimexpr.16150000000000002\linewidth-2\tabcolsep}p{\dimexpr.08569999999999999\linewidth-2\tabcolsep}p{\dimexpr.11440000000000001\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}QC level & Standard concentration (\ensuremath{\mu}g/ml) & \multicolumn{3}{p{\dimexpr(.3434\linewidth-2\tabcolsep)}}{\cAlignHack Intra-batch} & \multicolumn{3}{p{\dimexpr(.3616\linewidth-2\tabcolsep)}}{\cAlignHack Inter-batch}\\
\rowcolor{kwdboxcolor} &  & Amount found (\ensuremath{\mu}g/ml) & \%RSD & \%RE & Amount found (\ensuremath{\mu}g/ml) & \%RSD & \%RE\\
\tblmidrule 
LLOQ &
  0.05 &
  0.098 &
  2.8 &
  -2 &
  0.048 &
  4.21 &
  -4\\
LQC &
  0.14 &
  0.29 &
  3.54 &
  2.14 &
  0.144 &
  3.44 &
  2.85\\
MQC &
  1 &
  8 &
  2.85 &
  4 &
  1.038 &
  2.5 &
  3.8\\
HQC &
  1.5 &
  3.11 &
  4.01 &
  1.2 &
  1.52 &
  3.47 &
  1.33\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{RE: Relative error;  RSD: Relative standard deviation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\bgroup
\fixFloatSize{images/49de0d60-4c61-48e2-95f1-077ebac6e430-upicture7.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/49de0d60-4c61-48e2-95f1-077ebac6e430-upicture7.png}{\includegraphics{images/49de0d60-4c61-48e2-95f1-077ebac6e430-upicture7.png}}{}
\makeatother 
\caption{\boldmath {Calibration plot of ERGZ}}
\label{f-4d38ff0505dc}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/42ae485f-a7af-4527-a3fb-be88bc6d4a7e-upicture8.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/42ae485f-a7af-4527-a3fb-be88bc6d4a7e-upicture8.png}{\includegraphics{images/42ae485f-a7af-4527-a3fb-be88bc6d4a7e-upicture8.png}}{}
\makeatother 
\caption{\boldmath {Calibration plot of MTFN}}
\label{f-6a1894ec8fea}
\end{figure}
\egroup
\textbf{Phosphate Buffer of pH 3.5}

2.5 g of NaH\ensuremath{_{2}}PO\ensuremath{_{4\ }}was weighed accurately and made solubilized in 800 ml HPLC-grade water in a 1000 ml volumetric flask. The volume was made up to the mark with water and pH of the resulting solution was made 3.5 by the addition of 0.1\% orthophosphoric acid solution.

\textbf{Mobile Phase Preparation }

The mobile phase was processed by mixing 500ml of NaH\ensuremath{_{2}}PO\ensuremath{_{4\ }} buffer (pH-3.5), 100ml of methanol and 400 ml of acetonitrile in 1000ml capacity volumetric flask. The resultant solution was degasified by the application vacuum filtration thru 0.45\ensuremath{\mu} membrane filter and sonication techniques. 

\textbf{Diluent}

Diluent was processed by mixing NaH\ensuremath{_{2}}PO\ensuremath{_{4\ }} buffer (pH-3.5) and acetonitrile in the proportion of 50:50\% v/v. 

\textbf{UPLC Chromatographic System}

The chromatographic system utilized for the development and validation equipped with Acquity-UPLC of Waters consisting of PDA-Detector and auto-sampling system. The data generated during the research work was monitored with Waters Empower software. Chromatographic separation was obtained on Acquity BEH-C18 (1.7 \ensuremath{\mu }, 100\ensuremath{\times}2.1mm) non-polar column comprising NaH\ensuremath{_{2}}PO\ensuremath{_{4\ }} buffer (pH-3.5) \unskip~\citep{935386:20787268,935386:20787265}, methanol and acetonitrile in the ratio of 50:10:40\% \textit{v/v/v } as mobile phase. The detector response and flow of the mobile phase were monitored at 240nm and 0.5ml/min, respectively. Infusion volume of 5 \ensuremath{\mu }L and a column temperature of 25 \ensuremath{^\circ}C were maintained throughout the study.  

\textbf{Protocol for Calibration Curve (CC) and Quality Control (QC) Standards}

Individual primary stock (100 \ensuremath{\mu }g/ml) solutions of ERGZ and MTFN were processed by dissolving the required amount of reference drugs in the diluent separately. From the resulting solution, 5 ml of ERGZ and 10 ml of MTFN were transferred into a 100 ml volumetric flask to prepare a mixture and made the volume to 100 ml with diluent to get 10.0\ensuremath{\mu }g/ml of MTFN and 5.0 \ensuremath{\mu }g/ml of ERGZ. Further, serial dilutions were made by spiking to blank plasma to get the CC standards in the concentration range of 0.1 to 3.0 \ensuremath{\mu }g/ml for MTFN and 0.05 to 1.5 \ensuremath{\mu }g/ml for ERGZ. Similarly, low (LQC), medium (MQC) and high (HQC) QC samples were processed to get 0.28, 2.0, 3.0 \ensuremath{\mu }g/ml for MTFN and 0.14, 1.0, 1.5 \ensuremath{\mu }g/ml for ERGZ respectively. The lower limit of quantification (LLOQ) for ERGZ and MTFN were processed at 0.05 and 0.1 \ensuremath{\mu }g/ml, respectively.

