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\begin{document}


\def\RunningAuthor{Raja Sundararajan and Navya Singamsetty}
\firstPage{931}
\articleType{Original Article}
\receivedDate{18 Jan 2021}
\acceptedDate{24 Feb 2021}
\revisedDate{20 Feb 2021}
\journalVolume{2021, 12}
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\journalDoi{ijrps.v12i1.4471}
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\title{Analytical Method Development and Validation for Determination of Selpercatinib by Using RP-HPLC}
\author{Navya~Singamsetty, 
            Raja~Sundararajan\textsuperscript{*}~\\[5pt]{Department of Pharmaceutical Analysis, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam {\textendash} 530 045, Andhra Pradesh, India}}

\begin{abstract}
A novel, specific and precise RP-HPLC method has been developed and validated for the quantification of selpercatinib in pure and its pharmaceutical dosage form. Selpercatinib is a kinase interrupter with increased selectivity for rearranged during transfection (RET) tyrosine kinase receptors (RTKs) above the additional RTK classes. It is used for the treatment of RET fusion-positive non-small cell lung carcinoma (NSCLC). The segregation was accomplished on the Zorbax C18 column (150 x 4.6 mm) with a 5 \ensuremath{\mu} particle size. 0.1\% orthophosphoric acid and Acetonitrile (ACN) (60:40 v/v) was used as an optimized mobile phase at a flow rate of 1ml/min. The wavelength selected was 220.0nm. The retention time for selpercatinib was 2.653 min. The linearity of selpercatinib was detected to be 5- 30\ensuremath{\mu }g/ml. Linearity equations obtained for selpercatinib was y = 18428x + 2196.2 with correlation coefficient 0.999. The \% RSD for precision was found to be less than 2\%. \%Recovery was obtained as 99.74\% for selpercatinib. The LOD and LOQ for selpercatinib were obtained as 0.02 \ensuremath{\mu}g/ml and 0.05 \ensuremath{\mu}g/ml.  
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Selpercatinib,\newline Method Development,\newline Validation,\newline ICH guidelines,\newline RP-HPLC,\newline RTKs
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Raja~Sundararajan~\\ Phone:\ +91-9160508261~\\  Email:\ sraja61@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Selpercatinib (C\ensuremath{_{29}}H\ensuremath{_{31}}N\ensuremath{_{7}}O\ensuremath{_{3}}) is chemically 6-(2-hydroxy-2-methyl propoxy)-4-[6-[6-[(6-methoxy pyridin-3-yl)methyl]-3,6-diazabicyclo [3.1.1] heptan-3-yl]pyridin-3-yl] pyrazolo [1,5-a] pyridine-3-carbonitrile. The molecular weight of selpercatinib is 525.6 g/mol. It is a kinase inhibitor with improved selectivity for rearranged during transfection (RET) tyrosine kinase receptors (RTKs) above the additional RTK classes. Selpercatinib is indicated to treat non-small cell lung carcinoma \unskip~\citep{1027058:21592198} and 2 types of thyroid cancers with alterations in the RET gene together with advanced medullary thyroid cancer \unskip~\citep{1027058:21592202}. 

Selpercatinib (Retevmo) is an orally bioavailable wild-type selective inhibitor, fusion, and mutant products concerning the proto-oncogene receptor tyrosine kinase RET, with possible antineoplastic activity. When administered orally, it specifically binds to and targets different RET-containing fusion products and RET mutants which results in cell growth inhibition of tumor cells that show enhanced RET activity. 

The chemical structure of selpercatinib was shown in Figure~\ref{f-db961985bff6}. A literature survey on selpercatinib revealed that LC-MS \unskip~\citep{1027058:21592199} and LC-MS/MS \unskip~\citep{1027058:21592203} methods were reported so far for the determination of selpercatinib. The aim of the study is to establish novel, specific and precise reverse-phase high-performance liquid chromatography method for the quantification of selpercatinib in pure and its capsule dosage form.


