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\def\RunningAuthor{Pagidi Rajagopaludu}
\firstPage{7503}
\articleType{Original Article}
\receivedDate{09 Oct 2020}
\acceptedDate{30 Nov 2020}
\revisedDate{27 Nov 2020}
\journalVolume{2020, 11}
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\journalDoi{ijrps.v11i4.3954}
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\title{\textbf{Development and validation of LC-MS/MS method for the simultaneous estimation of tenofovir disoproxil fumarate and pioglitazone HCL in human plasma }}
\author{Pagidi~Rajagopaludu\textsuperscript{*}\textsuperscript{1},  
            Nimmakayala~Saritha\textsuperscript{2},  
            Devanna~N\textsuperscript{3}, 
            Srinivas~M\textsuperscript{4}~\\[5pt]{\textsuperscript{1}Research Scholar, Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur, Andhra Pradesh, India }~\\{\textsuperscript{2}Department of Chemistry, Jawaharlal Nehru Technological University College of Engineering, Kalikiri, Chittoor, Andhra Pradesh, India}~\\{\textsuperscript{3}Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur, Andhra Pradesh, India}~\\{\textsuperscript{4}Acubiosys Pvt. Ltd., Hyderabad-500078, Telangana State, India}}

\begin{abstract}
For the simultaneous detection of tenofovir and pioglitazone in a human plasma LC-MS/MS technique was utilized. API-4000 mass spectrometer was used. It should be operated in positive ion mode and contains ion source of turbo spray. Inverted C18 column was used which produces good resolution and symmetrical peaks. Metoprolol is used as internal standard. It requires acidic PH for elution but Tenofovir disoproxil fumarate and Pioglitazone requires neutral PH after extraction which is equity diluted with H\ensuremath{_{2}}O for supernate. Finally, it provides good resolution and elution. By using Acetonitrile 100mg of Tenofovir Disoproxil fumarate and Pioglitazone HCL were dissolved and the final volume is made with H\ensuremath{_{2}}O and Acetonitrile in 1:1 ratio to give 1mg/ml. standard stock solution are stored at -2 to \ensuremath{\pm} 2\ensuremath{^{0}}C till analysis is completed. The guidelines were provided by the FDA to fulfill the essentials requires for the technique. To regulate the concentration the procedure starts with standard curve which is built form the quantitative analysis standard ranges form 1-1000ng/mL. To determine the stability freeze-thaw technique were used. To determine the stability the analyte is kept at room temperature for 24 hours. The correlation coefficients for all calibration curves were more than 0.99.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Tenofovir Disoproxil Fumarate,\newline Pioglitazone,\newline Electron spray Ionization,\newline LC-MS,\newline Human Plasma
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Pagidi~Rajagopaludu~\\ Phone:\ 9000120129 ~\\  Email:\ gopal.gg54@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Tenofovir disoproxil fumarate is utilized in the treatment of Acquired Immuno Deficiency Syndrome \unskip~\citep{952955:20947124,952955:20947122} and chronic serum hepatitis. From the age of 2 it is used for HIV-1 treatment by combining with ART (antiretroviral). From the age of 12, it is used to treat chronic serum hepatitis \unskip~\citep{952955:20947113}. Tenofovir disoproxil is a nucleotide analogue reverse-transcriptase inhibitor (NtRTI). It is so-called as pro-drug. Concentrations in blood plasma in abstaining people is 25\% after an hour and in fatty food condition, it is 40\% subsequently two hours. Its hunks the efficiency of reverse transcriptase, which prevents from replicating. It results in a decrease in the content of virus in plasma. It is usually obtained in two forms as a tablet and as powder. Its melting point is 279\ensuremath{^\circ}C. Tenofovir disoproxil fumarate is a white to off-white crystal-like powder, i.e., dissolvable in methyl alcohol, a little dissolve in H\ensuremath{_{2}}O and very little dissolvable in methylene chloride. Tenofovir eliminates waste through kidneys. It acts as a substrate analogue in cellular pigments. Tenofovir is detected in body fluids by liquid chromatography. 

Pioglitazone is utilized for non-insulin-dependent diabetes type, which is Glucophage. Subsequent control of pioglitazone reduces insulin protest in the boundary and liver ensuing improvement in insulin subject to dextrose deed and reduces hepatic dextrose outcome. It is utilized together in monotherapy and in grouping with insulin in handling non-insulin-dependent diabetes type. Pioglitazone is also known as 5-[[4-[2-(5-ethyl pyridine-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione;hydrochloride. Its molecular formula is C\ensuremath{_{19}}H\ensuremath{_{21}}ClN\ensuremath{_{2}}O\ensuremath{_{3}}S. 

It is utilized to regulate hyperglycaemia and is used to avoid kidney harm, sightless, nervous difficulties and amputate difficulties. Controlling diabetes reduces myocardial infarction. It is used alone or together with other diabetes mellitus pills. Pioglitazone is utilized with proper diet and physical activities and from time to time with extra medicines which are used to treat diabetes type 2 \unskip~\citep{952955:20947115,952955:20947117}. Pioglitazone is a group of glitazones. It takes action to increase the ability to insulin, an organic element that serves to maintain the levels of sugar. It is not utilized for the treatment of diabetes type 1 and ketoacidosis.  

Structure of Tenofovir Disoproxil Fumarate is shown in Figure~\ref{f-d28e304526bb} and Pioglitazone HCL is shown in Figure~\ref{f-221d5ee586ef}. 

