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\def\RunningAuthor{Annapoorna V et al.}
\firstPage{2374}
\articleType{Original Article}
\receivedDate{09 Jun 2021}
\acceptedDate{12 Jul 2021}
\revisedDate{08 Jul 2021}
\journalVolume{2021, 12}
\journalIssue{4}
\journalDoi{ijrps.v12i4.4871}
\copyrightYear{2021}
\def\authorCount{5}
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\title{\textbf{Innovative Method Development Comprehensive Separation of Impurities and Validation for a novel Antipsychotic Drug Blonanserin}}
\author{Annapoorna~V\textsuperscript{*}\textsuperscript{1},  
            Ravindhranath~K\textsuperscript{1},  
            Sreenivasa~Rao~B\textsuperscript{2},  
            Rao~P~Y~G\textsuperscript{2}, 
            Venugopal~K\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Chemistry, Koneru Lakshmaiah Education Foundation, Green Fields, Vaddeswaram-522502, Andhra Pradesh, India}~\\{\textsuperscript{2}Department of Chemistry, GITAM Institute of Science, GITAM (Deemed to be University), Visakhapatnam - 530045, Andhra Pradesh, India}~\\{\textsuperscript{3}AU College of Pharmaceutical Sciences, Andhra University South Campus, Andhra University, Visakhapatnam-530003, Andhra Pradesh, India}}

\begin{abstract}
Blonanserin an antipsychotic novel drug used for the treatment of schizophrenia has antagonist properties for dopamine D2 and serotonin 5-HT2. On the other hand, it lacks adrenergic-\ensuremath{\alpha }1, muscarinic M1, and histamine H1 antagonist activities. Clinical studies demonstrated in Japan had shown to be more effective for treating negative as well as positive schizophrenic symptoms. This drug was accepted and approved worldwide in the treatment of schizophrenia. A new HPLC method was developed and validated for the estimation of Impurities of Blonanserin (BNS) to ensure that the methodology meets the requirements of the target analysis application. Active and efficient chromatographic separation was achieved on a Zorbax Bonus RP EP C18 column having a particle size of 5\ensuremath{\mu }m, with dimensions of 250mm \ensuremath{\times} 4.6 mm, mobile phase containing pH 2.4 buffer and Organic, with 1.0 ml/min flow rate, column oven temperature at 30\ensuremath{^{o}}C and the eluent detection at 245 nm. The method shows well-separated impurities, is specific without interference from blank solution with resolution more than 1.2 between any of the impurity, correlation coefficient more than 0.99 showing good linearity; mean recovery ranging from 97\% to 105\% and is very sensitive at lower detection and quantification limits. This method was well developed and has been applied successfully to monitor and estimate impurities in Blonanserin.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Antipsychotic,\newline Blonanserin Impurities,\newline Validated method,\newline HPLC
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Annapoorna~V~\\ Phone:\ +91-8121263969~\\  Email:\ annuvadlamani@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{ Introduction}
 Blonanserin, a new second-generation anti-psychotic drug \unskip~\citep{1207051:23150738}  used to treat schizophrenia, was approved in Korea and Japan by PMDA in 2008. The drug is now made more acceptable world wise as a promising antipsychotic drug for schizophrenia treatment. Blonanserin binds and acts as an antagonist and inhibits the serotonin 5-HT2A receptors, D2 and D3 receptors of Dopamine \unskip~\citep{1207051:23150737}. It is very effective in treating patients with schizophrenia, which is equivalent to risperidone and haloperidol for positive symptoms and is also higher than haloperidol for the development of negative symptoms \unskip~\citep{1207051:23150731}. Blonanserin, a heterocyclic compound belongs to the chemical series of 4-phenyl-2- (1-piperazinyl) pyridines, IUPAC nomenclature 1-ethyl-4- [4- (4-fluorophenyl) -5H, 6H, 7H, 8H, 9H, 10H-cycloocta [b] pyridin-2-yl] piperazine, metabolized mainly by CYP3A4 (Figure~\ref{f-6d0f1f009085}). The piperazine ring of BNS undergoes N-de-ethylation and N-oxidation as well as hydroxylation of the cyclooctane ring. The literature review of Blonanserin reveals that this molecule is not yet official in IP, BP, USP, or any pharmacopeia. In line with regulatory requirements and The International Conference of Harmonization (ICH), the FDA endorse identification, quantification, qualification and also laid control over impurities in pharmaceutical drug substances and their formulation. It has been found that there is no analytical method developed for the determination of impurities in Blonanserin. It is very important that there should be a simple, stable, sensitive, accurate, authentic, rapid and reliable method to determine the impurities of Blonanserin drug substance. Therefore, it was considered that Blonanserin impurities should be determined to ensure the quality, effectiveness, and safety of the final pharmaceutical drug. There is recognition of the UV Spectrophotometric method \unskip~\citep{1207051:23150733}, HPLC \unskip~\citep{1207051:23150740,1207051:23150732}  bioanalytical LCMS / MS method of Blonanserin and its human plasma and urine metabolites \unskip~\citep{1207051:23150736,1207051:23150735}, HPLC-FDA \unskip~\citep{1207051:23150739,1207051:23150734}, GC-MS \unskip~\citep{1207051:23150730}. The reported HPLC method has its limitations related to LOD, LOQ and analysis time. To date, there is no indicative, well-separated, fast analysis method available so far for the estimation of Blonanserin impurities. Keeping this fact in mind, the purpose of the present study is designed to develop a sensitive, fast, reliable method, demonstrating the stability of the analysis and separation of impurities in the Blonanserin pharmaceutical drug substances. This research demonstrates the novelty of the work by reduced runtime, well-separation of impurities, detection at lower LOD and LOQ levels, and proved the stability in different conditions.
    