\bgroup
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\centering \makeatletter\IfFileExists{images/2021409b-ddd5-4c2a-aaf2-b6f4595120b2-upicture1.png}{\includegraphics{images/2021409b-ddd5-4c2a-aaf2-b6f4595120b2-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Structure of Selpercatinib}}
\label{f-db961985bff6}
\end{figure}
\egroup

    
\section{Materials and Methods}
\textbf{Instruments}

Waters 2695 HPLC system equipped with quaternary pumps and photodiode array (PDA) detector was utilised. The pH of the solutions was calculated by a pH meter (BVK enterprises, India). All analytical measurements were done on Analytical balance (Denver).\textbf{\space }

\textbf{Chemicals and reagents}

Selpercatinib sample was obtained as a gift sample from Spectrum Pharma Research Solutions (Hyderabad). Ortho-phosphoric acid, methanol, potassium dihydrogen orthophosphate, acetonitrile were purchased from Rankem Laboratories Pvt Ltd. Millipore Milli Q water was prepared in the laboratory. 

\textbf{Preparation of solutions}

\textbf{Preparation of diluents}

Acetonitrile and water in a ratio of 50:50 v/v was utilised as a diluent 

\textbf{Preparation of standard stock solutions}

10mg of selpercatinib was weighed precisely and taken into a 50ml volumetric flask. The volume was then made up with diluent to obtain a drug standard stock solution of 200\ensuremath{\mu}g/ml concentration.

\textbf{Preparation of standard working solutions}

Drug standard stock solution (1ml) was transferred to five individual volumetric flasks (10ml). The volume was then made up with diluent to obtain a drug concentration ranging from 5\ensuremath{\mu}g/ml-30\ensuremath{\mu}g/ml.

\textbf{Preparation of sample stock solutions}

Ten capsules were weighed, the average weight of each capsule was determined. The weight identical to one capsule was then taken into a volumetric flask (100ml) and diluent (50 ml) was transferred to the flask. For 25 min flask was sonicated. The volume was then made up to the mark and filtered by using HPLC filters (200\ensuremath{\mu}g/ml of selpercatinib).

\textbf{\textbf{Preparation of sample working solutions}}

Filtered sample stock solution (0.5 ml) was taken into a volumetric flask (10ml) and made up to volume with diluent (20\ensuremath{\mu}g/ml of selpercatinib).

\textbf{Method Validation}

\textbf{System suitability parameters}

System suitability was considered under every validation parameter by introducing six replicate injections of the drug standard solution (20\ensuremath{\mu}g/ml). Parameters like tailing factor, plate count and column efficiency were noted \unskip~\citep{1027058:21592205}.

\textbf{Specificity}

The specificity was assessed by comparing the placebo solution with the drug solution by introducing the samples into the HPLC system. The resulting chromatograms were seen for the interference of placebo response with a drug peak response \unskip~\citep{1027058:21592204}.

\textbf{Linearity}

Linearity is the method capability to assemble test results that are proportionate to the concentration of an analyte in samples within a specified range. Dissimilar drug standard solutions were made to evaluate the linearity by diluting the drug stock solution with the diluents in different concentrations of selpercatinib ranging from 5\textit{ \ensuremath{\mu}}g/ml to 30 \textit{\ensuremath{\mu}}g/ml. The linearity plot of the calibration curve was assessed by linear regression analysis \unskip~\citep{1027058:21592201}.

\textbf{Limit of detection}

The LOD is the smallest concentration of the analyte in a sample that can be detected but not determined. The LOD was determined by using the following formula \unskip~\citep{1027058:21592208}. 

Limit of detection= 3.3 X Standard deviation of the response /slope of calibration curve of the analyte.

\textbf{Limit of quantification}

The LOQ is the smallest concentration of an analyte in a sample, which might be quantified with appropriate accuracy and precision. The LOQ was determined by using the following formula. 

Limit of quantification = 10 X Standard deviation of the response /slope of calibration curve of the analyte.

\textbf{Accuracy}

Accuracy is the degree of closeness of test results to the accurate value. Accuracy was performed at 50\%, 100\% and 150\% by adding an acknowledged amount of sample stock solution of selpercatinib (0.5,1,1.5 \ensuremath{\mu}g/ml) to the standard stock solution. The percentage of recoveries were calculated \unskip~\citep{1027058:21592200}.