According to literature survey several techniques such as RP-HPLC \unskip~\citep{952955:20947118,952955:20947111}, LC-MS \unskip~\citep{952955:20947120,952955:20947126} were used for the assessment of Tenofovir disoproxil fumarate and to assess Pioglitazone HPLC \unskip~\citep{952955:20947121,952955:20947116}, HPLC-UV \unskip~\citep{952955:20947123,952955:20947112}, RP-HPLC \unskip~\citep{952955:20947125,952955:20947114}, C-MS/MS \unskip~\citep{952955:20947119} were used. Our present work determines the simultaneous estimation of Tenofovir disoproxil fumarate and Pioglitazone HCL by LC-MS using human plasma. Parameters such as linearity, LOQ, accuracy and precision, Recovery and sustainability/stability are validated. 
    
\section{\textbf{EXPERIMENTAL }}
\textbf{Chemicals and reagents}

Working Standard of Tenofovir disoproxil fumarate and Pioglitazone was obtained from by Akshaya labs, Hyderabad, India. Tablets were acquired from the limited marketplace. Acetonitrile of HPLC grade by Merck, Ammonium Acetate AR grade obtained from Qualigens fine chemicals and Water HPLC grade from Milli-Q RO system was used. HPLC grade reagents were utilized. \textbf{\space }

\textbf{LC-MS/MS Instrumentation and Chromatographic Methods}

In positive ion mode, the LC-MS/MS instrument was functioned and equipped with ESI using nebulizer, curtain and collision gas which is embraced with AB Sciex API-4000 mass spectrometer. The HPLC setup contains online DGU-20A3 soluble degasser, segment boiler CTO20A, LC20AD drives-2, SIL-HTc sampler. C18-XBridge segment was split into 60:40 proportion with a stream rate of 1.0mL/min kept at 400C. Changeable phase contains 0.1\% methanoic acid(A) and ethanenitrile (B). In the primary phase up to 0.8min it is arranged as 95\% A/5\%B then it gets changed to 5 \% A/95\% B for 2.2 min. Later it slowly stimulated to 95\% A/5\% B by 2.4 min and remained the same at 3.5 min. 

\textbf{Optimization of MS/MS detection conditions and parameters}

Multi-reaction Monitoring settings were determined by using 500ng/mL of an analyte by distillation. Table~\ref{tw-0c784064d8a1} shows the optimum MS-parameters of Tenofovir Disoproxil fumarate and Pioglitazone HCL in positive ion mode.

\textbf{Preparation of stock solution}

By using Acetonitrile, 100mg of Tenofovir Disoproxil fumarate and Pioglitazone HCL were dissolved and the final volume is made with H\ensuremath{_{2}}O and Acetonitrile in 1:1 ratio to give 1mg/ml. The standard stock solution is stored at -2to \ensuremath{\pm} 20 C till analysis is completed. 

\textbf{Selection of HPLC column}

In HPLC column inverted C-18 columns were regularly used for the objective analyte. Primarily sio\ensuremath{_{2\ }} based C-8 columns are verified and these results in poor separation. Several aspects were taken, such as the composition of the mobile phase and PH stages were examined, yet no enhancement in chromatographic condition. C18 results in fine resolution but presents the uneven peaks. Finally, Phenomenex Luna C18 showed substantially good resolution, symmetrical peaks and less co-elution between target compounds due to its effectiveness in polarity and aromatic selectivity.

\textbf{Internal standard selection}

For positive mode, analysis metoprolol is used. It requires acidic PH for elution but Tenofovir disoproxil fumarate and Pioglitazone requires neutral PH after extraction, which is equity diluted with H\ensuremath{_{2}}O for supernate. Finally, it provides good resolution and elution.
    
\section{Results and Discussion}
\textbf{Validation}

It is a secondary phase. It is a very hard and important phase in quantitation and produces absolute results. The guidelines were provided by the FDA to fulfil the essentials requires for the technique. If the guidelines were not obeyed, then it seems to the improper analysis and not used for assent. To regulate the concentration, the procedure starts with a standard curve, which is built form the quantitative analysis standard ranges form 1-1000ng/mL. 

The standards were divided in the presence of Metoprolol as internal standard from Tenofovir disoproxil fumarate and Pioglitazone by LC-MS/MS using the MRM conversions. To determine the stability analyte is kept at room temperature for 24 hours.


