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\def\RunningAuthor{Vijey Aanandhi M et al.}
\firstPage{17}
\articleType{Original Article}
\receivedDate{02 Jul 2020}
\acceptedDate{03 Aug 2020}
\revisedDate{02 Aug 2020}
\journalVolume{2021, 12}
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\journalDoi{ijrps.v12i1.3907}
\copyrightYear{2021}
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\title{\textbf{Method Development and Validation for Quantification of Sulfamethoxazole in Human plasma by UPLC-MS/MS}}
\author{Vijey~Aanandhi~M\textsuperscript{*},  
            Yamini~R,  
            Elancheziyan~K,  
            Prakash~Chand~T,  
            Aysha~Jadeera~K~A, 
            Binoy~Varghese~Cheriyan~\\[5pt]{Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai- 600117, Tamil Nadu, India}}

\begin{abstract}
The main objective of the study aimed at developing and validating the advanced effective Liquid-Liquid Extraction method for evaluating Sulfamethoxazole in Human plasma, by following the FDA guidelines using UPLC-MS/MS and Sulfamethoxazole D4 as Internal Standard. The analyte was eluted using Acetonitrile: 10mM Ammonium Formate pH 4.5 (60:40 v/v) flows through the column and flow-rate of 0.400mL/min. The injected volume set was 5\ensuremath{\mu}l. Chromatogram obtained with isocratic elution with efficient separation. Spiking concentrations were fixed for the Calibration Curve and Quality Control samples that produced the  linearity over the range of 320.635ng/ml to 22009.900ng/ml for the standard curve. The slope 0.0000535324  and corresponding intercept was plotted using the developed method. The method has been validated according to all established parameters of inter and intra precision and accuracy and showing recovery of 71.39\% and 71.14\% for SM and SMD4 respectively. Reinjection and Reproducibility and Auto Sampler Carry Over Test were met with the specifications. The validated method drew accuracy and meets specifications for the Big Batch, Hemolyzed and Dilution Integrity samples. This assay method was developed that proves all the stability parameters for the matrices as well as solutions used for the study and suitable for the bioequivalence study. The proposed method may also be applicable for the study of combined formulations. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Sulfamethoxazole,\newline Method Development,\newline Chromatographic Conditions,\newline Method Validating Parameters
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Vijey~Aanandhi~M~\\ Phone:\ 9840959519~\\  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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\section{Introduction}
Sulfamethoxazole is the synthetic sulfonamide derivative, which has broad spectrum antibacterial activity. This bacteriostatic agent has a chemical name, 3-(4-Amino benzene sulphamido)-5-methyl isoxazole, which represents molecular formula C\ensuremath{_{10}}H\ensuremath{_{11}}N\ensuremath{_{3}}O\ensuremath{_{3}}S and carries a molecular weight as 253.278g/mol \unskip~\citep{866950:20154848}. It is well known for its effective action against urinary tract infection, intestinal infections and respiratory infections and for streptococcal pharyngitis, otitis and meningitis. The administration of Sulfamethoxazole as a drug alone has been limited nowadays due to the development of resistance. So to overcome this, it may be combined with Trimethoprim that may be effective in treating various bacterial infections \unskip~\citep{866950:20154847}. The serious adverse effect is found to be crystalluria \unskip~\citep{866950:20154862}. Sulfamethoxazole acts by inhibiting the enzyme, dihydropteroate synthase in bacteria that is essential for the production of Folic acid, which is structurally similar with para aminobenzoic acid which is shortly called PABA suppresses dihydropteroate diphosphate (DHPP) and PABA from combining to form dihydropteroic acid (an intermediate), which later forms dihydrofolic acid. Hence a number of essential metabolic functions are interrupted \unskip~\citep{866950:20154861}. Sulfamethoxazole is absorbed orally and topically and intermediate acting (8-12 hours), binds to 70\% plasma proteins. It is metabolized in the liver mainly by N4 acetylation and eliminates at half life of 10 hours. The bioavailability of Sulfamethoxazole observed to be 86.2\%{\textendash}99.1\% \unskip~\citep{866950:20154860}.

