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\def\RunningAuthor{Vijey Aanandhi M et al.}
\firstPage{1478}
\articleType{Original Article}
\receivedDate{30 Jan 2021}
\acceptedDate{17 Apr 2021}
\revisedDate{10 Apr 2021}
\journalVolume{2021, 12}
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\journalDoi{ijrps.v12i2.4718}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{\textbf{Estimation and Evaluation of Mexiletine for Bio-availability and Bio-equivalence studies by Liquid phase extraction using LC-MS/MS}}
\author{Elavarasi~E\textsuperscript{1},  
            Binoy~Varghese~Cherian\textsuperscript{1},  
            Shanmugasundaram~P\textsuperscript{2}, 
            Vijey~Aanandhi~M\textsuperscript{*}\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai, Tamil Nadu, India}~\\{\textsuperscript{2}Department of Chemistry, Thiruvalluvar Government Arts College, Rasipuram, Namakkal District, Tamil Nadu, India}}

\begin{abstract}
The work aims to develop an appropriate method for mexiletine with 35-65\% recovery by the LPE method with efficient and selective efficacy of the IS and analyte for the analysis under Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry. This method also reveals the bio-availability and bio-equivalence report for Internal Standard \& working standard. At first, the selection of proper IS. The Internal standard should be structurally more similar to mexiletine. The selection of method plays a major role in which extraction procedure is done either by LPE or SPE. The selection of separation procedure should be either isocratic or gradient. Selection of column on bases separation principle of the compound. Since separation is the major principle for chromatography. Argon and Nitrogen Gas is used as carries with a flow-rate of 2L min. Temperature at 20\ensuremath{^\circ}C, the pressure at 20psi. If the instrument doesn't show any peak or response in after loading sample, check the columns is an aqueous or reverse-phase and then submit the sample. Check all the solution and column and temperature and system stability before loading the sample. After loading the sample, must form calibration curve it must form linearity. The method found should possess the following parameters Specific \& Selectivity, Precision \& Accuracy. The work aims to develop a simple, elegant way for quantification of a molecule and the method determined will have recovery of 35-65\% worldwide. This quantification will be further utilized in Full-Method Validation.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Mexiletine,\newline Anti-arrhythmic agent,\newline Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry,\newline Liquid phase extraction (LPE)
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Vijey~Aanandhi~M~\\ Phone:\ ~\\  Email:\ hodpchemistry@velsuniv.ac.in
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
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        \makeatother
        
    
\section{Introduction}
Mexiletine is used as a local anesthetic, orally active class I~anti-arrythmic agent, and it is structurally more similar to lidocaine. Chemically it named \unskip~\citep{1071533:21966376} as 1-methyl-2-(2,6-xylyloxy)-ethylamine, as shown in Figure~\ref{f-9ddcd69e54e6}. Mexiletine is powder in nature and white in colour, solubility is about freely soluble in water and in organic solvent ethanol, partially soluble in acetonitrile(ACN). 

Mexiletine has a fast onset and offsets kinetics, meaning that they have little or no effect at slower heart rates and more effects at faster heart rates. It shortens the action potential duration, reduces refractoriness, and decreases \unskip~\citep{1071533:21966330} Cmax in partially depolarized cells with fast-response action potentials.

 Mexiletine either does not change the action potential duration or decreases the action potential duration. Mexiletine stops or controls the inward sodium current needed for the initial startup and conduction through impulses in the cardiac system. It doesn't directly affect sinus node, left ventricular action, systolic blood pressure, atrioventricular (AV) velocity, or QRS or QT intervals \unskip~\citep{1071533:21966378}. Half-life of this drug is 10-12 hours. The route of elimination is 10\% excreted through the kidney. 

Toxicity due to overdose are


  
  \begin{enumerate}
  \item \relax Nausea, 
  \item \relax Hypotension, 
  \item \relax Sinus bradycardia, 
  \item \relax Paresthesia, 
  \item \relax Seizures, 
  \item \relax Bundle branch block, 
  \item \relax Av heart block, 
  \item \relax Cardiovascular collapse and 
  \item \relax Coma.
  \end{enumerate}
  They are several methods for determining mexiletine in a biological matrix, specifically in plasma, using the instrument LC-MS/MS \unskip~\citep{1071533:21966377}. 

This method should possess parameters like Specific \& Selectivity, Precision \& Accuracy. The work aims to develop a simple and cost-efficient way for quantification of mexiletine and the method must have recovery of 35-65\% when tested in any worldwide CRO \unskip~\citep{1071533:21966379}.