\begin{table*}[!htbp]
\caption{\boldmath {Positive ion mode (for Tenofovir Disoproxil fumarate and Pioglitazone HCL): Waters TQD MS conditions } }
\label{tw-0c784064d8a1}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.4896\linewidth-2\tabcolsep}p{\dimexpr.5104\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Parameter & Value\\
\tblmidrule 
Ionization type and polarity &
  ESI, positive ion mode\\
Ion source &
  Turbo spray\\
Scan type &
  MRM\\
Ion spray voltage &
  5500V\\
Source Temp &
  150\\
Desolvation temp &
  400\\
Desolvation gas &
  750\\
Cone gas  &
  40\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Summaryof MRM transition} }
\label{tw-0e6d7da10ca5}
\def\arraystretch{1.1}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Compound & \cAlignHack Mode of ionization & \cAlignHack Q1 mass (m/z) & \cAlignHack Q3 mass (m/z) & \cAlignHack CE & \cAlignHack Cone Voltage\\
\tblmidrule 
\cAlignHack Tenofovir Disoproxil Fumarate  &
  \cAlignHack Positive &
  \cAlignHack 520.13 &
  \cAlignHack 270.12 &
  \cAlignHack  30 &
  \cAlignHack 38\\
\cAlignHack Pioglitazone HCL &
  \cAlignHack Positive &
  \cAlignHack 357.07 &
  \cAlignHack 134.17 &
  \cAlignHack  24 &
  \cAlignHack 44\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Back-calculated standard curve data for Tenofovir in human plasma} }
\label{tw-a245d2615327}
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\tbltoprule \rowcolor{kwdboxcolor} &  &  & \multicolumn{2}{p{\dimexpr(.24509999999999998\linewidth-2\tabcolsep)}}{\cAlignHack Concentration (ng/mL)} &  &  & \\
\rowcolor{kwdboxcolor}\cAlignHack Std conc. & \cAlignHack Batch-1 & \cAlignHack Batch-2 & \cAlignHack Batch-3 & \cAlignHack Mean & \cAlignHack SD & \cAlignHack \% CV & \cAlignHack \% Accuracy\\
\tblmidrule 
\cAlignHack 5.000 &
  \cAlignHack 0.94 &
  \cAlignHack 1.00 &
  \cAlignHack 1.00 &
  \cAlignHack 0.98 &
  \cAlignHack 0.035 &
  \cAlignHack 3.53 &
  \cAlignHack 98.0\\
\cAlignHack 10.000 &
  \cAlignHack 1.96 &
  \cAlignHack 2.01 &
  \cAlignHack 2.03 &
  \cAlignHack 2.00 &
  \cAlignHack 0.036 &
  \cAlignHack 1.80 &
  \cAlignHack 100.0\\
\cAlignHack 50.000 &
  \cAlignHack 9.78 &
  \cAlignHack 9.76 &
  \cAlignHack 9.77 &
  \cAlignHack 9.77 &
  \cAlignHack 0.010 &
  \cAlignHack 0.10 &
  \cAlignHack 97.7\\
\cAlignHack 250.000 &
  \cAlignHack 51.05 &
  \cAlignHack 50.67 &
  \cAlignHack 50.86 &
  \cAlignHack 50.86 &
  \cAlignHack 0.190 &
  \cAlignHack 0.37 &
  \cAlignHack 101.7\\
\cAlignHack 1000.000 &
  \cAlignHack 217.39 &
  \cAlignHack 215.51 &
  \cAlignHack 215.10 &
  \cAlignHack 216.00 &
  \cAlignHack 1.221 &
  \cAlignHack 0.57 &
  \cAlignHack 108.0\\
\cAlignHack 2500.000 &
  \cAlignHack 491.02 &
  \cAlignHack 486.71 &
  \cAlignHack 485.74 &
  \cAlignHack 487.82 &
  \cAlignHack 2.811 &
  \cAlignHack 0.58 &
  \cAlignHack 97.6\\
\cAlignHack 4000.000 &
  \cAlignHack 862.49 &
  \cAlignHack 854.86 &
  \cAlignHack 853.13 &
  \cAlignHack 856.83 &
  \cAlignHack 4.980 &
  \cAlignHack 0.58 &
  \cAlignHack 107.1\\
\cAlignHack 4500.000 &
  \cAlignHack 851.03 &
  \cAlignHack 888.39 &
  \cAlignHack 841.80 &
  \cAlignHack 860.41 &
  \cAlignHack 24.670 &
  \cAlignHack 2.87 &
  \cAlignHack 95.6\\
\cAlignHack 5000.000 &
  \cAlignHack 959.14 &
  \cAlignHack 950.65 &
  \cAlignHack 948.72 &
  \cAlignHack 952.84 &
  \cAlignHack 5.543 &
  \cAlignHack 0.58 &
  \cAlignHack 95.3\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Back-calculated standard curve data for Pioglitazone in human plasma} }
\label{tw-5d53153fa4eb}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1366\linewidth-2\tabcolsep}p{\dimexpr.11690000000000001\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.121\linewidth-2\tabcolsep}p{\dimexpr.12620000000000001\linewidth-2\tabcolsep}p{\dimexpr.1232\linewidth-2\tabcolsep}p{\dimexpr.123\linewidth-2\tabcolsep}p{\dimexpr.13309999999999999\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack  & \multicolumn{2}{p{\dimexpr(.2472\linewidth-2\tabcolsep)}}{\cAlignHack  Concentration (ng/mL)} & \cAlignHack  & \cAlignHack  & \cAlignHack \\