\section{ Materials and Methods}
\textbf{Reagents and chemicals}

In the present research, the following materials were used Blonanserin, Acetonitrile (Merck, HPLC Grade), Orthophosphoric acid (Qualigens, HPLC Grade), Triethylamine (Qualigens HPLC Grade), Tetrahydrofuran (Sigma Aldrich, HPLC Grade), Water (Millipore water system).

\textbf{Apparatus and Conditions for Chromatography}

Chromatography was performed on Waters, Alliance 2695 HPLC system connected with 2998 PDA detector with Empower software Version3. Zorbax Bonus RP EP C18 column having a particle size of 5\ensuremath{\mu }m, with dimensions of 250mm \ensuremath{\times} 4.6 mm, mobile phase containing pH 2.4 buffer and Organic with 1.0 ml /min flow rate, column oven temperature at 30\ensuremath{^{o}}C was used for separation and detected at 245nm using a PDA detector. The impurities and Blonanserin were separated in gradient mode of elution with Triethylamine (1ml in 1000ml water), pH adjustment was made to 2.4 with H\ensuremath{_{3}}PO\ensuremath{_{4}} (orthophosphoric acid) and a mixture of tetrahydrofuran and acetonitrile (6:94). Mobile phase A (75:25) and Mobile phase B (25:75) with the 10 \ensuremath{\mu }l injection volume. Water and acetonitrile mixed in an equal ratio is used as a diluent. The gradient elution program was designed as (0\ensuremath{\rightarrow}5, 95:5, 5\ensuremath{\rightarrow}30, 75:25, 30\ensuremath{\rightarrow}35, 5:95, 35\ensuremath{\rightarrow}40, 5:95, 40\ensuremath{\rightarrow}45, 95:5, 45\ensuremath{\rightarrow}48, 95:5).

\textbf{Resolution, Standards and sample preparation}

The resolution solution was prepared by accurately weighing and dissolving the Blonanserin sample solution containing all the impurities to get the concentration of 0.5mg/ml solution. The request was to have a resolution of a minimum of 1.2 between impurity B and C peaks and other impurities to confirm their relative retention times. 

The stock solution of Blonanserin standard was prepared by weighing an accurate amount of reference standard in a diluent, dissolved to get a concentration of 0.25ppm solution (equivalent to 0.05\% w.r.t test sample solution).

 Blonanserin test solution was prepared with diluent and diluted to an appropriate volume to get the concentration of 0.5mg/ml.Optimization of the Method.

\textbf{Optimization of the Method}

Several experimental trials have been taken to optimise the developed method by changing buffer concentration, pH, organic solution, and the ratio of buffer and organic solution. Finally, the mobile phase was optimized with gradient mode of elution, Zorbax Bonus RP EP C18 column with length 250 mm, internal diameter 4.6 mm and particle size 5\ensuremath{\mu }m, 30\ensuremath{^{o}}C temperature, the flow of mobile phase at 1.0ml/min and detected at 245nm.

\textbf{System Suitability}

System suitability parameter, which is an essential part of method parameters, is measured for verifying the system, method and column performance. Blank, Resolution solution, peak identification solution, Reference standard solution was prepared and