Some of the literatures reported have been studied considering the quantification methods for Sulfamethoxazole. An HPLC-MS/MS method was presented to determine Sulfamethoxazole and Trimethoprim in Dried Plasma and Urine Spot \unskip~\citep{866950:20154859} and a study was conducted to simultaneously determine Sulfadiazine and Sulfamethoxazole in formulations have been conducted in HPLC and the method carried along with the second order calibration method \unskip~\citep{866950:20154851}. HPLC method was performed to estimate Cotrimoxazole tablet as well as in human plasma, an invitro study \unskip~\citep{866950:20154854}. HPLC method was developed for simultaneously determined Sulfamethoxazole and Trimethoprim in Human plasma using different internal standards such as Antipyrine and Cefmetazole \unskip~\citep{866950:20154845,866950:20154844}. However another Internal standard Primidone was also used for the estimation of Sulfamethoxazole in Human Plasma \unskip~\citep{866950:20154856}. A multidimensional HPLC method and bidimensional HPLC method were proposed for estimating Sulfamethoxazole and Trimethoprim in Bovine milk \unskip~\citep{866950:20154842,866950:20154849}. A procedure for estimating Sulfamethoxazole and Trimethoprim from Cotrimoxazole tablet formulation was developed \unskip~\citep{866950:20154852}. Sulfamethoxazole, Trimethoprim, methyl and propylparaben were estimated using the HPLC method \unskip~\citep{866950:20154843}. Chromatographic method was developed to estimate Sulfamethoxazole, Trimethoprim, Sulfamethoxypyridazine, sulfadimethoxine, Bromhexine \unskip~\citep{866950:20154850}. Estimation of Sulfamethoxazole, Trimethoprim, and Sulphamethoxypyridazine has been applied with bivariate calibration algorithm \unskip~\citep{866950:20154853}. Spectrophotometric method was proposed to estimate Sulfamethoxazole and Trimethoprim \unskip~\citep{866950:20154846}. Capillary Electrophoresis method was proposed for estimating Sulfamethoxazole and Trimethoprim in plasma \unskip~\citep{866950:20154841}. A study was reported for the determination of Sulfonamides in milk samples using LCMS \unskip~\citep{866950:20154858}. However a method for determining Sulfamethoxazole and Trimethoprim in rat plasma using Estazolam as Internal standard was proposed in LCMS \unskip~\citep{866950:20154840}. In LCMS, ten sulfonamides in eggs were studied \unskip~\citep{866950:20154863}. Sulfamethoxazole and Trimethoprim were quantified by SPE (Solid Phase Extraction) method and compared two methods efficiency such as LC with UV and MS detection \unskip~\citep{866950:20154857}. A procedure was proposed on study in low volume plasma by SPE method for determining the Sulfamethoxazole and Trimethoprim in LCMS using Imipramine as Internal standard \unskip~\citep{866950:20154855}. 

Among these studies, determination of Sulfamethoxazole can be done almost in HPLC method majorly and one of them carried out in CPE method and the most efficient LCMS method also been done. So this study is performed to produce a simple, sensitive, rugged and reproducible UPLC (Ultra Performance Liquid Chromatography) Mass spectrometry method to quantify Sulfamethoxazole (SM) in Human plasma. The IS Sulfamethoxazole D4 (SMIS / SMD4) was procured for the analysis. Figure~\ref{f-01a1c68cecf5} shows structure of SM and SMD4.


\bgroup
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\centering \makeatletter\IfFileExists{images/ad2d2034-4070-4958-b18c-fdcf7490ca72-upicture1.png}{\includegraphics{images/ad2d2034-4070-4958-b18c-fdcf7490ca72-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Structure of (a) Sulfamethoxazole and (b) Sulfamethoxazole D4}}
\label{f-01a1c68cecf5}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Some of the mass parameters} }
\label{tw-054ac83a202d}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2657\linewidth-2\tabcolsep}p{\dimexpr.2343\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Compound & Collision energy  & Cone Voltage & Dwell time\\
\tblmidrule 
SM  &
  8.0  &
  10.0  &
  0.100 seconds \\
SMD4  &
  16.0  &
  40.0  &
  0.100 seconds \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

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\caption{\boldmath {Mass spectrum of Sulfamethoxazole (a) and Sulfamethoxazole D4 (b) }}
\label{f-9ad56eda911e}
\end{figure*}
\egroup

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\centering \makeatletter\IfFileExists{images/200571e2-e1dd-490e-bb0c-11bfd086decd-upicture3.png}{\includegraphics{images/200571e2-e1dd-490e-bb0c-11bfd086decd-upicture3.png}}{}
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\caption{\boldmath {Mass spectrum Sulfamethoxazole daughter ions }}
\label{f-1709e70f6477}
\end{figure*}
\egroup

