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\def\RunningAuthor{Rama Rao B et al.}
\firstPage{1747}
\articleType{Original Article}
\receivedDate{17 Aug 2020}
\acceptedDate{17 Sep 2020}
\revisedDate{14 Aug 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4366}
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\def\authorCount{3}
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\title{\textbf{Simultaneous Estimation of Bempedoic Acid and Ezetimibe by UPLC}}
\author{Rama~Rao~B\textsuperscript{*}\textsuperscript{1},  
            Venkateswarlu~BS\textsuperscript{2}, 
            Venkata~Rao~V\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Pharmaceutical Analysis, MAM College of Pharmacy, Narasaraopet, Andhra Pradesh 522601, India}~\\{\textsuperscript{2}Department of Pharmaceutics, Vinayaka Mission's College of Pharmacy, Kondappanaickenpatti, Salem-636008, Tamil Nadu, India}~\\{\textsuperscript{3}Department of Pharmaceutical Analysis, Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Chowdavaram, Andhra Pradesh-522019, India}}

\begin{abstract}
A selective and novel method has been optimized for the evaluation of Bempedoic Acid and Ezetimibe in bulk samples by U.P.L.C. The principle analyzes were eluted with the conditions of mobile phase having the 0.1\% orthophosphoric acid and acetonitrile (90:10 ratio) using the X bridge C18 (250 \ensuremath{\times} 4.6 mm, 5\ensuremath{\mu }m) analytical column with the 1.0 ml/min flow rate and 10 \ensuremath{\mu}l sample volume at 290 nm in a photodiode array detector. The retention times of Bempedoic Acid and Ezetimibe were 0.27 min and 0.73 min with the total run time of 2 min. The curve indicates the correlation coefficient (\textit{r\ensuremath{^{2}}}) was superior by having the value equal to 0.998 with a linear range of 30 n.g/m.l- 225 n.g/m.l for Bempedoic Acid and for Ezetimibe 150 n.g/m.l-1125 n.g/m.l. The correlation coefficient (\textit{r\ensuremath{^{2}}}) found linear. The LOQ and LOD for the Bempedoic Acid and Ezetimibe found 2.5 n.g/m.l and 7.5 n.g/m.l and 2.9 n.g/m.l and 8.9 n.g/m.l. After the method optimization, the method was validated as per ICH guidelines. As per the results obtained in the method validation, there was no interference of the blank and carryover problem even at the LOQ level quantification. Both LOQ and LOD of this method was verified practically in the instrument with S/N ratio criteria. The results were found satisfactory.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Bempedoic Acid,\newline Ezetimibe,\newline UPLC,\newline Validation,\newline Retention Time
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Rama~Rao~B~\\ Phone:\ 8106253172~\\  Email:\ rraoanalysis@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Bempedoic acid and ezetimibe is an inhibitor of adenosine triphosphateitrate lyase (ACL)\unskip~\citep{903355:20499290,903355:20499285} and a combination of cholesterol absorption inhibitors indicated as a dietary supplement and maximally tolerated statin therapy for the treatment of adults with heterozygous familial hypercholesterolemia or established atherosclerotic cardiovascular disease requiring additional lowering of LDL-C \unskip~\citep{903355:20499292,903355:20499287}.  Bempedoic acid is an adenosine triphosphate-citrate lyase (ACL) inhibitor. The molecular formula is C19H36O5, and the molecular weight is 344.5 grams per mole \unskip~\citep{903355:20499281,903355:20499282}. Ezetimibe is in a class of lipid-lowering compounds that inhibits the intestinal absorption of cholesterol and related phytosterols. The empirical formula is C24H21F2NO3. Its molecular weight is 409.4 \unskip~\citep{903355:20499286,903355:20499294}. Based on the literature survey there was no analytical method for this new formulation i.e. Bempedoic acid and Ezetimibe \unskip~\citep{903355:20499293,903355:20499283}. There are several methods were developed with other combination \unskip~\citep{903355:20499288,903355:20499295}. For the Bempedoic acid combination there was lack of sensitive analytical methods \unskip~\citep{903355:20499291,903355:20499289} for the identification and quantification in bulk samples and there was no sensitive analytical method having the low detectability to quantify the product \unskip~\citep{903355:20499284}.
\bgroup
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\makeatother 
\caption{\boldmath {Chemical structures of A) Bempedoic acid B) Ezetimibe.}}
\label{f-91b05d2ed5be}
\end{figure}
\egroup

    
\section{Materials and Methods}
Bempedoic acid and Ezetimibe Figure~\ref{f-91b05d2ed5be} were obtained as gift sample from MICRO LABS, Bangalore. 