\begin{table*}[!htbp]
\caption{\boldmath {System suitability results} }
\label{tw-f9c2f54e5c12}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.08190000000000003\linewidth-2\tabcolsep}p{\dimexpr.24380000000000003\linewidth-2\tabcolsep}p{\dimexpr.31230000000000008\linewidth-2\tabcolsep}p{\dimexpr.362\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Parameter & \%RSD of BNS standard solution from 6 injections & Resolution between Imp-B and Imp-C\\
\tblmidrule 
1 &
  System suitability &
  2.1 &
  1.4\\
\multicolumn{2}{p{\dimexpr(.3257000000000001\linewidth-2\tabcolsep)}}{Acceptance criteria} &
  Should not be more than 5 &
  Should not be less than 1.2\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Specificity results} }
\label{tw-165430e2d394}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.1005\linewidth-2\tabcolsep}p{\dimexpr.2995\linewidth-2\tabcolsep}p{\dimexpr.15690000000000001\linewidth-2\tabcolsep}p{\dimexpr.21159999999999997\linewidth-2\tabcolsep}p{\dimexpr.2315\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Peak names & RT & Purity angle & Purity threshold\\
\tblmidrule 
1 &
  Impurity-A &
  3.1 &
  1.93 &
  3.21\\
2 &
  Impurity-B &
  14.9 &
  2.63 &
  3.36\\
3 &
  Impurity-C &
  15.6 &
  1.82 &
  2.79\\
4 &
  BNS &
  17.2 &
  0.08 &
  2.51\\
5 &
  Impurity-D &
  24.0 &
  2.35 &
  3.22\\
 &
  Acceptance criteria &
  \multicolumn{3}{p{\dimexpr(.5999999999999999\linewidth-2\tabcolsep)}}{All impurities should resolve and peak purity should pass}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Forced Degradation results} }
\label{tw-54f8d9391e22}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.11387\linewidth-2\tabcolsep}p{\dimexpr.09847000000000001\linewidth-2\tabcolsep}p{\dimexpr.11417000000000002\linewidth-2\tabcolsep}p{\dimexpr.10137\linewidth-2\tabcolsep}p{\dimexpr.10057000000000002\linewidth-2\tabcolsep}p{\dimexpr.10527000000000001\linewidth-2\tabcolsep}p{\dimexpr.10247\linewidth-2\tabcolsep}p{\dimexpr.07447\linewidth-2\tabcolsep}p{\dimexpr.09007\linewidth-2\tabcolsep}p{\dimexpr.09927000000000001\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Condition & Purity angle of BNS & Purity Threshold of BNS & Imp-A  \mbox{}\protect\newline (\%W/W) & Imp-B \mbox{}\protect\newline (\%W/W) & Imp-C \mbox{}\protect\newline (\%W/W) & Imp-D  \mbox{}\protect\newline (\%W/W) & Total Imp & Assay & Mass Balance\\
\tblmidrule 
0.5N HCl at 50\ensuremath{^{O}}C for 20 hours &
  0.61 &
  1.98 &
  0.006 &
  0.01 &
  0.004 &
  0.01 &
  0.07 &
  99.2 &
  99.6\\
0.5N NaOH at 50\ensuremath{^{O}}C for 20 hours &
  1.21 &
  2.73 &
  0.003 &
  0.011 &
  0.001 &
  0.01 &
  0.05 &
  100.5 &
  100.4\\
1\%H\ensuremath{_{2}}O\ensuremath{_{2}}  at room temperature for 1hour &
  0.95 &
  2.36 &
  0.002 &
  0.018 &
  0.015 &
  0.007 &
  24.8 &
  76.5 &
  99.8\\
Heat at 50\ensuremath{^{O}}C for 20 hours &
  0.37 &
  2.91 &
  0.008 &
  0.013 &
  0.006 &
  0.01 &
  0.09 &
  99.1 &
  99.4\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begingroup
\makeatletter\if@twocolumn\@ifundefined{theposttbl}{\gdef\TwoColDocument{true}\onecolumn\onecolumn}{}\fi\makeatother \setlength\LTcapwidth{\textwidth}
\begin{longtable}{p{\dimexpr.2117\linewidth-2\tabcolsep}p{\dimexpr.4960000000000001\linewidth-2\tabcolsep}p{\dimexpr.29230000000000004\linewidth-2\tabcolsep}}
\caption{\boldmath {Linearity} }
\label{tw-aabd9cee98eb}
\def\arraystretch{1.1}\\\endfirsthead \hline \noalign{\vskip3pt} \noalign{\textit{Table \thetable\ continued}} \noalign{\vskip3pt} 
\tbltoprule \rowcolor{kwdboxcolor}Level  & \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{\cAlignHack Imp-A }\\
\rowcolor{kwdboxcolor} & Conc (\ensuremath{\mu}g/ml)  & Avg area counts (\ensuremath{\mu}V*Sec)\\
\tblmidrule \endhead \hline \noalign{\vskip3pt} \noalign{\textit{\hfill Continued on next page}} \noalign{\vskip3pt} \endfoot \endlastfoot 
\tbltoprule \rowcolor{kwdboxcolor}Level  & \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{\cAlignHack Imp-A }\\
\rowcolor{kwdboxcolor} & Conc (\ensuremath{\mu}g/ml)  & Avg area counts (\ensuremath{\mu}V*Sec)\\
\tblmidrule 
 &
   &
  \\
LOQ  &
  0.02715  &
  523.9 \\
50\%  &
  0.12360  &
  3438.1 \\
80\%  &
  0.19833  &
  5487.8 \\
90\%  &
  0.22290  &
  6127.3 \\
100\%  &