\textbf{Protocol for Sample Preparation}

Six different aliquots of spiked plasma samples of 1.0 ml were taken into polypropylene tubes and added 50 \ensuremath{\mu }L of 10\ensuremath{\mu }g/ml dapagliflozin solution. The resulting solution was subjected for vortex for 2.0 min, and to this 5.0 ml of ethyl, acetate solvent was added and vortexed for 25 min. Next, the solution was subjected for centrifugation at 3500 rpm for organic phase separation. The organic phase was separated and evaporated to produce a solid sample. The resulting solid was made solubilized with 200 \ensuremath{\mu }L of mobile phase and processed for UPLC-analysis.

\textbf{Method Validation}

Bioanalytical method for the quantification of ERGZ and MTFN was optimized, after the different method development trials. The optimized method was subjected for the method validation as per the US-FDA regulatory guidelines for bioanalytical method validation \unskip~\citep{935386:20787259,935386:20787263}. The developed method was validated for the parameters: selectivity, accuracy, precision, recovery, linearity and stability studies at bench-top, freeze and thaw, long-term and short-term conditions were executed \unskip~\citep{935386:20787258,935386:20787271,935386:20787277}.  
    
\section{Results and Discussion}
\textbf{Method Development} \textbf{ and Optimization}

With different mobile phase compositions and stationary phases, five different trials were executed, and the sixth trail was optimized. Optimized chromatographic separation was obtained on Acquity BEH-C18 (1.7 \ensuremath{\mu }, 100\ensuremath{\times}2.1mm) non-polar column comprising NaH\ensuremath{_{2}}PO\ensuremath{_{4\ }} buffer (pH-3.5), methanol and acetonitrile in the ratio of 50:10:40\% \textit{v/v/v } as mobile phase. The detector response and flow of the mobile phase were monitored at 240nm and 0.5ml/min, respectively. Infusion volume of 5 \ensuremath{\mu }L and a column temperature of 25 \ensuremath{^\circ}C was maintained throughout the study \unskip~\citep{935386:20787253,935386:20787257}.\textbf{\space }


\bgroup
\fixFloatSize{images/66073825-51b0-45b4-8515-23be76481ace-upicture9.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/66073825-51b0-45b4-8515-23be76481ace-upicture9.png}{\includegraphics{images/66073825-51b0-45b4-8515-23be76481ace-upicture9.png}}{}
\makeatother 
\caption{\boldmath {Precision and accuracy chromatograms of ERGZ and MTFN}}
\label{f-59c0d60199d6}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Stability data of ERGZ and MTFN at LQC and HQC level} }
\label{tw-52f5aca6ddd9}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1497\linewidth-2\tabcolsep}p{\dimexpr.0869\linewidth-2\tabcolsep}p{\dimexpr.0808\linewidth-2\tabcolsep}p{\dimexpr.0826\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}p{\dimexpr.1124\linewidth-2\tabcolsep}p{\dimexpr.0876\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Stability condition & QC level & \multicolumn{4}{p{\dimexpr(.3758\linewidth-2\tabcolsep)}}{\cAlignHack ERGZ} & \multicolumn{4}{p{\dimexpr(.3876\linewidth-2\tabcolsep)}}{\cAlignHack MTFN}\\
\rowcolor{kwdboxcolor} &  & X & Y & \%RSD & \%Stability & X & Y & \%RSD & \%Stability\\
\tblmidrule 
Freeze-thaw  &
  LQC &
  0.14 &
  0.135 &
  3.12 &
  96.83 &
  0.28 &
  0.273 &
  2.64 &
  97.84\\
stability &
  HQC &
  1.5 &
  1.52 &
  1.07 &
  101.35 &
  3 &
  2.88 &
  3.15 &
  96.23\\
Long-term  &
  LQC &
  0.14 &
  0.14 &
  4.12 &
  103.85 &
  0.28 &
  0.28 &
  4.24 &
  102.35\\
stability &
  HQC &
  1.5 &
  1.46 &
  2.84 &
  97.48 &
  3 &
  3.052 &
  2.64 &
  101.74\\
Short-term  &
  LQC &
  0.14 &
  0.135 &
  3.24 &
  96.741 &
  0.28 &
  0.28 &
  1.09 &
  102.64\\
stability &
  HQC &
  1.5 &
  1.52 &
  3.22 &
  101.38 &
  3 &
  3.135 &
  1.94 &
  104.5\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{X, original concentration (ng/ml); Y, average concentrations (ng/ml) of analytes}
\end{tablenotes}
\end{threeparttable}