\textbf{Precision}

Precision is the degree of closeness between the detector responses acquired by several individual estimations of the same sample under specified conditions. Precision was determined by introducing six replicates of selpercatinib standard solution into the HPLC system and \% RSD was calculated \unskip~\citep{1027058:21592207}. \textbf{\space }

\textbf{Robustness}

Robustness is an estimate of its capability to remain unchanged by little, but intentional changes in parameters of the analytical method and gives a suggestion of its consistency throughout usage. It was performed by varying the flow rate, temperature, ratio of the mobile phase \unskip~\citep{1027058:21592197}.\textbf{\space }

\textbf{Forced degradation studies}

\textbf{Oxidation}

One ml of 20\% hydrogen peroxide (H\ensuremath{_{2}}O\ensuremath{_{2}}) was added to one ml of selpercatinib stock solution\textbf{.} The solution was heated at 60\text{\textordmasculine}C for 30 minutes on a water bath. Then the solution was cooled and made up to the mark using diluent. 10\ensuremath{\mu }l of this solution was introduced into HPLC the system \unskip~\citep{1027058:21592196}.

\textbf{Acid degradation}

One ml of 2N hydrochloric acid (HCl) was added to a drug stock solution. For 30 minutes, the solution was heated at 60\text{\textordmasculine}C on a water bath. Then the solution was cooled and 1 ml 2N sodium hydroxide was added. The solution was made up to the mark using diluent. 10\ensuremath{\mu }l of the solution was introduced into the system.

\textbf{Alkali degradation}

One ml of 2N sodium hydroxide (NaOH) was added to the drug solution. The solution was heated for 30 minutes at 60\text{\textordmasculine}C on a water bath. Then the solution was cooled and 1 ml 2N HCl was added. The solution made up with diluent. Ten \ensuremath{\mu }l of this solution was introduced into the system.

\textbf{Dry heat degradation}

The drug standard solution was kept in the oven for 6hrs at 105\ensuremath{^\circ}C. The solution was made up with diluent. 10\ensuremath{\mu }l of the solution was introduced into the system. 

\textbf{Photolytic degradation}

200\ensuremath{\mu}g/ml selpercatinib solution was revealed to UV light by placing a beaker in UV Chamber for 200 Watt-hours/m\ensuremath{^{2}}or 7 days in a photostability chamber. Then the solution was diluted and made up to volume with the diluent. 10\ensuremath{\mu }l of this solution was introduced into the HPLC system \unskip~\citep{1027058:21592206}.

\textbf{Neutral degradation}

Neutral degradation was determined by refluxing the drug in the water at 60\text{\textordmasculine}C temperature for 6hrs. The solution was made up to volume with the diluent. 10\ensuremath{\mu }l of this solution was introduced into the HPLC system. 
    
\section{Results and Discussion}
Novel RP HPLC validated technique has been established for the quantification of selpercatinib. A Zorbax C18 column (150 x 4.6 mm, i.d. 5\ensuremath{\mu}m particle size) was selected with mobile phase composition 0.1\% orthophosphoric acid:ACN (60:40 v/v). The flow rate was kept at 1.0ml/min for the determination of selpercatinib. The developed chromatographic conditions were tabulated in Table~\ref{tw-971661d9c6c8}. The results for all the parameters were summarised in Table~\ref{tw-c8e751149fdc}.