  0.24690  &
  6986.6 \\
120\%  &
  0.29340  &
  8334.8 \\
150\%  &
  0.37290  &
  10288.8 \\
 &
   &
  \\
Slope  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{28436.4872 }\\
 &
   &
  \\
Intercept  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{-149.4289 }\\
 &
   &
  \\
C.C  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9994 }\\
 &
   &
  \\
R.Square  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9987 }\\
 &
   &
  \\
\cellcolor[HTML]{E5E5E5}{Level } &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{\cellcolor[HTML]{E5E5E5}\cAlignHack {Imp-B }}\\
\cellcolor[HTML]{E5E5E5}{} &
  \cellcolor[HTML]{E5E5E5}{Conc (\ensuremath{\mu}g/ml) } &
  \cellcolor[HTML]{E5E5E5}{Avg area counts (\ensuremath{\mu}V*Sec) }\\
 &
   &
  \\
LOQ  &
  0.02763  &
  1184.2 \\
50\%  &
  0.14527  &
  4354.1 \\
80\%  &
  0.23648  &
  6905.3 \\
90\%  &
  0.26745  &
  7749.4 \\
100\%  &
  0.29083  &
  8728.4 \\
120\%  &
  0.35697  &
  10345.7 \\
150\%  &
  0.44143  &
  12834.5 \\
 &
   &
  \\
Slope  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{28230.8627 }\\
 &
   &
  \\
Intercept  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{320.6004 }\\
 &
   &
  \\
C.C  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9996 }\\
 &
   &
  \\
R.Square  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9991 }\\
 &
   &
  \\
\cellcolor[HTML]{E5E5E5}{Level } &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{\cellcolor[HTML]{E5E5E5}\cAlignHack {Imp-C }}\\
\cellcolor[HTML]{E5E5E5}{} &
  \cellcolor[HTML]{E5E5E5}{Conc (\ensuremath{\mu}g/ml)\textbf{\space }} &
  \cellcolor[HTML]{E5E5E5}{Avg area counts (\ensuremath{\mu}V*Sec)\textbf{\space }}\\
 &
   &
  \\
LOQ  &
  0.03688  &
  1438.2 \\
50\%  &
  0.12995  &
  4416.7 \\
80\%  &
  0.20587  &
  6997.5 \\
90\%  &
  0.23952  &
  7962.9 \\
100\%  &
  0.25481  &
  8799.4 \\
120\%  &
  0.31629  &
  10657.4 \\
150\%  &
  0.38716  &
  13140.4 \\
 &
   &
  \\
Slope  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{33433.8834 }\\
 &
   &
  \\
Intercept  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{129.3221 }\\
 &
   &
  \\
C.C  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9996 }\\
 &
   &
  \\
R.Square  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9992 }\\
 &
   &
  \\
\cellcolor[HTML]{E5E5E5}{Level } &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{\cellcolor[HTML]{E5E5E5}\cAlignHack { BNS}}\\
\cellcolor[HTML]{E5E5E5}{} &
  \cellcolor[HTML]{E5E5E5}{Conc (\ensuremath{\mu}g/ml)\textbf{\space }} &
  \cellcolor[HTML]{E5E5E5}{Avg area counts (\ensuremath{\mu}V*Sec)\textbf{\space }}\\
 &
   &
  \\
LOQ  &
  0.03866  &
  1154.6 \\
50\%  &
  0.12846  &
  4367.8 \\
80\%  &
  0.20738  &
  6911.7 \\
90\%  &
  0.22693  &
  7847.3 \\
100\%  &
  0.25567  &
  8651.8 \\
120\%  &
  0.30369  &
  10608.9 \\
150\%  &
  0.37116  &
  13234.5 \\
 &
   &
  \\
Slope  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{36034.3154 }\\
 &
   &
  \\
Intercept  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{-346.5956 }\\
 &
   &
  \\
C.C  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9992 }\\
 &
   &
  \\
R.Square  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9984 }\\
 &
   &
  \\
\cellcolor[HTML]{E5E5E5}{Level } &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{\cellcolor[HTML]{E5E5E5}\cAlignHack {Imp-D }}\\
\cellcolor[HTML]{E5E5E5}{} &
  \cellcolor[HTML]{E5E5E5}{Conc (\ensuremath{\mu}g/ml) } &
  \cellcolor[HTML]{E5E5E5}{Avg area counts (\ensuremath{\mu}V*Sec)\textbf{\space }}\\
 &
   &
  \\
LOQ  &
  0.03768  &
  1186.6 \\
50\%  &
  0.12518  &
  4058.9 \\
80\%  &
  0.20786  &
  6544.7 \\
90\%  &
  0.22234  &
  7339.1 \\
100\%  &
  0.25936  &
  8152.7 \\
120\%  &
  0.30567  &
  9815.1 \\
150\% &
  0.37123  &
  12249.9 \\
 &
   &
  \\
Slope  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{32729.0351 }\\
 &
   &
  \\
Intercept  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{-100.8811 }\\
 &
   &
  \\
C.C  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9990 }\\
 &
   &
  \\
R.Square  &
  \multicolumn{2}{p{\dimexpr(.7883000000000001\linewidth-2\tabcolsep)}}{0.9980 }\\
\tblbottomrule 
\end{longtable}
\endgroup
\makeatletter\@ifundefined{TwoColDocument}{}{\twocolumn}\makeatother 