High purity acetonitrile, water (Milli {\textemdash} Q system, Millipore, Bedford, MA, USA), All chemicals were high purity grade.

\textbf{Conditions of chromatography}

Separation and estimation was performed using UPLC (Waters with PDA detector), X bridge C18 (250mm, 4.6 mm, 5\ensuremath{\mu }m) column used in the experiment.


\bgroup
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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/8e656190-88d5-46a6-ba0e-29746651a7e3-upicture2.png}{\includegraphics{images/8e656190-88d5-46a6-ba0e-29746651a7e3-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Linearity of Bempedoic Acid.}}
\label{f-9a640c0cf2b0}
\end{figure}
\egroup
The mobile phase was prepared by mixing 0.1 percent Ortho Phosphoric Acid pH adjusted to 2.5 buffer: the ratio of (90:10) acetonitrilein was filtered and degassed and detection was at 290 nm. 

The retention times of Bempedoic Acid and Ezetimibe hydro chloride were 0.27 min and 0.73 min with the total run time of 2 min.

\textbf{Solution Preparation for standards}

Standard stock solution of Bempedoic Acid and Ezetimibe pure drug (1mg / ml) was prepared by accurately weighing approximately 100 mg of the drug and transferred to a 100 ml volumetric flask and dissolved in a diluent.

\textbf{Buffer preparation and Mobile phase}

Accurately transferred 1ml of concentrated orthophosphoric acid in 1000ml of volumetric flask add about 900ml of milli water added to add 1ml of triethylamine and degas to the ultrasonic water bath for 10 minutes and finally make up the volume with water, then adjust the pH to 2.5 with dil. orthophosphoric acid. 0.1\% OrthoPhosphoric Acid buffer solution (pH 2.5) and Acetonitrile in the ratio of 90:10 (v/v). Prepared the diluent by taking 2000 ml of water in 4000 ml mobile phase bottle and added 2000 ml of methanol and degassed to get 4000 ml of diluent.


\bgroup
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\centering \makeatletter\IfFileExists{images/5e10d88d-516a-4b97-b8bd-562660b3b68d-upicture3.png}{\includegraphics{images/5e10d88d-516a-4b97-b8bd-562660b3b68d-upicture3.png}}{}
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\caption{\boldmath {Linearity of Ezetimibe.}}
\label{f-a27e3758f598}
\end{figure}
\egroup
\textbf{Method} \textbf{ Validation}

\textbf{Specificity}

To verify the method validation in terms of the selectivity and exactness injected triplicate preparations of 100 \% concentrations of Bempedoic Acid and Ezetimibe. Then injected one blank also to prove the method was not having the carryover issue. The limit for the specificity is, it should pass the system suitability criteria and there should not be RT shift for all the three preparations.\textit{\space }

\textbf{Performance of the instrument}

To verify the system producing the consistent results with the optimized method injected the standard for six times with the criteria of \% RSD for retention time and area NMT 2.0\%, theoretical plates NLT 3000 plates, tailing factor NMT 1.5 and resolution NLT 2.