\end{table*}
In all the five trials there was no baseline separation in trial-1 and trial-2, merged peaks with no baseline separation was observed in trail-3, peak shape and resolution was poor in trail-4, and two peaks were merged in the trial -5. Optimized chromatographic peaks (blank, LLOQ, LQC, MQC and HQC) were shown in Figures~\ref{f-02329a058367}, \ref{f-fbfefd9b42b2}, \ref{f-5d69f0ab49a2}, \ref{f-34afc332efbb} and~\ref{f-28fa24e6baca}.

\textbf{Method Validation}

\textbf{Selectivity and Specificity}

Method selectivity was executed at LLOQ level of ERGZ and MTFN by equating the blank plasma responses from 6 different batches with responses produced by the LLOQ standard. It was processed by infusing six blank plasma samples from different batches \unskip~\citep{935386:20787262,935386:20787264}. The findings were shown in Table~\ref{tw-c6a865a34e0f}.

\textbf{Calibration Curve}

Linearity plots for the ERGZ and MTFN were made by calculating peak area ratios for drug components concerning the internal standard (Oliver Scherf-Clavel et al., 2019). The area ratios were calculated for ERGZ and MTFN in the concentration range of 0.05-1.5 \ensuremath{\mu}g/ml and 0.1-3.0 \ensuremath{\mu}g/ml, respectively. The findings were represented in Table~\ref{tw-4486d4802bd3}. The regression equation of ERGZ and MTFN were found to be y = 2.232x - 0.0152 and y = 1.3955x - 0.0006 with correlation coefficient values of 0.9992 and 0.9996 respectively (Figures~\ref{f-4d38ff0505dc} and~\ref{f-6a1894ec8fea}).

\textbf{Precision and Accuracy}

Method precision and accuracy were processed in intra-batch and inter-batch, by infusing six spiked plasma samples at HQC, MQC, LQC and LLOQ levels in a particular batch and three consecutive batches, respectively \unskip~\citep{935386:20787251,935386:20787257}. Precision was determined the form of percentage relative standard deviation (RSD), and accuracy was estimated in the form of \%RE (relative error). Accuracy and precision findings were tabulated in Tables~\ref{tw-1e898fb44a30} and~\ref{tw-e2a25c3de29d}  (Figure~\ref{f-59c0d60199d6}). The findings of precision and accuracy were present in between 2.6 to 4.2 and -2 to 3.99, respectively.

\textbf{Stability Studies}

Stability protocol was processed for ERGZ and MTFN in human plasma for the estimation of stability of the drugs under different conditions like long term stability at -20\ensuremath{^\circ}C for 20 days, short term stability at 25\ensuremath{^\circ}C for 48 hrs and freeze (at -20\ensuremath{^\circ}C) and thaw (at room temperature) cycles for three times \unskip~\citep{935386:20787250,935386:20787256}. By equating the original fresh concentrations with the stability samples, \%RSD and \%stability was calculated for ERGZ and MTFN. The findings of the stability data were presented in Table~\ref{tw-52f5aca6ddd9}. 

The \% stability of ERGZ and MTFN were varying from 96 to 104 for ERGZ and 96 to 105 for MTFN.
    
\section{Conclusion}
New stability-indicating RP-UPLC chromatographic method was developed and validated for the quantification of ERGZ and MTFN in human plasma. The chromatographic elution was processed on Acquity BEH-C18 (1.7 \ensuremath{\mu }, 100\ensuremath{\times}2.1mm) non-polar column comprising NaH\ensuremath{_{2}}PO\ensuremath{_{4\ }} buffer (pH-3.5), methanol and acetonitrile in the ratio of 50:10:40\% \textit{v/v/v} as mobile phase. The detector response and flow of the mobile phase were monitored at 240nm and 0.5ml/min, respectively. The linearity plot was made in the concentration range of 0.1-3.0 \ensuremath{\mu}g/ml for MTFN and 0.05-1.5 \ensuremath{\mu}g/ml for ERGZ. The findings of precision and accuracy were present in between 2.6 to 4.2 \%RSD and -2 to 3.99 \%RE, respectively. The developed method was subjected for bench-top, freeze and thaw, long-term and short-term stability studies and the drug components were stable over the respective conditions. The \%stability of ERGZ and MTFN were varying from 96\% to 104\% for ERGZ and 96\% to 105\% for MTFN. The Lower limit of quantification (LLOQ) for ERGZ and MTFN were 0.05 and 0.1 \ensuremath{\mu}g/ml, respectively. The developed method can successfully apply for the estimation of ERGZ and MTFN in human plasma samples and also useful in the pharmacokinetic studies.

\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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