\begin{table*}[!htbp]
\caption{\boldmath {Recovery of Impurity-A} }
\label{tw-16caa69ca5d8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1891\linewidth-2\tabcolsep}p{\dimexpr.1835\linewidth-2\tabcolsep}p{\dimexpr.18630000000000003\linewidth-2\tabcolsep}p{\dimexpr.15569999999999997\linewidth-2\tabcolsep}p{\dimexpr.1767\linewidth-2\tabcolsep}p{\dimexpr.1087\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Amount Added(mcg/ml) & Amount Added(mcg/ml) & Amount Added(mcg/ml) & \%Recovered & Mean Recovery & \%RSD\\
\tblmidrule 
LOQ-1 &
  0.00002547 &
  0.00002730 &
  107.18 &
  \multicolumn{1}{p{\dimexpr(.1767\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{100.27}} &
  \multicolumn{1}{p{\dimexpr(.1087\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{5.97}}\\
LOQ-2 &
  0.00003272 &
  0.00003173 &
  96.97 &
   &
  \\
LOQ-3 &
  0.00002957 &
  0.00002858 &
  96.65 &
   &
  \\
50\% of Rec-1 &
  0.1358 &
  0.1398 &
  102.95 &
  \multicolumn{1}{p{\dimexpr(.1767\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{103.18}} &
  \multicolumn{1}{p{\dimexpr(.1087\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{4.09}}\\
50\% of Rec-2 &
  0.1313 &
  0.1301 &
  99.09 &
   &
  \\
50\% of Rec-3 &
  0.1292 &
  0.1389 &
  107.51 &
   &
  \\
100\% of Rec-1 &
  0.2913 &
  0.2861 &
  98.2 &
  \multicolumn{1}{p{\dimexpr(.1767\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{100.33}} &
  \multicolumn{1}{p{\dimexpr(.1087\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{3.08}}\\
100\% of Rec-2 &
  0.2823 &
  0.2792 &
  98.90 &
   &
  \\
100\% of Rec-3 &
  0.2346 &
  0.2437 &
  103.88 &
   &
  \\
120\% of Rec-1 &
  0.3389 &
  0.3375 &
  99.59 &
  \multicolumn{1}{p{\dimexpr(.1767\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{100.59}} &
  \multicolumn{1}{p{\dimexpr(.1087\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.01}}\\
120\% of Rec-2 &
  0.3810 &
  0.3872 &
  101.63 &
   &
  \\
120\% of Rec-3 &
  0.3675 &
  0.3705 &
  100.57 &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\%Recovery of Impurity-B} }
\label{tw-7975f2314221}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1794\linewidth-2\tabcolsep}p{\dimexpr.1793\linewidth-2\tabcolsep}p{\dimexpr.1781\linewidth-2\tabcolsep}p{\dimexpr.1599\linewidth-2\tabcolsep}p{\dimexpr.1849\linewidth-2\tabcolsep}p{\dimexpr.11839999999999998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Amount Added(mcg/ml) & Amount Added(mcg/ml) & Amount Added(mcg/ml) & \%Recovered & Mean Recovery & \%RSD\\
\tblmidrule 
LOQ-1 &
  0.00002744 &
  0.00002841 &
  103.53 &
  \multicolumn{1}{p{\dimexpr(.1849\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{98.77}} &
  \multicolumn{1}{p{\dimexpr(.11839999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{4.29}}\\
LOQ-2 &
  0.00003171 &
  0.00003026 &
  95.43 &
   &
  \\
LOQ-3 &
  0.00002957 &
  0.00002879 &
  97.36 &
   &
  \\
50\% of Rec-1 &
  0.1869 &
  0.1796 &
  96.09 &
  \multicolumn{1}{p{\dimexpr(.1849\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{97.70}} &
  \multicolumn{1}{p{\dimexpr(.11839999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.83}}\\
50\% of Rec-2 &
  0.1593 &
  0.1531 &
  96.11 &
   &
  \\
50\% of Rec-3 &
  0.1583 &
  0.1597 &
  100.88 &
   &
  \\
100\% of Rec-1 &
  0.2654 &
  0.2738 &
  103.2 &
  \multicolumn{1}{p{\dimexpr(.1849\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{104.67}} &