\rowcolor{kwdboxcolor}\cAlignHack Std conc. & \cAlignHack Batch-1 & \cAlignHack Batch-2 & \cAlignHack Batch-3 & \cAlignHack Mean & \cAlignHack SD & \cAlignHack \% CV & \cAlignHack \% Accuracy\\
\tblmidrule 
\cAlignHack 5.000 &
  \cAlignHack 1.00 &
  \cAlignHack 0.96 &
  \cAlignHack 0.97 &
  \cAlignHack 0.98 &
  \cAlignHack 0.021 &
  \cAlignHack 2.13 &
  \cAlignHack 97.7\\
\cAlignHack 10.000 &
  \cAlignHack 1.90 &
  \cAlignHack 2.06 &
  \cAlignHack 2.05 &
  \cAlignHack 2.00 &
  \cAlignHack 0.090 &
  \cAlignHack 4.47 &
  \cAlignHack 100.2\\
\cAlignHack 50.000 &
  \cAlignHack 11.77 &
  \cAlignHack 11.74 &
  \cAlignHack 11.55 &
  \cAlignHack 11.69 &
  \cAlignHack 0.119 &
  \cAlignHack 1.02 &
  \cAlignHack 116.9\\
\cAlignHack 250.000 &
  \cAlignHack 57.62 &
  \cAlignHack 56.75 &
  \cAlignHack 55.71 &
  \cAlignHack 56.69 &
  \cAlignHack 0.956 &
  \cAlignHack 1.69 &
  \cAlignHack 113.4\\
\cAlignHack 1000.000 &
  \cAlignHack 225.88 &
  \cAlignHack 221.88 &
  \cAlignHack 217.77 &
  \cAlignHack 221.84 &
  \cAlignHack 4.055 &
  \cAlignHack 1.83 &
  \cAlignHack 110.9\\
\cAlignHack 2500.000 &
  \cAlignHack 474.81 &
  \cAlignHack 466.19 &
  \cAlignHack 457.54 &
  \cAlignHack 466.18 &
  \cAlignHack 8.635 &
  \cAlignHack 1.85 &
  \cAlignHack 93.2\\
\cAlignHack 4000.000 &
  \cAlignHack 794.61 &
  \cAlignHack 793.17 &
  \cAlignHack 778.33 &
  \cAlignHack 788.70 &
  \cAlignHack 9.012 &
  \cAlignHack 1.14 &
  \cAlignHack 98.6\\
\cAlignHack 4500.000 &
  \cAlignHack 882.34 &
  \cAlignHack 858.19 &
  \cAlignHack 876.21 &
  \cAlignHack 872.25 &
  \cAlignHack 12.553 &
  \cAlignHack 1.44 &
  \cAlignHack 96.9\\
\cAlignHack 5000.000 &
  \cAlignHack 936.18 &
  \cAlignHack 970.12 &
  \cAlignHack 960.75 &
  \cAlignHack 955.68 &
  \cAlignHack 17.528 &
  \cAlignHack 1.83 &
  \cAlignHack 95.6\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Calculated concentrations obtained for precision and accuracy batches of Tenofovir Disoproxil Fumarate and pioglitazone HCL} }
\label{tw-f52a3ebe4f74}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0942\linewidth-2\tabcolsep}p{\dimexpr.0975\linewidth-2\tabcolsep}p{\dimexpr.1083\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}p{\dimexpr.10\linewidth-2\tabcolsep}p{\dimexpr.1017\linewidth-2\tabcolsep}p{\dimexpr.1049\linewidth-2\tabcolsep}p{\dimexpr.0934\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack  & \cAlignHack  & \multicolumn{4}{p{\dimexpr(.4017\linewidth-2\tabcolsep)}}{\cAlignHack Tenofovir Disoproxil Fumarate } & \cAlignHack  & \cAlignHack \\
\rowcolor{kwdboxcolor}\cAlignHack  & \multicolumn{3}{p{\dimexpr(.3058\linewidth-2\tabcolsep)}}{\cAlignHack Batch-1} & \multicolumn{3}{p{\dimexpr(.30\linewidth-2\tabcolsep)}}{\cAlignHack Batch-2} & \multicolumn{3}{p{\dimexpr(.30\linewidth-2\tabcolsep)}}{\cAlignHack Batch-3}\\
\rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack LQC & \cAlignHack MQC & \cAlignHack HQC & \cAlignHack LQC & \cAlignHack MQC & \cAlignHack HQC & \cAlignHack LQC & \cAlignHack MQC & \cAlignHack HQC\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 3.70 &
  \cAlignHack 441.93 &
  \cAlignHack 818.23 &
  \cAlignHack 4.39 &
  \cAlignHack 515.75 &
  \cAlignHack 908.50 &
  \cAlignHack 4.42 &
  \cAlignHack 511.24 &
  \cAlignHack 1080.56\\
\cAlignHack 2 &
  \cAlignHack 3.78 &
  \cAlignHack 437.98 &
  \cAlignHack 849.24 &
  \cAlignHack 4.31 &
  \cAlignHack 506.92 &
  \cAlignHack 981.44 &
  \cAlignHack 4.34 &
  \cAlignHack 502.47 &
  \cAlignHack 972.77\\
\cAlignHack 3 &
  \cAlignHack 4.25 &
  \cAlignHack 479.12 &
  \cAlignHack 852.34 &
  \cAlignHack 4.78 &
  \cAlignHack 570.50 &
  \cAlignHack 930.38 &
  \cAlignHack 4.31 &
  \cAlignHack 565.23 &
  \cAlignHack 999.44\\
\cAlignHack 4 &
  \cAlignHack 4.14 &
  \cAlignHack 475.45 &
  \cAlignHack 770.59 &
  \cAlignHack 4.41 &
  \cAlignHack 535.90 &
  \cAlignHack 1008.37 &
  \cAlignHack 4.81 &
  \cAlignHack 570.43 &
  \cAlignHack 941.42\\
\cAlignHack 5 &
  \cAlignHack 4.52 &
  \cAlignHack 414.43 &
  \cAlignHack 832.56 &
  \cAlignHack 4.45 &
  \cAlignHack 559.64 &
  \cAlignHack 949.82 &
  \cAlignHack 4.44 &