\begin{table*}[!htbp]
\caption{\boldmath {Optimized chromatographic conditions} }
\label{tw-971661d9c6c8}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Parameter & \cAlignHack Chromatographic conditions\\
\tblmidrule 
\cAlignHack Stationary Phase &
  \cAlignHack ZorbaxC18 150x4.6 mm, 5\ensuremath{\mu }\\
\cAlignHack Mobile Phase &
  \cAlignHack 0.1\% OPA: acetonitrile (60:40 v/v) \\
\cAlignHack Column temperature &
  \cAlignHack 30\ensuremath{^\circ}C\\
\cAlignHack Injection Volume &
  \cAlignHack 10 \ensuremath{\mu}L\\
\cAlignHack Total Run Time &
  \cAlignHack 6 min\\
\cAlignHack Detector &
  \cAlignHack Photodiode array detector\\
\cAlignHack Elution &
  \cAlignHack Isocratic mode\\
\cAlignHack Flow Rate &
  \cAlignHack 1.0ml/min\\
\cAlignHack \ensuremath{\lambda }\ensuremath{_{max}} &
  \cAlignHack 220 nm\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {System suitability data} }
\label{tw-5babd9a0a412}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Parameter & \cAlignHack Selpercatinib\\
\tblmidrule 
\cAlignHack Retention time &
  \cAlignHack 2.653\\
\cAlignHack Theoretical plates &
  \cAlignHack 3712\\
\cAlignHack Tailing factor &
  \cAlignHack 1.47\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Linearity data of selpercatinib} }
\label{tw-02c843168ad9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3258\linewidth-2\tabcolsep}p{\dimexpr.3342\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Linearity level (\%) & \cAlignHack Conc. (\ensuremath{\mu }g/ml)  & \cAlignHack Peak area \\
\tblmidrule 
\cAlignHack 25 &
  \cAlignHack 5 &
  \cAlignHack 95634\\
\cAlignHack 50 &
  \cAlignHack 10 &
  \cAlignHack 189370\\
\cAlignHack 75 &
  \cAlignHack 15 &
  \cAlignHack 274716\\
\cAlignHack 100 &
  \cAlignHack 20 &
  \cAlignHack 373485\\
\cAlignHack 125 &
  \cAlignHack 25 &
  \cAlignHack 464247\\
\cAlignHack 150 &
  \cAlignHack 30 &
  \cAlignHack 552859\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Accuracy data of selpercatinib} }
\label{tw-ecfde60d5741}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.16179999999999996\linewidth-2\tabcolsep}p{\dimexpr.22160000000000004\linewidth-2\tabcolsep}p{\dimexpr.2232\linewidth-2\tabcolsep}p{\dimexpr.1934\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack \% Level  & \cAlignHack Amount Spiked  \mbox{}\protect\newline (\ensuremath{\mu }g/ml)  & \cAlignHack Amount recovered  \mbox{}\protect\newline (\ensuremath{\mu }g/ml)  & \cAlignHack \% Recovery  & \cAlignHack Mean \mbox{}\protect\newline \\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.16179999999999996\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 50\% }} &
  \cAlignHack 10 &
  \cAlignHack 9.98 &
  \cAlignHack 99.78 &
  \multicolumn{1}{p{\dimexpr(.20\linewidth-2\tabcolsep)}}{\multirow{9}{\linewidth}{\cAlignHack 99.74\%}}\\
 &
  \cAlignHack 10 &
  \cAlignHack 10.09 &
  \cAlignHack 100.87 &
  \\
 &
  \cAlignHack 10 &
  \cAlignHack 9.96 &
  \cAlignHack 99.59 &
  \\
\multicolumn{1}{p{\dimexpr(.16179999999999996\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100\% }} &
  \cAlignHack 20 &
  \cAlignHack 19.72 &
  \cAlignHack 98.61 &
  \\
 &
  \cAlignHack 20 &
  \cAlignHack 20.03 &
  \cAlignHack 100.15 &
  \\
 &
  \cAlignHack 20 &
  \cAlignHack 19.98 &
  \cAlignHack 99.89 &
  \\
\multicolumn{1}{p{\dimexpr(.16179999999999996\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 150\% }} &
  \cAlignHack 30 &
  \cAlignHack 29.83 &
  \cAlignHack 99.42 &
  \\
 &
  \cAlignHack 30 &
  \cAlignHack 29.72 &
  \cAlignHack 99.05 &
  \\
 &
  \cAlignHack 30 &
  \cAlignHack 30.09 &