  \multicolumn{1}{p{\dimexpr(.11839999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.26}}\\
100\% of Rec-2 &
  0.2558 &
  0.2692 &
  105.24 &
   &
  \\
100\% of Rec-3 &
  0.2446 &
  0.2583 &
  105.60 &
   &
  \\
120\% of Rec-1 &
  0.3286 &
  0.3363 &
  102.34 &
  \multicolumn{1}{p{\dimexpr(.1849\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{102.70}} &
  \multicolumn{1}{p{\dimexpr(.11839999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.49}}\\
120\% of Rec-2 &
  0.3268 &
  0.3279 &
  100.34 &
   &
  \\
120\% of Rec-3  &
  0.3375 &
  0.3558 &
  105.42 &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\%Recovery of Impurity-C} }
\label{tw-3e7df1c93ae2}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.17669999999999998\linewidth-2\tabcolsep}p{\dimexpr.1931\linewidth-2\tabcolsep}p{\dimexpr.1752\linewidth-2\tabcolsep}p{\dimexpr.1724\linewidth-2\tabcolsep}p{\dimexpr.17810000000000002\linewidth-2\tabcolsep}p{\dimexpr.1045\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Amount Added(mcg/ml) & Amount Added(mcg/ml) & Amount Added(mcg/ml) & \%Recovered & Mean Recovery & \%RSD\\
\tblmidrule 
LOQ-1 &
  0.00002693 &
  0.00002586 &
  96.03 &
  \multicolumn{1}{p{\dimexpr(.17810000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{97.55}} &
  \multicolumn{1}{p{\dimexpr(.1045\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.46}}\\
LOQ-2 &
  0.00002525 &
  0.00002496 &
  98.85 &
   &
  \\
LOQ-3 &
  0.00002591 &
  0.00002533 &
  97.76 &
   &
  \\
50\% of Rec-1 &
  0.1869 &
  0.1796 &
  96.09 &
  \multicolumn{1}{p{\dimexpr(.17810000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{97.70}} &
  \multicolumn{1}{p{\dimexpr(.1045\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.83}}\\
50\% of Rec-2 &
  0.1593 &
  0.1531 &
  96.11 &
   &
  \\
50\% of Rec-3 &
  0.1583 &
  0.1597 &
  100.88 &
   &
  \\
100\% of Rec-1 &
  0.2654 &
  0.2738 &
  103.2 &
  \multicolumn{1}{p{\dimexpr(.17810000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{104.67}} &
  \multicolumn{1}{p{\dimexpr(.1045\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.26}}\\
100\% of Rec-2 &
  0.2558 &
  0.2692 &
  105.24 &
   &
  \\
100\% of Rec-3 &
  0.2446 &
  0.2583 &
  105.60 &
   &
  \\
120\% of Rec-1 &
  0.3184 &
  0.3081 &
  96.77 &
  \multicolumn{1}{p{\dimexpr(.17810000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{99.59}} &
  \multicolumn{1}{p{\dimexpr(.1045\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{3.23}}\\
120\% of Rec-2 &
  0.3133 &
  0.3099 &
  98.91 &
   &
  \\
120\% of Rec-3 &
  0.3201 &
  0.3300 &
  103.09 &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\%Recovery of Impurity-D} }
\label{tw-7ee30482e27c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1752\linewidth-2\tabcolsep}p{\dimexpr.1725\linewidth-2\tabcolsep}p{\dimexpr.18900000000000002\linewidth-2\tabcolsep}p{\dimexpr.1656\linewidth-2\tabcolsep}p{\dimexpr.18349999999999998\linewidth-2\tabcolsep}p{\dimexpr.11420000000000002\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Amount Added(mcg/ml) & Amount Added(mcg/ml) & Amount Added(mcg/ml) & \%Recovered & Mean Recovery & \%RSD\\
\tblmidrule 
LOQ-1 &
  0.00002485 &
  0.00002503 &
  100.72 &
  \multicolumn{1}{p{\dimexpr(.18349999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{98.39}} &
  \multicolumn{1}{p{\dimexpr(.11420000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.29}}\\
LOQ-2 &