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\centering \makeatletter\IfFileExists{images/887216a7-1da7-4a1d-9827-ebd5f3668c86-upicture4.png}{\includegraphics{images/887216a7-1da7-4a1d-9827-ebd5f3668c86-upicture4.png}}{}
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\caption{\boldmath {Mass spectrum Sulfamethoxazole D4 daughter ions}}
\label{f-6c6f9d019ef0}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Matrix Factor with Peak area of analyte and IS} }
\label{tw-a6a319f311bd}
\def\arraystretch{1.1}
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\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.20720000000000002\linewidth-2\tabcolsep}p{\dimexpr.2054\linewidth-2\tabcolsep}p{\dimexpr.1874\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Concentration & Considerations & SM & SMD4 & MF\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.20720000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Low QC Level }} &
  Average  &
  13899.0000  &
  230885.0000  &
  1.0150 \\
 &
  Std D  &
  581.24177  &
  7865.85583  &
  0.00707 \\
 &
  \%COVs  &
  4.18  &
  3.41  &
  0.70 \\
\multicolumn{1}{p{\dimexpr(.20720000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{High QC Level }} &
  Average  &
  142503.0000  &
  160434.5000  &
  0.9900 \\
 &
  Std D  &
  1258.65007  &
  1929.69441  &
  0.02828 \\
 &
  \%COV  &
  0.88  &
  1.20  &
  2.86 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Calibration Standard Curve} }
\label{tw-f6072cbbb3f3}
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\tbltoprule \rowcolor{kwdboxcolor}Concentration (ng/mL) & Average  \mbox{}\protect\newline (ng/mL) & \% Coefficient of Variation & \% Accuracy  & \% Deviation\\
\tblmidrule 
STD 1 (320.635)  &
  314.7374 &
  2.41 &
  98.16 &
  -1.84\\
STD 2 (641.269)  &
  653.6740 &
  4.06 &
  101.93 &
  1.93\\
STD 3 (1105.637)  &
  1116.1710 &
  1.11 &
  100.95 &
  0.95\\
STD 4 (2211.273)  &
  2298.0712 &
  4.18 &
  103.93 &
  3.93\\
STD 5 (4422.546)  &
  4514.6096 &
  2.57 &
  102.08 &
  2.08\\
STD 6 (8845.092)  &
  8857.4976 &
  4.81 &
  100.14 &
  0.14\\
STD 7 (13241.156)  &
  12995.7108 &
  1.66 &
  98.15 &
  -1.85\\
STD 8 (17605.920)  &
  17288.4950 &
  0.89 &
  98.19 &
  -1.81\\
STD 9 (22009.900)  &
  21233.5502 &
  0.8 &
  96.47 &
  -3.53\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Precision and Accuracy} }
\label{tw-5b3091800fcf}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.14421111111111111\linewidth-2\tabcolsep}p{\dimexpr.1149111111111111\linewidth-2\tabcolsep}p{\dimexpr.1092111111111111\linewidth-2\tabcolsep}p{\dimexpr.0945111111111111\linewidth-2\tabcolsep}p{\dimexpr.09271111111111114\linewidth-2\tabcolsep}p{\dimexpr.12678888888888894\linewidth-2\tabcolsep}p{\dimexpr.12400000000000002\linewidth-2\tabcolsep}p{\dimexpr.09911111111111115\linewidth-2\tabcolsep}p{\dimexpr.09454444444444448\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Conc. of QC & \multicolumn{4}{p{\dimexpr(.4113444444444444\linewidth-2\tabcolsep)}}{\cAlignHack Intra Run {\textendash} P\&A } & \multicolumn{4}{p{\dimexpr(.4444444444444446\linewidth-2\tabcolsep)}}{\cAlignHack Inter Run {\textendash} P\&A}\\
\rowcolor{kwdboxcolor}\cAlignHack (ng/mL) & \cAlignHack Average (ng/mL) & \cAlignHack Std D  \mbox{}\protect\newline (ng/mL) & \cAlignHack \% COV & \cAlignHack \% A & \cAlignHack Average  \mbox{}\protect\newline (ng/mL)  & \cAlignHack Std D  \mbox{}\protect\newline (ng/mL) & \cAlignHack \% COV & \cAlignHack \% A\\
\tblmidrule 
LLOQQC 328.868  &
  333.66 &
  61.14 &
  18.32 &
  101.4 &
  313.98 &
  37.42 &
  11.92 &
  95.47\\
LQC  \mbox{}\protect\newline 893.664  &
  895.46 &
  13.10 &
  1.46 &
  100.2 &
  878.13 &
  32.09 &
  3.65 &
  98.26\\
MQC  \mbox{}\protect\newline 7680.000  &
  7907.63 &
  74.35 &
  0.94 &
  102.9 &
  7869.10 &
  82.51 &
  1.05 &
  102.42\\
HQC  \mbox{}\protect\newline 16006.406  &
  16505.07 &
  181.16 &
  1.10 &
  103.1 &
  16152.07 &
  389.73 &
  2.41 &
  100.91\\
 &
  \multicolumn{4}{p{\dimexpr(.4113444444444444\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Intra Run {\textendash} Non P\&A}} &
  \multicolumn{4}{p{\dimexpr(.4444444444444446\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Inter Run {\textendash} Non P\&A}}\\
\cAlignHack  &
  \cAlignHack \textbf{Average} \textbf{(ng/mL)} &
  \cAlignHack \textbf{Std D} \mbox{}\protect\newline \textbf{(ng/mL)} &
  \cAlignHack \textbf{\% COV} &
  \cAlignHack \textbf{\% A} &
  \cAlignHack \textbf{Average } \mbox{}\protect\newline \textbf{(ng/mL)} &
  \cAlignHack \textbf{Std D} \mbox{}\protect\newline \textbf{(ng/mL)} &
  \cAlignHack \textbf{\% COV} &
  \cAlignHack \textbf{\% A}\\
LQC  \mbox{}\protect\newline 893.664  &
  934.268 &
  17.36 &
  1.86 &
  104.5 &
  900.35 &
  41.82 &
  4.65 &
  100.75\\
MQC  \mbox{}\protect\newline 7680.000  &
  3203.28 &
  75.46 &
  2.36 &
  96.78 &
  3110.58 &
  124.54 &
  4.00 &
  93.98\\
HQC  \mbox{}\protect\newline 16006.400  &
  15982.06 &
  163.83 &
  2.24 &
  95.17 &
  15964.98 &
  273.84 &
  3.86 &
  92.40\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{The accuracy of all the standards must be \ensuremath{\pm}15\% to meet with specifications. Accuracy \ensuremath{\pm} 20.0 \%for LLOQQC and \ensuremath{\pm} 15.0 \% for low mid and high QCs}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Recovery of Sulfamethoxazole} }