  \cAlignHack 531.19 &
  \cAlignHack 969.64\\
\cAlignHack 6 &
  \cAlignHack 3.97 &
  \cAlignHack 448.59 &
  \cAlignHack 860.21 &
  \cAlignHack 4.59 &
  \cAlignHack 575.50 &
  \cAlignHack 965.34 &
  \cAlignHack 4.48 &
  \cAlignHack 554.71 &
  \cAlignHack 978.56\\
\cAlignHack Mean &
  \cAlignHack 4.06 &
  \cAlignHack 449.58 &
  \cAlignHack 830.53 &
  \cAlignHack 4.49 &
  \cAlignHack 544.04 &
  \cAlignHack 957.31 &
  \cAlignHack 4.47 &
  \cAlignHack 539.21 &
  \cAlignHack 990.40\\
\cAlignHack SD &
  \cAlignHack 0.31 &
  \cAlignHack 24.38 &
  \cAlignHack 33.03 &
  \cAlignHack 0.17 &
  \cAlignHack 28.91 &
  \cAlignHack 35.83 &
  \cAlignHack 0.18 &
  \cAlignHack 28.60 &
  \cAlignHack 47.94\\
\cAlignHack \%CV &
  \cAlignHack 7.55 &
  \cAlignHack 5.42 &
  \cAlignHack 3.98 &
  \cAlignHack 3.79 &
  \cAlignHack 5.31 &
  \cAlignHack 3.74 &
  \cAlignHack 4.02 &
  \cAlignHack 5.30 &
  \cAlignHack 4.84\\
\multicolumn{10}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack \textbf{Pioglitazone HCL}}\\
\cAlignHack 1 &
  \cAlignHack 4.45 &
  \cAlignHack 510.19 &
  \cAlignHack 1078.37 &
  \cAlignHack 3.82 &
  \cAlignHack 433.92 &
  \cAlignHack 860.84 &
  \cAlignHack 3.77 &
  \cAlignHack 425.86 &
  \cAlignHack 844.83\\
\cAlignHack 2 &
  \cAlignHack 4.37 &
  \cAlignHack 501.46 &
  \cAlignHack 970.8 &
  \cAlignHack 3.91 &
  \cAlignHack 431.87 &
  \cAlignHack 834.23 &
  \cAlignHack 3.86 &
  \cAlignHack 423.85 &
  \cAlignHack 788.35\\
\cAlignHack 3 &
  \cAlignHack 4.83 &
  \cAlignHack 564.09 &
  \cAlignHack 997.41 &
  \cAlignHack 4.37 &
  \cAlignHack 470.42 &
  \cAlignHack 919.14 &
  \cAlignHack 4.31 &
  \cAlignHack 461.68 &
  \cAlignHack 818.72\\
\cAlignHack 4 &
  \cAlignHack 4.47 &
  \cAlignHack 569.29 &
  \cAlignHack 939.51 &
  \cAlignHack 4.26 &
  \cAlignHack 466.82 &
  \cAlignHack 873.68 &
  \cAlignHack 4.2 &
  \cAlignHack 458.15 &
  \cAlignHack 881.22\\
\cAlignHack 5 &
  \cAlignHack 4.5 &
  \cAlignHack 530.13 &
  \cAlignHack 951.18 &
  \cAlignHack 4.63 &
  \cAlignHack 406.93 &
  \cAlignHack 932.67 &
  \cAlignHack 4.57 &
  \cAlignHack 399.38 &
  \cAlignHack 863.85\\
\cAlignHack 6 &
  \cAlignHack 4.66 &
  \cAlignHack 553.6 &
  \cAlignHack 1020.21 &
  \cAlignHack 4 &
  \cAlignHack 440.46 &
  \cAlignHack 908.86 &
  \cAlignHack 3.95 &
  \cAlignHack 432.28 &
  \cAlignHack 858.21\\
\cAlignHack Mean  &
  \cAlignHack 4.55 &
  \cAlignHack 538.13 &
  \cAlignHack 992.91 &
  \cAlignHack 4.17 &
  \cAlignHack 441.74 &
  \cAlignHack 888.24 &
  \cAlignHack 4.11 &
  \cAlignHack 433.53 &
  \cAlignHack 842.53\\
\cAlignHack SD  &
  \cAlignHack 0.17 &
  \cAlignHack 28.54 &
  \cAlignHack 51.29 &
  \cAlignHack 0.31 &
  \cAlignHack 23.76 &
  \cAlignHack 38.04 &
  \cAlignHack 0.30 &
  \cAlignHack 23.32 &
  \cAlignHack 33.78\\
\cAlignHack  \% CV   &
  \cAlignHack 3.70 &
  \cAlignHack 5.30 &
  \cAlignHack 5.17 &
  \cAlignHack 7.43 &
  \cAlignHack 5.38 &
  \cAlignHack 4.28 &
  \cAlignHack 7.41 &
  \cAlignHack 5.38 &
  \cAlignHack 4.01\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Intra- and inter-run precision and accuracy of Tenofovir Disoproxil Fumarate and Pioglitazone HCL inhuman plasma} }
\label{tw-428d3ebb701f}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1117\linewidth-2\tabcolsep}p{\dimexpr.2031\linewidth-2\tabcolsep}p{\dimexpr.11569999999999997\linewidth-2\tabcolsep}p{\dimexpr.11379999999999999\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.1096\linewidth-2\tabcolsep}p{\dimexpr.11790000000000003\linewidth-2\tabcolsep}p{\dimexpr.1082\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \multicolumn{3}{p{\dimexpr(.34949999999999996\linewidth-2\tabcolsep)}}{\cAlignHack Tenofovir} & \multicolumn{3}{p{\dimexpr(.3357000000000001\linewidth-2\tabcolsep)}}{\cAlignHack Pioglitazone HCL}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack LQC & \cAlignHack MOC & \cAlignHack HQC & \cAlignHack LQC  & \cAlignHack MQC & \cAlignHack HQC\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack (4.2) & \cAlignHack (500) & \cAlignHack (900) & \cAlignHack (4.2) & \cAlignHack (500) & \cAlignHack (900)\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.1117\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{\cAlignHack BATCH-1 \mbox{}\protect\newline (N=6)}} &