  0.00002596 &
  0.00002498 &
  96.22 &
   &
  \\
LOQ-3 &
  0.00002544 &
  0.00002499 &
  98.23 &
   &
  \\
50\% of Rec-1 &
  0.1363 &
  0.1355 &
  99.41 &
  \multicolumn{1}{p{\dimexpr(.18349999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{101.66}} &
  \multicolumn{1}{p{\dimexpr(.11420000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.74}}\\
50\% of Rec-2 &
  0.1423 &
  0.1491 &
  104.78 &
   &
  \\
50\% of Rec-3 &
  0.1385 &
  0.1396 &
  100.79 &
   &
  \\
100\% of Rec-1 &
  0.2592 &
  0.2582 &
  99.6 &
  \multicolumn{1}{p{\dimexpr(.18349999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{99.61}} &
  \multicolumn{1}{p{\dimexpr(.11420000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{0.51}}\\
100\% of Rec-2 &
  0.2588 &
  0.2591 &
  100.12 &
   &
  \\
100\% of Rec-3 &
  0.2553 &
  0.253 &
  99.10 &
   &
  \\
120\% of Rec-1 &
  0.3555 &
  0.3421 &
  96.23 &
  \multicolumn{1}{p{\dimexpr(.18349999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{97.57}} &
  \multicolumn{1}{p{\dimexpr(.11420000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.19}}\\
120\% of Rec-2 &
  0.3483 &
  0.34199 &
  98.19 &
   &
  \\
120\% of Rec-3 &
  0.3363 &
  0.3306 &
  98.31 &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Effect of Robustness} }
\label{tw-74b4ae02ef17}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1577\linewidth-2\tabcolsep}p{\dimexpr.06776923076923075\linewidth-2\tabcolsep}p{\dimexpr.07608461538461536\linewidth-2\tabcolsep}p{\dimexpr.07348461538461536\linewidth-2\tabcolsep}p{\dimexpr.06638461538461537\linewidth-2\tabcolsep}p{\dimexpr.08108461538461537\linewidth-2\tabcolsep}p{\dimexpr.06862307692307693\linewidth-2\tabcolsep}p{\dimexpr.06862307692307692\linewidth-2\tabcolsep}p{\dimexpr.07252307692307691\linewidth-2\tabcolsep}p{\dimexpr.06612307692307692\linewidth-2\tabcolsep}p{\dimexpr.05952307692307694\linewidth-2\tabcolsep}p{\dimexpr.06552307692307691\linewidth-2\tabcolsep}p{\dimexpr.07655384615384615\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Parameters & \multicolumn{2}{p{\dimexpr(.14385384615384611\linewidth-2\tabcolsep)}}{\cAlignHack \%RSD} & \multicolumn{2}{p{\dimexpr(.13986923076923073\linewidth-2\tabcolsep)}}{\cAlignHack Resolution} & \multicolumn{2}{p{\dimexpr(.1497076923076923\linewidth-2\tabcolsep)}}{\cAlignHack  Imp-A \mbox{}\protect\newline  (\%w/w)} & \multicolumn{2}{p{\dimexpr(.14114615384615384\linewidth-2\tabcolsep)}}{\cAlignHack  Imp-B \mbox{}\protect\newline (\%w/w)} & \multicolumn{2}{p{\dimexpr(.12564615384615387\linewidth-2\tabcolsep)}}{\cAlignHack Imp-C \mbox{}\protect\newline (\%w/w)} & \multicolumn{2}{p{\dimexpr(.14207692307692305\linewidth-2\tabcolsep)}}{\cAlignHack Imp-D \mbox{}\protect\newline (\%w/w)}\\
\rowcolor{kwdboxcolor} & Low & High & Low & High & Low & High & Low & High & Low & High & Low & High\\
\tblmidrule 
Flow(ml/min) &
  1.5 &
  0.4 &
  1.5 &
  1.7 &
  0.05 &
  0.06 &
  0.04 &
  0.06 &
  0.05 &
  0.05 &
  0.05 &
  0.05\\
Wave length (nm) &
  2.7 &
  0.9 &
  1.9 &
  2.3 &
  0.06 &
  0.05 &
  0.05 &
  0.05 &
  0.05 &
  0.04 &
  0.05 &
  0.05\\
Column Temperature(\ensuremath{^{o}}C) &
  1.7 &
  1.1 &
  1.3 &
  1.5 &
  0.05 &
  0.06 &
  0.05 &
  0.04 &
  0.05 &
  0.05 &
  0.04 &
  0.05\\
Buffer (pH) &
  2.7 &
  1.8 &
  2.4 &
  2.1 &
  0.05 &
  0.05 &
  0.05 &
  0.05 &
  0.05 &
  0.05 &
  0.05 &
  0.05\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