\label{tw-8ccb36f025fb}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1188\linewidth-2\tabcolsep}p{\dimexpr.14849999999999998\linewidth-2\tabcolsep}p{\dimexpr.14759999999999998\linewidth-2\tabcolsep}p{\dimexpr.28259999999999998\linewidth-2\tabcolsep}p{\dimexpr.3025\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}QC level & \multicolumn{4}{p{\dimexpr(.8811999999999999\linewidth-2\tabcolsep)}}{\cAlignHack Sulfamethoxazole }\\
\rowcolor{kwdboxcolor} & \% Recovery  & \% Average  & \% Standard Deviation & \% Coefficient of Variation \\
\tblmidrule 
High   &
  70.35  &
  \multicolumn{1}{p{\dimexpr(.14759999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{71.39 }} &
  \multicolumn{1}{p{\dimexpr(.28259999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 1.07 }} &
  \multicolumn{1}{p{\dimexpr(.3025\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 1.51 }}\\
Mid  &
  72.50  &
   &
   &
  \\
Low  &
  71.32  &
   &
   &
  \\
 &
  \multicolumn{4}{p{\dimexpr(.8811999999999999\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Sulfamethoxazole D4}}\\
 &
  \textbf{\% Recovery } &
  \textbf{\% Average } &
  \textbf{\% Standard Deviation} &
  \textbf{\% Coefficient of Variation}\\
High  &
  69.56  &
  \multicolumn{1}{p{\dimexpr(.14759999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{71.14 }} &
  \multicolumn{1}{p{\dimexpr(.28259999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 2.09 }} &
  \multicolumn{1}{p{\dimexpr(.3025\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 2.95 }}\\
Mid  &
  70.35  &
   &
   &
  \\
Low  &
  73.52  &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{\%COV- Percentage Coefficient of Variation; \% A - Percentage Accuracy; \%Dev- Percentage Deviation; Std D - Standard Deviation}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\bgroup
\fixFloatSize{images/400d7b01-152e-4313-b6ae-81f7ecfaab69-upicture5.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/400d7b01-152e-4313-b6ae-81f7ecfaab69-upicture5.png}{\includegraphics{images/400d7b01-152e-4313-b6ae-81f7ecfaab69-upicture5.png}}{}
\makeatother 
\caption{\boldmath {(A) Standard Curve, Representative Chromatogram of (B) ZC, (C) LLOQ, (D) ULOQ level}}
\label{f-2078f9a7a15f}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {This table shows results for Big batch, Hemolysis Effect, Dilution Integrity} }
\label{tw-2e3646ccc067}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.11445714285714285\linewidth-2\tabcolsep}p{\dimexpr.17715714285714284\linewidth-2\tabcolsep}p{\dimexpr.1712571428571429\linewidth-2\tabcolsep}p{\dimexpr.1796571428571429\linewidth-2\tabcolsep}p{\dimexpr.11675714285714292\linewidth-2\tabcolsep}p{\dimexpr.11915714285714294\linewidth-2\tabcolsep}p{\dimexpr.12155714285714287\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Sample  & Big Batch &  & \cAlignHack  & \cAlignHack  & \cAlignHack  & \cAlignHack \\
\rowcolor{kwdboxcolor}level & \cAlignHack Conc. (ng/mL) & \cAlignHack Accuracy (ng/mL) & \cAlignHack Std D (ng/mL) & \cAlignHack \% COV & \cAlignHack \% Accuracy & \cAlignHack \%Dev\\
\tblmidrule 
LQC &
  893.664 &
  863.2980 &
  15.43112 &
  1.79 &
  96.60 &
  -3.40\\
MQC &
  7680.000 &
  7288.4495 &
  85.26012 &
  1.17 &
  94.86 &
  -5.14\\
HQC &
  16006.400 &
  15256.4230 &
  205.61180 &
  1.35 &
  35.31 &
  -4.69\\
 &
  \multicolumn{2}{p{\dimexpr(.3484142857142858\linewidth-2\tabcolsep)}}{\textbf{Hemolysis} \textbf{Effect}} &
   &
   &
   &
  \\
 &
  \textbf{Conc. (ng/mL)} &
  \textbf{Mean(ng/mL)} &
  \textbf{Std D (ng/mL)} &
  \textbf{\% COV} &
  \textbf{\% Accuracy} &
  \textbf{\%Dev}\\
Hemo-Low &
  893.664 &
  855.9961 &
  32.03813 &
  3.74 &
  95.79 &
  -4.21\\
Hemo-High &
  16006.400 &
  15757.5005 &
  204.86004 &
  1.30 &
  98.45 &
  -1.55\\
 &
  \multicolumn{2}{p{\dimexpr(.3484142857142858\linewidth-2\tabcolsep)}}{\textbf{Dilution Integrity}} &
   &
   &
   &
  \\
 &
  \textbf{Conc. (ng/mL)} &
  \textbf{Dilution Factor} &
  \textbf{Average (ng/mL)} &
  \textbf{Std D (ng/mL)} &
  \textbf{\% COV} &
  \textbf{\% Accuracy}\\
DIS-150  &
  33013.200 &
  2 &
  30859.7180 &
  462.82119 &
  1.50 &
  93.48\\
DIS-150 &
  33013.200 &
  4 &
  30729.0380 &
  239.49848 &
  0.78 &
  93.08\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Refg - Refrigerator, ng - Nano gram, \ensuremath{\mu}g- Micro gram, \ensuremath{\mu}L- Micro litre, mL- Milli litre, mm- Millimetre}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Matrix Stabilities} }
\label{tw-26b25fef18aa}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2002\linewidth-2\tabcolsep}p{\dimexpr.074399999999999995\linewidth-2\tabcolsep}p{\dimexpr.04280000000000001\linewidth-2\tabcolsep}p{\dimexpr.0834\linewidth-2\tabcolsep}p{\dimexpr.11900000000000002\linewidth-2\tabcolsep}p{\dimexpr.040600000000000005\linewidth-2\tabcolsep}p{\dimexpr.0925\linewidth-2\tabcolsep}p{\dimexpr.0875\linewidth-2\tabcolsep}p{\dimexpr.0696\linewidth-2\tabcolsep}p{\dimexpr.0439\linewidth-2\tabcolsep}p{\dimexpr.1461\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{Stability \& Storage } & \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{Sample Level } & Average  \mbox{}\protect\newline (ng/mL) & \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{Std D  \mbox{}\protect\newline (ng/mL)} & \%COV  & \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{\%A } & \%Dev \\