\begin{table*}[!htbp]
\caption{\boldmath {Outcomes of precision.} }
\label{tw-bff0fb2111eb}
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\begin{tabulary}{\linewidth}{p{\dimexpr.26360000000000003\linewidth-2\tabcolsep}p{\dimexpr.4254\linewidth-2\tabcolsep}p{\dimexpr.31099999999999998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack \textbf{Sample No} & \cAlignHack \textbf{Area of Bempedoic Acid } & \cAlignHack \textbf{Area of Ezetimibe }\\
\tblmidrule 
\cAlignHack Mean &
  \cAlignHack 221195 &
  \cAlignHack 455388.5\\
\cAlignHack SD &
  \cAlignHack 194.57 &
  \cAlignHack 4889.43\\
\cAlignHack \% RSD &
  \cAlignHack 0.09 &
  \cAlignHack 1.07\\
\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Interday precision data}}\\
\cAlignHack Mean &
  \cAlignHack 219216.33 &
  \cAlignHack 458224.83\\
\cAlignHack SD &
  \cAlignHack 4112.48 &
  \cAlignHack 7111.48\\
\cAlignHack \% RSD &
  \cAlignHack 1.88 &
  \cAlignHack 1.55\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Outcomes of Accuracy (Recovery).} }
\label{tw-d69675279dba}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1242\linewidth-2\tabcolsep}p{\dimexpr.13229999999999997\linewidth-2\tabcolsep}p{\dimexpr.15329999999999998\linewidth-2\tabcolsep}p{\dimexpr.1573\linewidth-2\tabcolsep}p{\dimexpr.12654444444444442\linewidth-2\tabcolsep}p{\dimexpr.13350000000000001\linewidth-2\tabcolsep}p{\dimexpr.06500000000000001\linewidth-2\tabcolsep}p{\dimexpr.10785555555555558\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack \textbf{Level of \% recovery} & \cAlignHack \textbf{Target Conc } \textbf{(n.g/m.l)} & \cAlignHack \textbf{Amount of drug Spiked (n.g/m.l)} & \cAlignHack \textbf{Amount found (n.g/m.l)} & \cAlignHack \textbf{\%Recovery} & \cAlignHack \textbf{Mean Value} & \cAlignHack \textbf{S.D} & \cAlignHack \textbf{\% R.S.D}\\
\tblmidrule 
\multicolumn{8}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Bempedoic Acid }}\\
\multicolumn{1}{p{\dimexpr(.1242\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 80}} &
  \multicolumn{1}{p{\dimexpr(.13229999999999997\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 180}} &
  \multicolumn{1}{p{\dimexpr(.15329999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 144}} &
  \cAlignHack 144.29 &
  \cAlignHack 100.20 &
  \multicolumn{1}{p{\dimexpr(.13350000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 99.62}} &
  \multicolumn{1}{p{\dimexpr(.06500000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.86}} &
  \multicolumn{1}{p{\dimexpr(.10785555555555558\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.86}}\\
 &
   &
   &
  \cAlignHack 144.04 &
  \cAlignHack 100.03 &
   &
   &
  \\
 &
   &
   &
  \cAlignHack 142.03 &
  \cAlignHack 98.63 &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.1242\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100}} &
  \multicolumn{1}{p{\dimexpr(.13229999999999997\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 180}} &
  \multicolumn{1}{p{\dimexpr(.15329999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 180}} &
  \cAlignHack 180.04 &
  \cAlignHack 100.02 &
  \multicolumn{1}{p{\dimexpr(.13350000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100.04}} &
  \multicolumn{1}{p{\dimexpr(.06500000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.02}} &
  \multicolumn{1}{p{\dimexpr(.10785555555555558\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.02}}\\
 &
   &
   &
  \cAlignHack 180.08 &
  \cAlignHack 100.04 &
   &
   &
  \\
 &
   &
   &
  \cAlignHack 180.11 &
  \cAlignHack 100.06 &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.1242\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 120}} &
  \multicolumn{1}{p{\dimexpr(.13229999999999997\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 180}} &
  \multicolumn{1}{p{\dimexpr(.15329999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 216}} &
  \cAlignHack 214.68 &
  \cAlignHack 99.39 &
  \multicolumn{1}{p{\dimexpr(.13350000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100.20}} &
  \multicolumn{1}{p{\dimexpr(.06500000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.85}} &
  \multicolumn{1}{p{\dimexpr(.10785555555555558\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.85}}\\
 &
   &
   &
  \cAlignHack 218.36 &
  \cAlignHack 101.09 &
   &
   &
  \\
 &
   &
   &
  \cAlignHack 216.25 &
  \cAlignHack 100.12 &
   &
   &
  \\
\multicolumn{8}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Ezetimibe}}\\
\multicolumn{1}{p{\dimexpr(.1242\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 80}} &
  \multicolumn{1}{p{\dimexpr(.13229999999999997\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 900}} &
  \multicolumn{1}{p{\dimexpr(.15329999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 720}} &
  \cAlignHack 721.45 &
  \cAlignHack 100.20 &
  \multicolumn{1}{p{\dimexpr(.13350000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 99.62}} &
  \multicolumn{1}{p{\dimexpr(.06500000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.86}} &
  \multicolumn{1}{p{\dimexpr(.10785555555555558\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.87}}\\
 &
   &
   &
  \cAlignHack 720.23 &
  \cAlignHack 100.03 &
   &
   &
  \\
 &
   &
   &
  \cAlignHack 710.14 &
  \cAlignHack 98.63 &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.1242\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100}} &
  \multicolumn{1}{p{\dimexpr(.13229999999999997\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 900}} &
  \multicolumn{1}{p{\dimexpr(.15329999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 900}} &
  \cAlignHack 900.23 &
  \cAlignHack 100.03 &
  \multicolumn{1}{p{\dimexpr(.13350000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100.05}} &
  \multicolumn{1}{p{\dimexpr(.06500000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.02}} &
  \multicolumn{1}{p{\dimexpr(.10785555555555558\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.02}}\\
 &
   &
   &
  \cAlignHack 900.43 &
  \cAlignHack 100.05 &
   &
   &
  \\
 &
   &
   &
  \cAlignHack 900.56 &
  \cAlignHack 100.06 &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.1242\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 120}} &
  \multicolumn{1}{p{\dimexpr(.13229999999999997\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 900}} &
  \multicolumn{1}{p{\dimexpr(.15329999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 1080}} &
  \cAlignHack 1078.32 &
  \cAlignHack 99.84 &
  \multicolumn{1}{p{\dimexpr(.13350000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100.35}} &
  \multicolumn{1}{p{\dimexpr(.06500000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.99}} &
  \multicolumn{1}{p{\dimexpr(.10785555555555558\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 0.99}}\\
 &
   &
   &
  \cAlignHack 1096.18 &
  \cAlignHack 101.50 &
   &
   &
  \\
 &
   &
   &
  \cAlignHack 1076.97 &
  \cAlignHack 99.72 &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Linearity} \textbf{ \& Range}