  Intra-run mean &
  \cAlignHack 4.06 &
  \cAlignHack 449.58 &
  \cAlignHack 830.53 &
  \cAlignHack 4.55 &
  \cAlignHack 538.13 &
  \cAlignHack 992.91\\
 &
  Intra-run SD &
  \cAlignHack 0.31 &
  \cAlignHack 24.38 &
  \cAlignHack 33.03 &
  \cAlignHack 0.17 &
  \cAlignHack 28.54 &
  \cAlignHack 51.29\\
 &
  Intra-run \% CV &
  \cAlignHack 7.55 &
  \cAlignHack 5.42 &
  \cAlignHack 3.98 &
  \cAlignHack 3.70 &
  \cAlignHack 5.30 &
  \cAlignHack 5.17\\
 &
  Intra-run \% Accuracy &
  \cAlignHack 97 &
  \cAlignHack 90 &
  \cAlignHack 92 &
  \cAlignHack 108 &
  \cAlignHack 107 &
  \cAlignHack 110\\
\multicolumn{1}{p{\dimexpr(.1117\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{\cAlignHack BATCH-2 \mbox{}\protect\newline (N=6)}} &
  Intra-run mean &
  \cAlignHack 4.49 &
  \cAlignHack 544.04 &
  \cAlignHack 957.31 &
  \cAlignHack 4.17 &
  \cAlignHack 441.74 &
  \cAlignHack 888.24\\
 &
  Intra-run SD &
  \cAlignHack 0.17 &
  \cAlignHack 28.91 &
  \cAlignHack 35.83 &
  \cAlignHack 0.31 &
  \cAlignHack 23.76 &
  \cAlignHack 38.04\\
 &
  Intra-run \% CV &
  \cAlignHack 3.79 &
  \cAlignHack 5.31 &
  \cAlignHack 3.74 &
  \cAlignHack 7.43 &
  \cAlignHack 5.38 &
  \cAlignHack 4.28\\
 &
  Intra-run \% Accuracy &
  \cAlignHack 107 &
  \cAlignHack 109 &
  \cAlignHack 106 &
  \cAlignHack 99 &
  \cAlignHack 88 &
  \cAlignHack 89\\
\multicolumn{1}{p{\dimexpr(.1117\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{\cAlignHack BATCH-3 \mbox{}\protect\newline (N=6)}} &
  Intra-run mean &
  \cAlignHack 4.47 &
  \cAlignHack 539.21 &
  \cAlignHack 990.40 &
  \cAlignHack 4.11 &
  \cAlignHack 433.53 &
  \cAlignHack 842.53\\
 &
  Intra-run SD &
  \cAlignHack 0.18 &
  \cAlignHack 28.60 &
  \cAlignHack 47.94 &
  \cAlignHack 0.30 &
  \cAlignHack 23.32 &
  \cAlignHack 33.78\\
 &
  Intra-run \% CV &
  \cAlignHack 4.02 &
  \cAlignHack 5.30 &
  \cAlignHack 4.84 &
  \cAlignHack 7.41 &
  \cAlignHack 5.38 &
  \cAlignHack 4.01\\
 &
  Intra-run \% Accuracy &
  \cAlignHack 106 &
  \cAlignHack 108 &
  \cAlignHack 110 &
  \cAlignHack 98 &
  \cAlignHack 87 &
  \cAlignHack 94\\
\multicolumn{1}{p{\dimexpr(.1117\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{\cAlignHack INTER-BATCH \mbox{}\protect\newline (N=18)}} &
  Inter-run mean &
  \cAlignHack 4.30 &
  \cAlignHack 487.27 &
  \cAlignHack 878.80 &
  \cAlignHack 3.97 &
  \cAlignHack 458.91 &
  \cAlignHack 885.63\\
 &
  Inter-run SD &
  \cAlignHack 0.12 &
  \cAlignHack 22.6 &
  \cAlignHack 53.45 &
  \cAlignHack 0.26 &
  \cAlignHack 28.93 &
  \cAlignHack 42.67\\
 &
  Inter-run \% CV &
  \cAlignHack 2.79 &
  \cAlignHack 4.63 &
  \cAlignHack 6.0 &
  \cAlignHack 6.5 &
  \cAlignHack 6.3 &
  \cAlignHack 4.81\\
 &
  Inter-run \% Accuracy &
  \cAlignHack 102 &
  \cAlignHack 97 &
  \cAlignHack 98 &
  \cAlignHack 95 &
  \cAlignHack 92 &
  \cAlignHack 98\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Tenofovir Recovery Data} }
\label{tw-676927fddfd3}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1957\linewidth-2\tabcolsep}p{\dimexpr.15429999999999998\linewidth-2\tabcolsep}p{\dimexpr.13290000000000001\linewidth-2\tabcolsep}p{\dimexpr.12099999999999998\linewidth-2\tabcolsep}p{\dimexpr.1305\linewidth-2\tabcolsep}p{\dimexpr.1139\linewidth-2\tabcolsep}p{\dimexpr.1517\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No. & \multicolumn{2}{p{\dimexpr(.2872\linewidth-2\tabcolsep)}}{\cAlignHack LQC Area (Counts)} & \multicolumn{2}{p{\dimexpr(.2515\linewidth-2\tabcolsep)}}{\cAlignHack MQC Area (Counts)} & \multicolumn{2}{p{\dimexpr(.26560000000000002\linewidth-2\tabcolsep)}}{\cAlignHack HQC Area (Counts)}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack Aqueous & \cAlignHack Extracted & \cAlignHack Aqueous & Extracted & \cAlignHack Aqueous & \cAlignHack Extracted\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 2714 &
  \cAlignHack 2687 &
  \cAlignHack 47892 &
  \cAlignHack 38421 &
  \cAlignHack 98123 &
  \cAlignHack 91588\\