\tblmidrule 
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{FT for 5 cycles }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{FT-Low } &
  857.66  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{36.55 } &
  4.26  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{95.97 } &
  -4.03 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{FT-High } &
  14959.16  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{230.68 } &
  1.54  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{93.46 } &
  -6.54 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{BT Stability for 21.5hrs }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{BT-Low } &
  887.52  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{12.60 } &
  1.42  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{99.31 } &
  -0.69 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{BT-High } &
  14538.09  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{305.80 } &
  2.10  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{90.83 } &
  -9.17 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{PS Stability (RT) for 26.5hrs }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{PS-RT-Low } &
  883.76  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{2.32 } &
  0.26  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{98.89 } &
  -1.11 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{PS-RT-High } &
  14809.89  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{176.09 } &
  1.19  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{92.52 } &
  -7.48 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{PS Stability(Autosampler) for 57hrs }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{PS-Low } &
  843.23  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{3.19 } &
  0.38  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{94.36 } &
  -5.64 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{PS-High } &
  14444.28  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{34.35 } &
  0.24  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{90.24 } &
  -9.76 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{RI stability  \mbox{}\protect\newline (Re-injected CC) }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{RIR-Low } &
  897.90  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{16.24 } &
  1.81  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{100.47 } &
  0.47 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{RIR-High } &
  16159.29  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{26.33 } &
  0.16  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{100.96 } &
  0.96 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{RI Stability  \mbox{}\protect\newline (Original CC) }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{RIR-Low  } &
  844.70  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{15.83 } &
  1.88  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{94.52 } &
  -5.48 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{RIR-High } &
  15726.31  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{25.68 } &
  0.16  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{98.25 } &
  -1.75 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{LT Stability -200C for 40 days }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{LT-Low(-20) } &
  891.19  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{7.95 } &
  0.89  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{99.72 } &
  -0.28 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{LT-High(-20) } &
  15064.06  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{263.68 } &
  1.75  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{94.11 } &
  -5.89 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{LT Stability -700C for 40 days }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{LT-Low(-70) } &
  866.37  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{14.48 } &
  1.67  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{96.95 } &
  -3.05 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{LT-High(-70) } &
  14879.21  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{515.91 } &
  3.47  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{92.96 } &
  -7.04 \\
\multicolumn{2}{p{\dimexpr(.2746\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{ DE Stability for 26hrs }} &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{DE-Low } &
  889.74  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{54.481 } &
  6.12  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{99.56 } &
  -0.44 \\
 &
   &
  \multicolumn{2}{p{\dimexpr(.12620000000000001\linewidth-2\tabcolsep)}}{DE-High } &
  14772.27  &
  \multicolumn{2}{p{\dimexpr(.1331\linewidth-2\tabcolsep)}}{535.46 } &
  3.62  &
  \multicolumn{2}{p{\dimexpr(.1135\linewidth-2\tabcolsep)}}{92.29 } &
  -7.71 \\
\textbf{Sample Level } &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{\textbf{Test/} \mbox{}\protect\newline \textbf{Ref }} &
  \textbf{Average} \textbf{(ng/mL)} &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{\textbf{Std D  } \mbox{}\protect\newline \textbf{(ng/mL)}} &