The method linearity was verified with the five concentrations of 100\% concentration as 30, 75, 105, 150, 180, 225 n.g/m.l of Bempedoic Acid (30 to 225 n.g/m.l) and 50, 75 525, 750, 900, 1125 n.g/m.l of Ezetimibe (50 to 1125 n.g/m.l)\textit{\space }with the acceptance criteria of regression coefficient (R\ensuremath{^{2}}) NLT 0.99.

\textbf{Precision}

After passing the specificity and system suitability criteria the method was verified for the system precision and method precision with the limit of \% RSD for the retention time and area NMT 2\%. The intermediate precision was verified on the next day with another column by following the limit as \% RSD for the retention time and area should be NMT 2\%.\textit{\space }

\textbf{Accuracy}\textbf{ (Recovery)} \textbf{\space }

To verify the method accuracy triplicate preparations were prepared at 80\% and 100\% and 120\% level of 100\% concentration (180 n.g/m.l of Bempedoic Acid and 900 n.g/m.l of Ezetimibe) by spiking the standard into the diluent. Calculated the recovery with the acceptance criteria 95-105\%.

\textbf{Robustness}

To verify the method efficiency when the minor changes happened in the optimized method parameters like mobile phase composition and column temperature and flow and buffer pH this parameter was performed with the criteria, it should pass the system suitability criteria.