\cAlignHack 2 &
  \cAlignHack 2766 &
  \cAlignHack 2680 &
  \cAlignHack 48429 &
  \cAlignHack 43568 &
  \cAlignHack 96982 &
  \cAlignHack 86088\\
\cAlignHack 3 &
  \cAlignHack 2727 &
  \cAlignHack 2549 &
  \cAlignHack 46231 &
  \cAlignHack 41098 &
  \cAlignHack 94543 &
  \cAlignHack 92702\\
\cAlignHack 4 &
  \cAlignHack 2754 &
  \cAlignHack 2628 &
  \cAlignHack 49920 &
  \cAlignHack 46532 &
  \cAlignHack 93421 &
  \cAlignHack 91676\\
\cAlignHack 5 &
  \cAlignHack 2806 &
  \cAlignHack 2782 &
  \cAlignHack 47793 &
  \cAlignHack 45874 &
  \cAlignHack 92135 &
  \cAlignHack 86165\\
\cAlignHack 6 &
  \cAlignHack 2700 &
  \cAlignHack 2547 &
  \cAlignHack 45774 &
  \cAlignHack 43217 &
  \cAlignHack 90762 &
  \cAlignHack 88362\\
\cAlignHack Mean Area &
  \cAlignHack 2744.5 &
  \cAlignHack 2645.5 &
  \cAlignHack 47673.2 &
  \cAlignHack 43118.3 &
  \cAlignHack 94327.6 &
  \cAlignHack 89430.2\\
\cAlignHack \% Recovery &
  \multicolumn{2}{p{\dimexpr(.2872\linewidth-2\tabcolsep)}}{\cAlignHack 96.4} &
  \multicolumn{2}{p{\dimexpr(.2515\linewidth-2\tabcolsep)}}{\cAlignHack 90.4} &
  \multicolumn{2}{p{\dimexpr(.26560000000000002\linewidth-2\tabcolsep)}}{\cAlignHack 94.8}\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Avg. \% Recovery= 93.9; SD= 2.5; \% CV= 2.7}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Pioglitazone Recovery Data} }
\label{tw-dee7deb3387f}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.17205714285714286\linewidth-2\tabcolsep}p{\dimexpr.15425714285714285\linewidth-2\tabcolsep}p{\dimexpr.14495714285714287\linewidth-2\tabcolsep}p{\dimexpr.13215714285714284\linewidth-2\tabcolsep}p{\dimexpr.12985714285714284\linewidth-2\tabcolsep}p{\dimexpr.12505714285714285\linewidth-2\tabcolsep}p{\dimexpr.14165714285714285\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \multicolumn{2}{p{\dimexpr(.2992142857142857\linewidth-2\tabcolsep)}}{\cAlignHack LQC Area (Counts)} & \multicolumn{2}{p{\dimexpr(.2620142857142857\linewidth-2\tabcolsep)}}{\cAlignHack MQC Area (Counts)} & \multicolumn{2}{p{\dimexpr(.2667142857142857\linewidth-2\tabcolsep)}}{\cAlignHack HQC Area (Counts)}\\
\rowcolor{kwdboxcolor} & \cAlignHack Aqueous & \cAlignHack Extracted & \cAlignHack Aqueous & \cAlignHack Extracted & \cAlignHack Aqueous & \cAlignHack Extracted\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 35298 &
  \cAlignHack 37018 &
  \cAlignHack 857071 &
  \cAlignHack 845961 &
  \cAlignHack 1428851 &
  \cAlignHack 1518928\\
\cAlignHack 2 &
  \cAlignHack 35440 &
  \cAlignHack 36785 &
  \cAlignHack 853245 &
  \cAlignHack 847928 &
  \cAlignHack 1456969 &
  \cAlignHack 1463473\\
\cAlignHack 3 &
  \cAlignHack 34354 &
  \cAlignHack 36845 &
  \cAlignHack 861064 &
  \cAlignHack 888406 &
  \cAlignHack 1476750 &
  \cAlignHack 1509093\\
\cAlignHack 4 &
  \cAlignHack 36789 &
  \cAlignHack 36345 &
  \cAlignHack 867852 &
  \cAlignHack 864807 &
  \cAlignHack 1483957 &
  \cAlignHack 1469930\\
\cAlignHack 5 &
  \cAlignHack 37296 &
  \cAlignHack 37531 &
  \cAlignHack 875386 &
  \cAlignHack 884702 &
  \cAlignHack 1466265 &
  \cAlignHack 1361762\\
\cAlignHack 6 &
  \cAlignHack 37807 &
  \cAlignHack 37512 &
  \cAlignHack 862120 &
  \cAlignHack 881363 &
  \cAlignHack 1452747 &
  \cAlignHack 1445909\\
\cAlignHack Mean Area &
  \cAlignHack 27330.67 &
  \cAlignHack 27006.00 &
  \cAlignHack 862789.7 &
  \cAlignHack 868861.2 &
  \cAlignHack 1460923 &
  \cAlignHack 1461516\\
\cAlignHack \% Recovery &
  \multicolumn{2}{p{\dimexpr(.2992142857142857\linewidth-2\tabcolsep)}}{\cAlignHack 98.81} &
  \multicolumn{2}{p{\dimexpr(.2620142857142857\linewidth-2\tabcolsep)}}{\cAlignHack 100.70} &
  \multicolumn{2}{p{\dimexpr(.2667142857142857\linewidth-2\tabcolsep)}}{\cAlignHack 100.04}\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{Avg. \% Recovery=99.85; SD= 0.96; \% CV= 0.96}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Stability data showing the \% CV of analytes} }