  \textbf{\%COV} &
  \multicolumn{4}{p{\dimexpr(.3471\linewidth-2\tabcolsep)}}{\textbf{Average Test/ Ref (\%)}}\\
\multicolumn{1}{p{\dimexpr(.2002\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{WB-LOW-RT(1.5 hrs) }} &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{TEST } &
  0.0749  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.00107 } &
  1.43  &
  \multicolumn{2}{p{\dimexpr(.1571\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{100.67 }}\\
 &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{REF } &
  0.0744  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.00151 } &
  2.03  &
   &
  \\
\multicolumn{1}{p{\dimexpr(.2002\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{WB-HIGH-RT(1.5hrs) }} &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{TEST } &
  1.2209  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.02352 } &
  1.93  &
  \multicolumn{2}{p{\dimexpr(.1571\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{100.82 }} &
  \multicolumn{2}{p{\dimexpr(.19\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{}}\\
 &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{REF } &
  1.2110  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.02704 } &
  2.23  &
   &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.2002\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{WB-LOW-IB(1.5 hrs) }} &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{TEST } &
  0.0563  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.00206 } &
  3.66  &
  \multicolumn{2}{p{\dimexpr(.1571\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{100.54 }} &
  \multicolumn{2}{p{\dimexpr(.19\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{}}\\
 &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{REF } &
  0.0560  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.00095 } &
  1.70  &
   &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.2002\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{WB-HIGH-IB(1.5 hrs) }} &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{TEST } &
  0.8951  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.03628 } &
  4.05  &
  \multicolumn{2}{p{\dimexpr(.1571\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{100.55 }} &
  \multicolumn{2}{p{\dimexpr(.19\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{}}\\
 &
  \multicolumn{2}{p{\dimexpr(.1172\linewidth-2\tabcolsep)}}{REF } &
  0.8902  &
  \multicolumn{2}{p{\dimexpr(.15960000000000003\linewidth-2\tabcolsep)}}{0.03636 } &
  4.08  &
   &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{FT- Freeze Thaw, BT-Bench Top, PS- Processed Sample, RI- Reinjection, LT- Long Term, DE- Dry Extract, WB- Whole Blood}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Solution Stability} }
\label{tw-574ebc1f6236}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2935\linewidth-2\tabcolsep}p{\dimexpr.1675\linewidth-2\tabcolsep}p{\dimexpr.13\linewidth-2\tabcolsep}p{\dimexpr.121\linewidth-2\tabcolsep}p{\dimexpr.13600000000000001\linewidth-2\tabcolsep}p{\dimexpr.152\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Solution stability & Test/Reference  & Average Area & Std D & \%COV  & Average (\%) \\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.2935\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Master Stock LTSS for 10 days at Refg }} &
  LTSS-TEST  &
  69109.00  &
  1739.48  &
  2.52  &
  \multicolumn{1}{p{\dimexpr(.152\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{99.39 }}\\
 &
  LTSS-REF  &
  69530.50  &
  555.07  &
  0.80  &
  \\
\multicolumn{1}{p{\dimexpr(.2935\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Master Stock STSS for 6.5hrs at RT }} &
  STSS-TEST  &
  71142.00  &
  861.25  &
  1.21  &
  \multicolumn{1}{p{\dimexpr(.152\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{102.32 }}\\
 &
  STSS-REF  &
  69530.50  &
  555.07  &
  0.80  &
  \\
\multicolumn{1}{p{\dimexpr(.2935\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Neat standard LTSS for 30days at Refg }} &
  NS-TEST  &
  64602.50  &
  207.18  &
  0.32  &
  \multicolumn{1}{p{\dimexpr(.152\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{96.22 }}\\
 &
  NS-REF  &
  67139.00  &
  188.09  &
  0.28  &
  \\
\multicolumn{1}{p{\dimexpr(.2935\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{ASS for 11 days at Refg }} &
  ASS-TEST  &
  69738.00  &
  216.37  &
  0.31  &
  \multicolumn{1}{p{\dimexpr(.152\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{103.99}}\\
 &
  ASS-REF  &
  67062.00  &
  90.50  &
  0.13  &
  \\
\multicolumn{1}{p{\dimexpr(.2935\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{ASS for 6.5hrs at Refg }} &
  ASS-TEST  &
  70010.00  &
  287.08  &
  0.41  &
  \multicolumn{1}{p{\dimexpr(.152\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{104.40}}\\
 &
  ASS-REF  &
  67062.00  &
  90.50  &
  0.13  &
  \\
\multicolumn{1}{p{\dimexpr(.2935\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{IS Spiking Solution for 12 days at Refg }} &
  IS-TEST  &
  181587.0000  &
  743.87633  &
  0.41  &
  \multicolumn{1}{p{\dimexpr(.152\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{99.11 }}\\
 &
  IS-REF  &
  183219.5000  &
  1397.95011  &
  0.76  &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