  \cAlignHack 100.31 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*Mean of three replicates}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {System precision data of selpercatinib} }
\label{tw-a8c548c9035a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.24890000000000004\linewidth-2\tabcolsep}p{\dimexpr.7511\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No.  & \cAlignHack Area of selpercatinib \\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 369172\\
\cAlignHack 2 &
  \cAlignHack 368855\\
\cAlignHack 3 &
  \cAlignHack 366301\\
\cAlignHack 4 &
  \cAlignHack 370186\\
\cAlignHack 5 &
  \cAlignHack 372102\\
\cAlignHack 6 &
  \cAlignHack 366276\\
\cAlignHack Mean  &
  \cAlignHack 368815\\
\cAlignHack S.D  &
  \cAlignHack 2261.8\\
\cAlignHack \% RSD  &
  \cAlignHack 0.6 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Repeatability data of selpercatinib} }
\label{tw-4b632e0562af}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2801\linewidth-2\tabcolsep}p{\dimexpr.7199\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No.  & \cAlignHack Area of selpercatinib\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 369650\\
\cAlignHack 2 &
  \cAlignHack 365025\\
\cAlignHack 3 &
  \cAlignHack 367566\\
\cAlignHack 4 &
  \cAlignHack 365914\\
\cAlignHack 5 &
  \cAlignHack 367584\\
\cAlignHack 6 &
  \cAlignHack 363307\\
\cAlignHack Mean &
  \cAlignHack 366508\\
\cAlignHack S.D &
  \cAlignHack 2233.7\\
\cAlignHack \% RSD &
  \cAlignHack 0.6\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Intermediate precision data of selpercatinib} }
\label{tw-e745221d95b9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3236\linewidth-2\tabcolsep}p{\dimexpr.6764\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No.  & \cAlignHack Area of selpercatinib\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 375744\\
\cAlignHack 2 &
  \cAlignHack 371751\\
\cAlignHack 3 &
  \cAlignHack 374954\\
\cAlignHack 4 &
  \cAlignHack 372564\\
\cAlignHack 5 &
  \cAlignHack 373376\\
\cAlignHack 6 &
  \cAlignHack 365756\\
\cAlignHack Mean &
  \cAlignHack 372358\\
\cAlignHack S.D &
  \cAlignHack 3556.3\\
\cAlignHack \% RSD &
  \cAlignHack 1.0\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Robustness data for Selpercatinib} }
\label{tw-c4f825d00bdc}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.18689999999999998\linewidth-2\tabcolsep}p{\dimexpr.5167\linewidth-2\tabcolsep}p{\dimexpr.2964\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Condition & \cAlignHack \% RSD\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack Mobile phase (+)55A:45B &
  \cAlignHack 1.1\\
\cAlignHack 2 &
  \cAlignHack Temp. (+) 35\ensuremath{^\circ}C &
  \cAlignHack 0.3\\
\cAlignHack 3 &
  \cAlignHack Flow rate (+) 1.1ml/min &
  \cAlignHack 0.3\\
\cAlignHack 4 &
  \cAlignHack Mobile phase (-)35A:65B &
  \cAlignHack 0.9\\
\cAlignHack 5 &
  \cAlignHack Temp. (-) 25\ensuremath{^\circ}C &
  \cAlignHack 0.3\\
\cAlignHack 6 &
  \cAlignHack Flow rate (-) 0.9ml/min &
  \cAlignHack 0.3\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Specificity data of Selpercatinib} }
\label{tw-654bfdbde0e4}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.18469999999999995\linewidth-2\tabcolsep}p{\dimexpr.4359\linewidth-2\tabcolsep}p{\dimexpr.3794\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No. & \cAlignHack Sample details & \cAlignHack Retention time (min)\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack Placebo solution &
  \cAlignHack Interference is not detected\\
\cAlignHack 2 &