\textbf{Methodology}

For general baseline start up the study should flow the BA flow with sample preparation and solution preparation \unskip~\citep{1071533:21966382}. First defined an appropriate method for sample process and major equipment used in sample processing in Bio-analytical is cyclomixer \& vortex for LPE. 

Then followed by vibramax for mixing the sample and then generally proceed with centrifuge for collecting clear supernatant and take for injection if the response is not up to the level then proceed with LV-evaporator and reconstitute with mobile phase and re-inject it checks the response \unskip~\citep{1071533:21966381}. This is the general method development flow in BA as per the nature of the compound.

\textbf{Major requirement}


  
  \begin{enumerate}
  \item \relax Drug or Analyte,
  \item \relax IS standards, 
  \item \relax Chemicals \& extraction solvent, 
  \item \relax Solution mobile phase \& wash, 
  \item \relax Columns, 
  \item \relax Biological matrix.
  \end{enumerate}
  Collect all the required materials for the process \unskip~\citep{1071533:21966383}.

\textbf{Preparation of working standard and CC samples}

The stock solution must be dilute in acetonitrile for 100mg mL\ensuremath{^{-1}} concentration of the solution mexiletine and keep stored at -2\ensuremath{^{0}}C to -10 \ensuremath{^{0}}C in glass flasks \unskip~\citep{1071533:21966384}. Working standard is prepared from the stock solution stored. A calibration graph was obtained performing serial dilution by diluting from higher to lower level. 

\textbf{Preparation standard solutions}

Weigh Mexiletine 1mg and dilute in 1ml organic solvent so that the concentration is linear at all parts for 1\ensuremath{\mu}g per \ensuremath{\mu}l and this solution is used for sample processing and its \unskip~\citep{1071533:21966446}  also must be maintained and stored at -2\ensuremath{^{0}}C to -10 \ensuremath{^{0}}C in glass flasks. 


\bgroup
\fixFloatSize{images/975d04e8-3774-4d3c-a8d5-83ff3c69cc61-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/975d04e8-3774-4d3c-a8d5-83ff3c69cc61-upicture1.png}{\includegraphics{images/975d04e8-3774-4d3c-a8d5-83ff3c69cc61-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Mexiletine Structure}}
\label{f-9ddcd69e54e6}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/f33f35a9-648f-485d-83c2-7580a102293c-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/f33f35a9-648f-485d-83c2-7580a102293c-upicture2.png}{\includegraphics{images/f33f35a9-648f-485d-83c2-7580a102293c-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Steps for Liquid phase extraction (LPE)}}
\label{f-dc40544c2390}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/f9c68fe1-8399-43f7-a859-a579a2b04788-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/f9c68fe1-8399-43f7-a859-a579a2b04788-upicture3.png}{\includegraphics{images/f9c68fe1-8399-43f7-a859-a579a2b04788-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Steps for Liquid phase extraction (LPE) }}
\label{f-87e6625dc944}
\end{figure*}
\egroup
\textbf{Quality Control for validation}

LOQQC- Lowest lower quality control the analyte area must 5\% of zero 

LQC - Lower quality control 2 times the value of LLOQC

INTQC - Intermediate quality control should hold 30 \% of a calibration curve.

MQC - Middle-quality control it is the equilibrium point.

HQC - Highest quality control is the highest point at the calibration curve \unskip~\citep{1071533:21966445}.

\textbf{Bulk Spiking}

The general calculation for a number of subject samples must be made and for that samples required amount of QC samples must be spiked under bulk spiking activity \unskip~\citep{1071533:21966448}. If the subject sample has 15-time points, the means number of QC spiked should be multiplication of 10 times, so 150 QC must be spiked. And moreover, before going for bulk spiking, at least one TRPA and PA-01 must be passed as per acceptance criteria. 

\textbf{Acceptance Criteria for CC Standards and QC Sample}

\textbf{Calibration Curve Standards Acceptance Criteria}

It is based on the linearity of the value obtained for study 2 standards can fail, but consecutive failure is not accepted, and in MD A and H can be failed or excluded as shown in Figure~\ref{f-dc40544c2390}. And before accepting r\ensuremath{^2} value must be evaluated by a statistical method by applying value in the provided template \unskip~\citep{1071533:21966449}. Atleast 75\% of a sample must pass the criteria; then only the batch comes under acceptance, or else the whole batch will be rejected.