\begin{table*}[!htbp]
\caption{\boldmath {Outcomes of the Robustness.} }
\label{tw-7d65ee3e23f8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.05729999999999999\linewidth-2\tabcolsep}p{\dimexpr.1811000000000001\linewidth-2\tabcolsep}p{\dimexpr.2386\linewidth-2\tabcolsep}p{\dimexpr.11629999999999999\linewidth-2\tabcolsep}p{\dimexpr.14579999999999998\linewidth-2\tabcolsep}p{\dimexpr.1222\linewidth-2\tabcolsep}p{\dimexpr.1387\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{\cAlignHack \textbf{S No}}} & {\multirow{2}{*}{\textbf{Parameter}}} & {\multirow{2}{*}{\textbf{Condition }}} & \multicolumn{2}{p{\dimexpr(.26209999999999997\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Bempedoic Acid}} & \multicolumn{2}{p{\dimexpr(.2609\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Ezetimibe}}\\
\rowcolor{kwdboxcolor} &  &  & \cAlignHack \textbf{\textbf{Area(n=3)}} & \cAlignHack \textbf{\textbf{\% change}} & \cAlignHack \textbf{Area(n=3)} & \cAlignHack \textbf{\% change }\\
\tblmidrule 
\cAlignHack 1  &
   Standard &
  Standard conditions &
  \cAlignHack 221455 &
  \cAlignHack 0  &
  \cAlignHack 451134 &
  \cAlignHack 0 \\
\multicolumn{1}{p{\dimexpr(.05729999999999999\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{\cAlignHack 2 }} &
  \multicolumn{1}{p{\dimexpr(.1811000000000001\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Mobile Phase composition}} &
  92:8, \%v/v &
  \cAlignHack 221351 &
  \cAlignHack 0.1  &
  \cAlignHack 450637 &
  \cAlignHack 0.14\\
 &
   &
  88:12, \%v/v &
  \cAlignHack 221247 &
  \cAlignHack 1.33 &
  \cAlignHack 470140 &
  \cAlignHack 0.55\\
\multicolumn{1}{p{\dimexpr(.05729999999999999\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{\cAlignHack 3 }} &
  \multicolumn{1}{p{\dimexpr(.1811000000000001\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Mobile phase pH}} &
  2.7  &
  \cAlignHack 221143 &
  \cAlignHack 0.83 &
  \cAlignHack 459643 &
  \cAlignHack 0.11\\
 &
   &
  2.3  &
  \cAlignHack 221039 &
  \cAlignHack 0.52 &
  \cAlignHack 459146 &
  \cAlignHack 0.56\\
\multicolumn{1}{p{\dimexpr(.05729999999999999\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{\cAlignHack 4 }} &
  \multicolumn{1}{p{\dimexpr(.1811000000000001\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Wavelength}} &
  288 nm &
  \cAlignHack 220935 &
  \cAlignHack 0.19 &
  \cAlignHack 458649 &
  \cAlignHack 0.07\\
 &
   &
  292 nm &
  \cAlignHack 220831 &
  \cAlignHack 0.23 &
  \cAlignHack 463485 &
  \cAlignHack 1.51\\
\multicolumn{1}{p{\dimexpr(.05729999999999999\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{\cAlignHack 5}} &
  \multicolumn{1}{p{\dimexpr(.1811000000000001\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Flow rate}} &
  1.2 &
  \cAlignHack 220727 &
  \cAlignHack 0.25 &
  \cAlignHack 464988 &
  \cAlignHack 0.07\\
 &
   &
  0.8 &
  \cAlignHack 220623 &
  \cAlignHack 0.18 &
  \cAlignHack 466491 &
  \cAlignHack 1.51\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Limit} \textbf{ of detection and Limit of quantification}

The detection limit (LOD) is defined as the lowest analyte concentration that can be reliably distinguished from the background levels. The quantification limit (LOQ) of an individual analytical procedure is the lowest amount of analyze which can be quantitatively determined with appropriate accuracy and precision. 


\bgroup
\fixFloatSize{images/c3ffe888-83e2-4144-80aa-5d7f01499f6d-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c3ffe888-83e2-4144-80aa-5d7f01499f6d-upicture4.png}{\includegraphics{images/c3ffe888-83e2-4144-80aa-5d7f01499f6d-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Blank chromatogram of Bempedoic Acid \& Ezetimibe.}}
\label{f-0e8a6397e593}
\end{figure}
\egroup
\textbf{Solution} \textbf{ Stability}

Stability of the solution was assured using standard solutions and test stock. These stocks were prepared and stored for 36 hrs under room temperature and refrigerated conditions and differences were calculated for percentage.

\textbf{Filter} \textbf{ validation}

To evaluate the impact of PVDF and Nylon filters on the assay results the samples were analyzed after passing through the filters.
    