  \cAlignHack Blank solution &
  \cAlignHack Interference is not detected\\
\cAlignHack 3 &
  \cAlignHack Selpercatinib &
  \cAlignHack 2.647\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Assay data of Selpercatinib} }
\label{tw-b3f1a0d4a4af}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.196\linewidth-2\tabcolsep}p{\dimexpr.2542\linewidth-2\tabcolsep}p{\dimexpr.2873\linewidth-2\tabcolsep}p{\dimexpr.2625\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No. & \cAlignHack Sample area & \cAlignHack Standard area & \cAlignHack \% recovery\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 369650 &
  \cAlignHack 369172 &
  \cAlignHack 99.93\\
\cAlignHack 2 &
  \cAlignHack 365025 &
  \cAlignHack 368855 &
  \cAlignHack 98.68\\
\cAlignHack 3 &
  \cAlignHack 367566 &
  \cAlignHack 366301 &
  \cAlignHack 99.36\\
\cAlignHack 4 &
  \cAlignHack 365914 &
  \cAlignHack 370186 &
  \cAlignHack 98.92\\
\cAlignHack 5 &
  \cAlignHack 367584 &
  \cAlignHack 372102 &
  \cAlignHack 99.37\\
\cAlignHack 6 &
  \cAlignHack 363307 &
  \cAlignHack 366276 &
  \cAlignHack 98.21\\
\cAlignHack Avg &
  \cAlignHack 366508 &
  \cAlignHack 368815 &
  \cAlignHack 99.08\\
\cAlignHack Std dev &
  \cAlignHack 2233.7 &
  \cAlignHack 2261.8 &
  \cAlignHack 0.604\\
\cAlignHack \% RSD &
  \cAlignHack 0.6 &
  \cAlignHack 0.6 &
  \cAlignHack 0.61\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Degradation data of Selpercatinib} }
\label{tw-0f22dd41027f}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0914\linewidth-2\tabcolsep}p{\dimexpr.1708\linewidth-2\tabcolsep}p{\dimexpr.1412\linewidth-2\tabcolsep}p{\dimexpr.1341\linewidth-2\tabcolsep}p{\dimexpr.1506\linewidth-2\tabcolsep}p{\dimexpr.1424\linewidth-2\tabcolsep}p{\dimexpr.1695\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Degradation condition & \cAlignHack \% Drug \mbox{}\protect\newline degraded & \cAlignHack \% Drug \mbox{}\protect\newline Undegraded & \cAlignHack Retention time & \cAlignHack Peak Area & \cAlignHack Theoretical plates ({\textgreater}2000)\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack Acid &
  \cAlignHack 5.87 &
  \cAlignHack 94.13 &
  \cAlignHack 2.540 &
  \cAlignHack 348221 &
  \cAlignHack 3637\\
\cAlignHack 2 &
  \cAlignHack Alkali &
  \cAlignHack 4.99 &
  \cAlignHack 95.01 &
  \cAlignHack 2.630 &
  \cAlignHack 351458 &
  \cAlignHack 4027\\
\cAlignHack 3 &
  \cAlignHack Oxidation &
  \cAlignHack 4.06 &
  \cAlignHack 95.94 &
  \cAlignHack 2.623 &
  \cAlignHack 354902 &
  \cAlignHack 3668\\
\cAlignHack 4 &
  \cAlignHack Thermal &
  \cAlignHack 2.95 &
  \cAlignHack 97.05 &
  \cAlignHack 2.618 &
  \cAlignHack 359023 &
  \cAlignHack 3873\\
\cAlignHack 5 &
  \cAlignHack Photostability &
  \cAlignHack 1.77 &
  \cAlignHack 98.23 &
  \cAlignHack 2.622 &
  \cAlignHack 363384 &
  \cAlignHack 3575\\
\cAlignHack 6 &
  \cAlignHack Neutral &
  \cAlignHack 1.11 &
  \cAlignHack 98.89 &
  \cAlignHack 2.628 &
  \cAlignHack 365814 &
  \cAlignHack 4002\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Summary Table} }
\label{tw-c8e751149fdc}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1877\linewidth-2\tabcolsep}p{\dimexpr.25430000000000003\linewidth-2\tabcolsep}p{\dimexpr.2582\linewidth-2\tabcolsep}p{\dimexpr.2998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{\cAlignHack Parameters} & \cAlignHack Values & \cAlignHack Limit\\
\tblmidrule 
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Linearity (\ensuremath{\mu }g/ml)} &
  \cAlignHack 5-30\ensuremath{\mu}g/ml  &
  \cAlignHack \\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Regression coefficient } &