    
\section{\textbf{METHODOLOGY}}
\textbf{Chemicals}

Human blank plasma (K\ensuremath{_{2}}EDTA) maintained at -20\ensuremath{^{0}}C was bought from Symbiosis, Ahmedabad, India. Sulfamethoxazole (SM) and Sulfamethoxazole D4 (SMD4) were procured from Clearsynth Labs Limited for which purity remains {\textgreater}98, Methanol and Acetonitrile from Finar, Ammonium Formate obtained from Fisher Scientific, Formic acid (FA) and Ortho Phosphoric Acid (OPA) from Merck, Mumbai, India. Methyl Tert-Butyl Ether (TBME) from Spectrochem, and Dichloromethane bought from Honeywell, Chennai, India. MilliQ pure water was made use for the whole study. 

\textbf{Instruments and Conditions}

Waters Acquity Ultra Performance LC system (Massachusetts, US) connected to the Mass Lynx V4.1. The Mass spectrometer used Waters XEVO Triple Quadrupole Detector (TQD) along with Electrospray Ionization. 

\textbf{Chromatographic conditions}

The mobile phase Acetonitrile and 10mM Ammonium Formate with FA to pH 4.5 at the ratio 60:40 flows at 0.400mL/min through the analytical column ACE 3 C18 5cm X 4.6mm that was maintained at 40\ensuremath{^{0}}C. The autosampler maintained at 5\ensuremath{^{0}}C with the volume for injection to be 5\ensuremath{\mu}L. The whole run takes place at 2.5mintues.

\textbf{Mass parameters optimization}

Electrospray ionization carried out with the ions in positive mode and source temperature 150\ensuremath{^{0}}C. The compound ionization and tuning were carried out by which the Sulfamethoxazole parent ion (M.W 253.28g/mol) and Sulfamethoxazole D4 (M.W 257.3g/mol) both ionised to produce daughter ions and set different fragmentation patterns which are shown in the Figures~\ref{f-1709e70f6477} and~\ref{f-6c6f9d019ef0}. The mass spectrum of SM and SMD4 produced product ions at mass charge ratio among which the major product ion at m/z was 155.96 for SM and 159.99 for SMD4. Table~\ref{tw-054ac83a202d} shows mass parameters. 


  
  \begin{enumerate}
  \item \relax Desolvation gas flow - 1100L/hr
  \item \relax Probe flow rate - 400\ensuremath{\mu}L/min
  \item \relax Capillary voltage - 2.0 kV 
  \item \relax Cone gas flow - 50 L/hr
  \end{enumerate}
  \textbf{Calibration Curve (CC) Standards and Quality Controls (QC)}

The calibration range was selected based on the Cmax value. The analytical curve range was 320.635 to 22009.900ng/mL. The master stock solutions were prepared by weighing Sulfamethoxazole equivalent to 2000.00\ensuremath{\mu}g/mL and dissolved in methanol in suitable volumetric flasks. The IS stock solution prepared by weighing of SMD4 equivalent to 100.00\ensuremath{\mu}g/mL and dissolved in HPLC grade Methanol.  The spiking solutions for IS (5000.00ng /ml) dissolved in 50\% Methanol both kept at (-2 to -8\ensuremath{^\circ}C). Concentrations of SM from Lower Limit of Quantification (LLOQ) 320.635, 641.269, 1105.637, 2211.273, 4422.546, 8845.092, 13241.156, 17607.920, 22009.900ng/mL Upper Limit of Quantification (ULOQ) for analytical standards and 328.868, 893.664, 7680.000, 16006.400ng/mL of Lower Limit of Quantification, Low, Mid, High QC samples maintained at 70\ensuremath{^{0}}C). 

\textbf{Sample Preparation}

Blank plasma samples that are spiked with different concentrations of SM and SMD4 are extracted using Liquid-liquid extraction method. 50\ensuremath{\mu}L SMD4 (5000.0ng/ml) and 100\ensuremath{\mu}L plasma added using micropipette into vials, 100\ensuremath{\mu}L of 0.1\%OPA was added and mixed using platform shaker. Add 2.5 ml of TBME: DCM (70:30), vortex at 2000rpm for 10mins followed by refrigerated centrifugation at 4000rpm for 10mins. Then the organic phase was separated and subjected to Low volume evaporator maintained at 40\ensuremath{^\circ}C and let it dry completely. The residues were then mixed with 500 \ensuremath{\mu}L mobile phase composition solution to reconstitute the molecules and the samples were loaded for injection.  

\textbf{Method Validation (MV)} \textbf{\space }

MV consists of series of experiments that can be performed to get the quantitative measurements in a particular matrix type that shows the method is reliable and reproducible whenever conducted in another LC system and by analyst that should be constant. Parameters may include Selectivity and Matrix factor, Ruggedness, Sensitivity, Precision and Accuracy (PA), Recovery, Big batch, Hemolysis effect, Dilution factor, Whole Blood Stability and Matrix and Solution Stabilities. 
    
\section{\textbf{RESULTS AND DISCUSSIONS}}
\textbf{Tuning, Optimization and Method Development}

Sulfamethoxazole and Sulfamethoxazole D4 were tuned according to the mass parameters. Daughter ions that were formed by Positive mode of ESI where the molecules were bombarded based on the mass charge ratio. The Figures~\ref{f-9ad56eda911e} and~\ref{f-1709e70f6477} and Figure~\ref{f-6c6f9d019ef0} represents peak of the parent and daughter ions. 

The Major product ions for SM and SMD4 were selected. Multiple Monitoring Reactions was setup in the software and their parameters were optimized.