\section{Results and Discussion}
There was a clear separation and good resolution and without any carryover was achieved with this method as shown in the Figures~\ref{f-0e8a6397e593} and~\ref{f-693f5a1997f3}. The system suitability acceptance criteria also found satisfactory as ICH guidelines. For the system precision parameter the \%RSD of area for the Bempedoic acid and Ezetimibe achieved less than 2\% against the limit. For the method precision parameter the \%RSD of RT and area for the Bempedoic acid and Ezetimibe achieved against the limit NMT 2.0\%. 

The linearity parameter was quantified by peak area Vs concentration methodology. Different concentrations from 50 to 1125 n.g/m.l standard solutions and 30 to 225 n.g/m.l for Bempedoic acid and Ezetimibe were prepared and injected into the system. The calculated regression coefficient for Bempedoic acid and Ezetimibe are nearer to 0.998 as shown in the Figures~\ref{f-9a640c0cf2b0} and~\ref{f-a27e3758f598}.


\bgroup
\fixFloatSize{images/455630ca-cb66-4d35-bae4-6c4262110da5-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/455630ca-cb66-4d35-bae4-6c4262110da5-upicture5.png}{\includegraphics{images/455630ca-cb66-4d35-bae4-6c4262110da5-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of Bempedoic Acid \& Ezetimibe.}}
\label{f-693f5a1997f3}
\end{figure}
\egroup
The method was verified for the ruggedness as inter day and intra day precision. For the intermediate precision parameter the \%RSD of area for the Bempedoic acid and Ezetimibe achieved in day -1 as 0.617, 0.664 and on the next day 1.80, 1.50 against the limit NMT 2.0\% as shown in the Table~\ref{tw-bff0fb2111eb}. The recovery for the 80\%, 100\% and 120\% was more than 99\% against the acceptance criteria 95-105\% as shown in the Table~\ref{tw-d69675279dba}. 

To evaluate the method capability of producing precise results with the minor variations of flow, mobile phase composition, pH, column temperature variations as a robustness was performed. The results were shown in the Table~\ref{tw-7d65ee3e23f8}. 

The results proving that the method was stable to produce consistent results with the minor variation of the method parameters. The compatibility of the filters were verified with the PVDF and Nylon filters.

The LOQ and LOD were identified by injecting the lower concentrations with the S/N ratio criteria,2.5n.g / ml for Bempedoic Acid and 7.5n.g/ m.l for Ezetimibe and 2.9n.g/ m.l for Bempedoic Acid and 8.9n.g/ m.l for Ezetimibe. 

Standard solution are stable for 36 hours at 5\ensuremath{^\circ}C as the percentage difference was found to be less than 2.0 percent in the area. Filter interference was performed of 0.45\ensuremath{\mu } filters (Nylon, PVDF), and the percentage difference for sample solutions and standard solutions calculated against centrifuged samples and standard was found to be below 2.0 percent.

During method optimization initially organic solvents used as mobile phase with the water in different composition. But both compounds were not detected. Then started usage of buffer with organic solvent such as acetonitrile in different ratios and pH with the C\ensuremath{_{18}} column. Finally, the method was found optimized with the conditions of mobile phase 0.1 percent ortho Phosphoric Acid buffer system: Acetonitrile (90:10 v / v), wavelength 290 nm, flow rate of 1.0 ml/min, column temperature of 25\ensuremath{^\circ}C, sample compartment temperature of 10\ensuremath{^\circ}C, sample volume of 10 \ensuremath{\mu}l. With this method both actives i.e. Bempedoic Acid and Ezetimibe were having good resolution and symmetry.
    
\section{Conclusions}
Based on the results obtained in the developed method was very sensitive, accurate, linear and economical. Due to short time of chromatographic program more samples can be analyzed within the short period of time, which will be helpful in the industry at the time of multiple products manufacturing continuously. The method was met all the predefined acceptance criteria. With this method the sample of bulk and formulation samples can be analyzed. As the method having the capability of detecting the formulation, bulk shelf life samples also can be analyzed by using this method.
\section*{Acknowledgement}The authors are thankful to Principal and chairman, MAM College of Pharmacy, Narasaraopet, Andhra Pradesh, India, for providing necessary research facilities for carrying 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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