  \cAlignHack 0.999  &
  \cAlignHack \\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Slope(m)} &
  \cAlignHack 18428  &
  \cAlignHack R\textless\ 1\\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Intercept(c)} &
  \cAlignHack 2196.2  &
  \\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Regression equation (Y=mx+c)} &
  \cAlignHack y = 18428x + 2196.2  &
  \\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Specificity} &
  \cAlignHack Specific &
  \cAlignHack No intrusion of any peak\\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Accuracy (\%recovery) } &
  \cAlignHack 99.74\%  &
  \cAlignHack 98-102\% \\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{System precision (\%RSD) } &
  \cAlignHack 0.6  &
  \multicolumn{1}{p{\dimexpr(.2998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack NMT 2.0\%}}\\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Intermediate precision (\%RSD)} &
  \cAlignHack 1  &
  \\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Repeatability (\%RSD)} &
  \cAlignHack 0.6 &
  \\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{Assay (\% mean assay)} &
  \cAlignHack 99.08\% &
  \cAlignHack 90-110\%\\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{LOD } &
  \cAlignHack 0.02 \ensuremath{\mu}g/ml  &
  \cAlignHack NMT 3\\
\multicolumn{2}{p{\dimexpr(.442\linewidth-2\tabcolsep)}}{LOQ } &
  \cAlignHack 0.05 \ensuremath{\mu}g/ml  &
  \cAlignHack NMT 10\\
\multicolumn{1}{p{\dimexpr(.1877\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{Robustness}} &
  Mobile phase (+) 45B: 55A  &
  \cAlignHack 1.1 &
  \multicolumn{1}{p{\dimexpr(.2998\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{\cAlignHack \%RSD NMT 2.0}}\\
 &
  Flow rate (+) 1.1ml/min  &
  \cAlignHack 0.3 &
  \\
 &
  Temperature (+) 35\ensuremath{^\circ}C  &
  \cAlignHack 0.3 &
  \\
 &
  Mobile phase (-) 65B: 35A &
  \cAlignHack 0.9 &
  \\
 &
  Flow rate (-) 0.9ml/min &
  \cAlignHack 0.3 &
  \\
 &
  Temperature (-) 25\ensuremath{^\circ}C  &
  \cAlignHack 0.3 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/6671f0a8-8ec5-4778-8353-3866ebab71cd-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/6671f0a8-8ec5-4778-8353-3866ebab71cd-upicture2.png}{\includegraphics{images/6671f0a8-8ec5-4778-8353-3866ebab71cd-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Optimized chromatogram of Selpercatinib}}
\label{f-d86a47f5fe6a}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/63999f66-06d1-4474-8f1d-7702b1059362-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/63999f66-06d1-4474-8f1d-7702b1059362-upicture3.png}{\includegraphics{images/63999f66-06d1-4474-8f1d-7702b1059362-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Calibration curve of Selpercatinib}}
\label{f-d2c410fad88d}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/42aaa800-709a-435e-b76f-6e88f974e750-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/42aaa800-709a-435e-b76f-6e88f974e750-upicture4.png}{\includegraphics{images/42aaa800-709a-435e-b76f-6e88f974e750-upicture4.png}}{}
\makeatother 
\caption{\boldmath {LOD of Selpercatinib}}
\label{f-91f4b542021b}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/1f671f1c-af29-4ba5-bedd-e02ffb8d7a35-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/1f671f1c-af29-4ba5-bedd-e02ffb8d7a35-upicture5.png}{\includegraphics{images/1f671f1c-af29-4ba5-bedd-e02ffb8d7a35-upicture5.png}}{}
\makeatother 
\caption{\boldmath {LOQ of Selpercatinib}}
\label{f-795699e7b78e}
\end{figure}
\egroup
\textbf{System suitability parameters}