For QC samples, the peak must sharp and without any split and interference. If any unwanted things caused and some parameters must be tuned as molecule nature \unskip~\citep{1071533:21966455}. QC samples should not possess IS variation. These are the acceptance criteria for CC \& QC samples \unskip~\citep{1071533:21966453}.

\textbf{Solution Preparation like Buffer, Mobile Phase, Reagents and Solutions}


  
  \begin{enumerate}
  \item \relax Make sure the sample processing area and glassware are clean before preparation of the solution.
  \item \relax Weigh the sample for the required amount and transfer it to the reagent bottle with a specified label \unskip~\citep{1071533:21966465}.
  \item \relax Sonicate the solution for de-gasification and proper solubility of the solution 
  \end{enumerate}
  \textbf{ Mobile Phase Preparation}


  
  \begin{enumerate}
  \item \relax Measure the required amount of buffer and add with organic solvent for the preparation of the mobile phase.
  \item \relax Then filter the mobile phase with a membrane filter to filter the un-dissolved and colloidal particle \unskip~\citep{1071533:21966469}.
  \end{enumerate}
  \textbf{Chromatography}

LCMS/MS plays a major role in the bio-analytical department for BA/BE studies.

\textbf{LCMS/MS console}

The starting point of the instrument is HPLC which runs on the major principle of separation on the bases of separating compounds \unskip~\citep{1071533:21966460}. It holds the sample manager where the samples are loaded and placed, as shown in Figure~\ref{f-87e6625dc944}. Then pump, which pushes the sample to a column for separation. LC column is generally made of silica gel. The silica bed plays a major in the separation of the compound based on their polarity. A column can be checked as either an aqueous or RP column by adding water and if the RT swift for positive points, it is said to be an aqueous column. And if the RT swift towards negative points while adding water, it is said to be an RP column. Thus the selection of column is much necessary since it separates the compounds. Solvent manager holds mobile phase, weak wash, strong wash; needle wash should be placed in the appropriate solvent dispenser \unskip~\citep{1071533:21966459}.

Then the sample is taken to a mass spectrometer. It is combined with MRM and the ions are verified and the mass is calculated. And from the analyzer, the value are noted in readout data.

\textbf{Electrospray ionization (ESI) }

ESI is used for minute polar compounds to separate in a column. The liquid from the tube is nebulized and thrown as fine spray of a charged particle. Here the temperature created is responsible for contamination of the tube. The process excites the ion and it evaluates as per MRM fixed in an instrument. It can find ion with some bigger fragments and it can hold a certain amount of pressure and molecule \unskip~\citep{1071533:21966464}.

\textbf{LC-MS/MS tuning with suitable solutions}

Tune the instrument with suitable parameters as given in MRM and the addition and reduction of one ion can be evaluated by acid or alcohol nature. Mix IS and working standard and give an injection to get a baseline for the compound so that we can plan for RT fixation. Implement Q1/Q3 parameters for proper ionization and readout data. Optimize the compound parameters for both Ql and Q3 Ions. In case of system is not a suitable condition, give an Aqueous Std to verify the system condition. And perform system suitability for the instrument before starting a parameter.

\textbf{Precision and accuracy}

The precision of the method was done with repeatability within-day and also intermediate precision parameter done between-day. Three variants of concentrations of quality control samples were run six times per day for valuate precision and once a three days run in between-day precision. The RSD values for precision must be repeatable and must have the same precision \unskip~\citep{1071533:21966458}. 

\textbf{Recovery}

Here recovery is found using QC samples the recovery must be 35-65\% for LPE and 90-105\% for the SPE method \unskip~\citep{1071533:21966456}. Recovery is an individual parameter that shows the efficacy and potency of the drug in action.

\textbf{Limit of detection and quantitation}

The limit of detection (LOD) and limit of quantitation (LOQ) were determined by method run across each other as standard for other this \unskip~\citep{1071533:21966457} method always should be performed after all study, MD and MV  Development and validation.
    
\section{Conclusions}
This method is simple, precise and accurate for finding bio-availability and bio-equivalence studies. Using this method CC\&QC spiking, GCV, Trial PA, bulk spiking, and PA- 01 \& 02 must be performed for pre-method validation. We should determine. Hence this method is very much suitable for determining the mexilitine in the biological matrix. Therefore, further full-method validation with 24 parameters should be performed for reporting the proper method development for mexilitine with proper acceptance recovery, which will be suitable for worldwide parameter determination.
\section*{Acknowledgement}The authors are grateful to the management of Vels Institute of Science, Technology and Advanced Studies (VISTAS) for the facilities provided.

\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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