During the method development different mobile phases at various compositions, different analytical columns, flow rates and injection volumes were studied to fix suitable chromatographic conditions. After the trails mobile phase composition fixed was Acetonitrile: 10mM Ammonium Formate with FA to pH 4.5 flowing at a rate of 0.400mL/min through the analytical column ACE 3 C18 5cm X 4.6mm showed perfect peak shape. Extraction trails were carried out for Protein precipitation observed very less recovery. So trails were made with Liquid-Liquid Extraction technique that showed 70\% recovery. PA batch was performed check and fix the method. 

\textbf{Pre Method Validation (PMV) } \textbf{\space }

PMV was performed with three PA and the other three non PA batch. The recovery was determined along with PA batch. The SM and SMD4 recovery were 73.55\% and 74.10\%. The matrix factor experiment showed no suppression or enhancement. Some stability parameters were determined in PMV. Blood Stability for 1.5hrs, Freeze and Thaw stability for three cycles of 24, 36 and 48hrs, Benchtop stability for 15.5hrs, Processed samples at RT condition for 44.5hrs and autosampler condition for 53.5hrs and the Dry extract stability at 23hrs were all proved. The ruggedness showed promising results from analyst to analyst. 

\textbf{Full Method Validation } \textbf{\space }

\textbf{Selectivity \& Matrix factor} \textbf{\space }

All the blank samples of eight lots which contain hemolyzed and lipemic were selected and observed for no interferences. Zero Calibrator (ZC) and LLOQ meet specifications. The Rt for SM and SMD4 showed 1.52 and 1.52. Here the precision represented as Coefficient of Variation (\%CV) for Matrix Factor (MF) should be \ensuremath{\leq } 15\%, for results refer Table~\ref{tw-a6a319f311bd}.\textbf{\space }

\textbf{Precision, Accuracy and Recovery}

The CC showed linearity with the slope of 0.0000535324 intercepts at 0.00167398. They remain reliable and reproducible with the CC range of 320.635 to 22009.900ng/mL. Inter and Intra assay were conducted for both PA and Non PA batches. Blank samples were clear with no interference and Zero Calibrator (ZC) meets specification. The standard curve, ZC, LLOQ and ULOQ level were represented in the Figure~\ref{f-2078f9a7a15f}. All the QC samples meet with the specifications. The mean recovery for SM and SMD4 calculated and were found to be 71.39\% and 71.14\%. Refer Table~\ref{tw-f6072cbbb3f3}, Table~\ref{tw-5b3091800fcf} and Table~\ref{tw-8ccb36f025fb}.

\textbf{ASCOT, Sensitivity and Ruggedness}

The Auto Sampler Carry over Test (ASCOT) results showed that LLOQ and ULOQ did not carry the analyte to the corresponding injection of a blank. The blank sample was clearly represented as no carryover. The instrument is sensitive to the analyte that detects LOQ based on signal to noise ratio that was found to be 1540.000.  Ruggedness was observed for the analyte when tested with different column to prove that the method not affected by any small variations. 

\textbf{Big Batch, Dilution Integrity and Hemolysis effect }

All the Big batch QC samples meet with the specifications and the percentage of acceptance showed to be 100\%. The minimum limit requirement to meet specifications should be 67\%. The minimum requirement of four from six dilutions 150 samples must pass. Here, all the DIS-150 and QC samples meet with the specifications and the Non PA batch passed. The minimum requirement of four from six hemolysed samples must pass. All the hemolysed QC samples meet with the specifications, for results refer Table~\ref{tw-2e3646ccc067}. 

\textbf{Solution and Matrix stabilities}

Solution Stability of Master Stock Sulfamethoxazole for a Long Term (LTSS) at 2-8\ensuremath{^{0}}C for 10 days and a Short Term (STSS) for 6.5 hours at RT condition, Neat standard solution stability for 30 days, Analyte Spiking Solution Stability (ASS) at refrigerator condition (Refg) for 11 days and RT condition for 6.5hours and IS Spiking Solution stability for 12 days of test were compared with references (REF). All were observed to meet specified range 85-115\%. The results were represented in Table~\ref{tw-26b25fef18aa} and Table~\ref{tw-574ebc1f6236}. 
    
\section{Conclusion}
By using LCMS, a simple and cost effective extraction technique was developed to provide rugged and suitable analytical method to quantify Sulfamethoxazole in human plasma that adapts with Food and Drug Administration guidelines. The method was optimized with the chromatographic and mass parameters. This method of extraction would be selective and easy when compared to the Solid phase extraction. The chromatographic separation from LC and the analytes ionised and detected would be accurate and efficient. The values from the experiment were concluded that the analyte SM may capable to undergo stressed conditions (from stability). The validation results proved to be reliable when the quantification method may be carried out with different system or different analyst, outcome would be reproducible and specific when the method adapted to subject sample analysis for the bioequivalence study.  
    
\section{\ensuremath{^{}}Acknowledgements}
The authors are thankful to Vels Institute of Science, Technology \& Advanced Sciences (VISTAS) and its management for the research support.

\textbf{Funding Support}

The authors declare that there is no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that there is no conflict of interest for this study.
    

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