    
\section{Results and Discussion}

\bgroup
\fixFloatSize{images/a6c783f0-fb81-4f48-8537-70d6c1bfa4ff-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/a6c783f0-fb81-4f48-8537-70d6c1bfa4ff-upicture1.png}{\includegraphics{images/a6c783f0-fb81-4f48-8537-70d6c1bfa4ff-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Chemical Structure of Blonanserin}}
\label{f-6d0f1f009085}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/1b4fe0c4-bc68-4af0-8ad8-fafa6292a7ea-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/1b4fe0c4-bc68-4af0-8ad8-fafa6292a7ea-upicture2.png}{\includegraphics{images/1b4fe0c4-bc68-4af0-8ad8-fafa6292a7ea-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatogram of Blank}}
\label{f-e1916c2e0002}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/4157da6f-4769-4225-9b7a-4518854cf57f-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/4157da6f-4769-4225-9b7a-4518854cf57f-upicture3.png}{\includegraphics{images/4157da6f-4769-4225-9b7a-4518854cf57f-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatogram of Control sample}}
\label{f-075999fe4747}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/fcad4c01-a6c3-49da-8dbf-500c4c376bf4-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/fcad4c01-a6c3-49da-8dbf-500c4c376bf4-upicture4.png}{\includegraphics{images/fcad4c01-a6c3-49da-8dbf-500c4c376bf4-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Typical chromatogram of spiked sample}}
\label{f-59854378debf}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/3321a668-8724-422a-b5cb-bfc2cb226b59-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/3321a668-8724-422a-b5cb-bfc2cb226b59-upicture5.png}{\includegraphics{images/3321a668-8724-422a-b5cb-bfc2cb226b59-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Linearity of Impurity-A}}
\label{f-28d274ce4822}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/3b24bdfc-4e95-49da-81c8-0b46e239e5ac-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/3b24bdfc-4e95-49da-81c8-0b46e239e5ac-upicture6.png}{\includegraphics{images/3b24bdfc-4e95-49da-81c8-0b46e239e5ac-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Linearity of Impurity-B}}
\label{f-d9df6364309b}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/f54ac8cf-a7b8-4f4e-8dc0-3ca5e0cdf8ab-upicture7.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/f54ac8cf-a7b8-4f4e-8dc0-3ca5e0cdf8ab-upicture7.png}{\includegraphics{images/f54ac8cf-a7b8-4f4e-8dc0-3ca5e0cdf8ab-upicture7.png}}{}
\makeatother 
\caption{\boldmath {Linearity of Impurity-C}}
\label{f-1d3e85182b2c}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/e354fb02-dd0b-4f4c-8806-a2179ee15b19-upicture8.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/e354fb02-dd0b-4f4c-8806-a2179ee15b19-upicture8.png}{\includegraphics{images/e354fb02-dd0b-4f4c-8806-a2179ee15b19-upicture8.png}}{}
\makeatother 
\caption{\boldmath {Linearity of BNC}}
\label{f-cc1bb8588888}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/4b146ae7-aee0-487f-984f-6a029a91bdf1-upicture9.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/4b146ae7-aee0-487f-984f-6a029a91bdf1-upicture9.png}{\includegraphics{images/4b146ae7-aee0-487f-984f-6a029a91bdf1-upicture9.png}}{}
\makeatother 
\caption{\boldmath {Linearity of Impurity-D}}
\label{f-f4e2f222c5a5}
\end{figure}
\egroup
 analysed as per the method. Results of system suitability parameters such as \%RSD of the standard and resolution between Imp-B and Imp-C, results of Suitability of the system are compiled in Table~\ref{tw-f9c2f54e5c12}.

\textbf{Specificity}

A research study was conducted to detect interference. A blank solution was injected three times, According to the test method. A spiked sample and individual impurities were prepared and injected. The blank chromatogram did not show any interference at BNS and impurities retention time. This indicates that the blank does not interfere with the BNS and related impurities (Figures~\ref{f-e1916c2e0002}, \ref{f-075999fe4747} and~\ref{f-59854378debf}). The results are tabulated in Table~\ref{tw-165430e2d394}. All forced degradation samples were stressed by 0.5N HCl, 0.5N NaOH, 1\%H\ensuremath{_{2}}O\ensuremath{_{2}}, Heat and analyzed as per the method and the resulted are tabulated in Tables~\ref{tw-165430e2d394} and~\ref{tw-54f8d9391e22} \textbf{.}

\textbf{Linearity}

The results obtained between concentration and Analyte peak area infers that a very good correlation exists. The calibration plot, which is linear, was obtained over the tested measured values, i.e., from LOQ to 150\% for BNS and its impurities (Figures~\ref{f-28d274ce4822}, \ref{f-d9df6364309b}, \ref{f-1d3e85182b2c}, \ref{f-cc1bb8588888} and~\ref{f-f4e2f222c5a5}), the correlation coefficient obtained was greater than 0.99 and tabulated in Table~\ref{tw-aabd9cee98eb}.

\textbf{Accuracy}

The recovery of BNS and impurities (in percentage) was determined using a sample solution containing all impurities spiked at LOQ, 50\%, 100\%, and 150\% of the sample concentration. Percentage acquisition of the recovery sample is calculated and tabulated in Tables~\ref{tw-16caa69ca5d8}, \ref{tw-7975f2314221}, \ref{tw-3e7df1c93ae2} and~\ref{tw-7ee30482e27c}. 

Percentage recovery for each impurity should be between 90\% and 110\%.

\textbf{Robustness}

Robustness is a deliberate varied chromatographic condition for parameters like flow (\ensuremath{\pm}0.1\%), wavelength (\ensuremath{\pm}2nm), column oven temperature (\ensuremath{\pm}2\ensuremath{^{o}}C) and Buffer pH (\ensuremath{\pm}1), the resolution between critical pairs was more than 1.2, illustrating the robustness of the method as mentioned in Table~\ref{tw-74b4ae02ef17}.
    
\section{Conclusions}
A gradient method was developed for the separation and estimation of Impurities in Blonanserin active pharmaceutical ingredients. The validated method is linear, precise and accurate, selective and specific and shows ruggedness. The method can be effectively used to monitor impurities for stability analysis of controlled samples in Blonanserin API.

\textbf{Funding Support}

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

\textbf{Conflict of interest}

The authors report that they have no conflict of interest in this work.
    

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