A system suitability test was an essential part of the method development to confirm that the system is satisfactory for the analysis of selpercatinib. The parameters for the selpercatinib have revealed that the theoretical plates were \textit{{\textgreater} }2000, and the tailing factor was \textit{{\textless}} 2 (Table~\ref{tw-5babd9a0a412}), (Figure~\ref{f-d86a47f5fe6a}). 

\textbf{Linearity}

The developed method demonstrated linearity in the concentration range of 5- 30\ensuremath{\mu }g/ml. Linearity equations obtained for selpercatinib was found to be y = 18428x + 2196.2 with a correlation coefficient of 0.999. The high value of the correlation coefficient indicates good linearity. Results were tabulated in Table 3, graphically depicted in Figure~\ref{f-d2c410fad88d}.

\textbf{Accuracy}

Three levels (50\%, 100\%and 150\%) of accuracy samples were made using the standard addition method. The \% recovery was obtained in the range of 98.61\%-100.87\%. High recovery results obtained from the method shows that the suggested method can be utilised for QC analysis of capsule dosage forms. Results were represented in Table~\ref{tw-ecfde60d5741}.

\textbf{Precision}

Chromatogram data for system precision (Table~\ref{tw-a8c548c9035a}) revealed that \% RSD was found to be 0.6, which was within the limit specified (\%RSD NMT 2.0\%). Chromatogram data for repeatability (Table~\ref{tw-4b632e0562af}) revealed that \% RSD was found to be 0.6, which was within the limit specified (\%RSD NMT 2.0\%). Chromatogram data for intermediate precision (Table~\ref{tw-e745221d95b9}) revealed that \% RSD was found to be one which was within the limit specified (\%RSD NMT 2.0\%). Hence, it proved the method was found to be precise.


\bgroup
\fixFloatSize{images/ca2d0302-37fa-4589-9ab1-d0e8f51c2076-upicture6.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/ca2d0302-37fa-4589-9ab1-d0e8f51c2076-upicture6.png}{\includegraphics{images/ca2d0302-37fa-4589-9ab1-d0e8f51c2076-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Chromatograms of blank, placebo, standard and sample}}
\label{f-b56312fba4a4}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/ed86cd61-bb01-413d-98a3-2d42f3e54e18-upicture7.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/ed86cd61-bb01-413d-98a3-2d42f3e54e18-upicture7.png}{\includegraphics{images/ed86cd61-bb01-413d-98a3-2d42f3e54e18-upicture7.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatograms of forced degradation studies}}
\label{f-2ef759b3280f}
\end{figure*}
\egroup
\textbf{Sensitivity}

LOD of selpercatinib was detected to be 0.02 \ensuremath{\mu }g/ml. LOQ of selpercatinib was detected to be 0.05\ensuremath{\mu }g/ml. Chromatograms of Limit of Detection and Limit of Quantification were demonstrated in Figure~\ref{f-91f4b542021b} and Figure~\ref{f-795699e7b78e}, respectively.

\textbf{Robustness}

The robustness was assessed by introducing little, intentional variations in the chromatographic conditions, which comprise the flow rate of mobile (\ensuremath{\pm} 0.1 ml/min), \% of acetonitrile in the mobile phase (35 \& 45\%) and temperature(\ensuremath{\pm}5). 

Robustness was carried out using 10 \ensuremath{\mu }g/ml of selpercatinib and the \% RSD was found to be 0.3-1.1. Data was represented in Table~\ref{tw-c4f825d00bdc}.

\textbf{Specificity}

Specificity was performed by introducing a blank solution, placebo solution, selpercatinib standard and sample solutions. Data represented in Table~\ref{tw-654bfdbde0e4} (Figure~\ref{f-b56312fba4a4}) indicates that was no interference in the placebo and blank sample at the retention time of the standard selpercatinib sample. Therefore, the method was specific.

\textbf{Assay}

Retevmo (label claim 40mg) was used for the assay. The percentage purity of selpercatinib was found to be 99.08-99.93 in pharmaceutical formulations. Data was represented in Table~\ref{tw-b3f1a0d4a4af}.

\textbf{Forced degradation studies}

Selpercatinib was eluted at 2.647 min. Selpercatinib has undergone acid degradation (5.87\%), alkali degradation (4.99\%), oxidation (4.06\%), thermal degradation (2.95\%), photolytic degradation (1.77\%) and neutral degradation (1.11\%), which is less than 10\%, indicating that the selpercatinib is more resistant towards all forced degradation conditions applied. 

During acid degradation and alkali degradation, an extra peak was observed at 1.1857 min and 1.1873, along with the drug peak at 2.640 min (Table~\ref{tw-0f22dd41027f}; Figure~\ref{f-2ef759b3280f}). The system suitability parameters were within limits.
    
\section{Conclusion }
Validated RP-HPLC method has been established for the quantification of selpercatinib in pure and its capsule dosage forms. The developed method was found to be precise, specific and accurate. Hence, the method can be utilised in the quality control analysis of selpercatinib.  
\section*{Acknowledgement}The authors are thankful to the management of GITAM (Deemed to be University), Visakhapatnam, for providing the research facilities. 

\textbf{Conflict of Interest}

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

\textbf{Funding Support}

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

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