\label{tw-e00763a6fa52}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1883\linewidth-2\tabcolsep}p{\dimexpr.18850000000000005\linewidth-2\tabcolsep}p{\dimexpr.20159999999999997\linewidth-2\tabcolsep}p{\dimexpr.2066\linewidth-2\tabcolsep}p{\dimexpr.215\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Analyte name & \cAlignHack Fresh samples & \cAlignHack Freeze-thaw stability  & \cAlignHack Auto-sampler stability  & \cAlignHack Bench-top stability \\
\tblmidrule 
\cAlignHack Tenofovir &
  \cAlignHack  &
  \cAlignHack  &
  \cAlignHack  &
  \cAlignHack \\
\cAlignHack LQC &
  \cAlignHack 9.12 &
  \cAlignHack 12.09 &
  \cAlignHack 8.98 &
  \cAlignHack 4.19\\
\cAlignHack MQC &
  \cAlignHack 5.06 &
  \cAlignHack 9.12 &
  \cAlignHack 5.42 &
  \cAlignHack 9.13\\
\cAlignHack  HQC &
  \cAlignHack 8.63 &
  \cAlignHack 6.34 &
  \cAlignHack 3.09 &
  \cAlignHack 8.01\\
\cAlignHack Pioglitazone &
  \cAlignHack  &
  \cAlignHack  &
  \cAlignHack  &
  \cAlignHack \\
\cAlignHack  LQC &
  \cAlignHack 3.21 &
  \cAlignHack 9.53 &
  \cAlignHack 9.23 &
  \cAlignHack 9.87\\
\cAlignHack  MQC &
  \cAlignHack 7.55 &
  \cAlignHack 6.07 &
  \cAlignHack 7.54 &
  \cAlignHack 6.31\\
\cAlignHack  HQC &
  \cAlignHack 6.21 &
  \cAlignHack 6.89 &
  \cAlignHack 6.92 &
  \cAlignHack 5.03\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/039000ba-c067-47f9-8c48-34f3c9f967c7-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/039000ba-c067-47f9-8c48-34f3c9f967c7-upicture1.png}{\includegraphics{images/039000ba-c067-47f9-8c48-34f3c9f967c7-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Tenofovir Disoproxil Fumarate}}
\label{f-d28e304526bb}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/cb1fd451-4205-4f9e-b660-698af0799aba-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/cb1fd451-4205-4f9e-b660-698af0799aba-upicture2.png}{\includegraphics{images/cb1fd451-4205-4f9e-b660-698af0799aba-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Pioglitazone HCL}}
\label{f-221d5ee586ef}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/b31977c5-8c45-4a96-9ba6-746c0495211f-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/b31977c5-8c45-4a96-9ba6-746c0495211f-upicture3.png}{\includegraphics{images/b31977c5-8c45-4a96-9ba6-746c0495211f-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Calibration curve of Tenofovir }}
\label{f-1effbdc803a7}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/609edfaf-9b2c-4448-a41d-f0ca4db1c7ff-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/609edfaf-9b2c-4448-a41d-f0ca4db1c7ff-upicture4.png}{\includegraphics{images/609edfaf-9b2c-4448-a41d-f0ca4db1c7ff-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Calibration curve of Pioglitazone }}
\label{f-258d766a768c}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/fb914ae4-a9dd-4c94-8019-4da95ead5c98-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/fb914ae4-a9dd-4c94-8019-4da95ead5c98-upicture5.png}{\includegraphics{images/fb914ae4-a9dd-4c94-8019-4da95ead5c98-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Product ion mass spectra of Tenofovir Disoproxil Fumarate in positive ionization mode }}
\label{f-a9dc5df594ad}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/cea0bbe2-d62b-42ae-9297-3504432ccf12-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/cea0bbe2-d62b-42ae-9297-3504432ccf12-upicture6.png}{\includegraphics{images/cea0bbe2-d62b-42ae-9297-3504432ccf12-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Product ion mass spectra of Pioglitazone HCL in positive ionization mode }}
\label{f-be95af910ed7}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/1830e712-417a-4c42-9a2f-16999f1aee5e-upicture7.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/1830e712-417a-4c42-9a2f-16999f1aee5e-upicture7.png}{\includegraphics{images/1830e712-417a-4c42-9a2f-16999f1aee5e-upicture7.png}}{}
\makeatother 
\caption{\boldmath {Representative example of the blank chromatogram for Tenofovir}}
\label{f-2f3dc937ffdd}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/b05b9dad-31d8-429f-b9bd-2e21ea41e93d-upicture8.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/b05b9dad-31d8-429f-b9bd-2e21ea41e93d-upicture8.png}{\includegraphics{images/b05b9dad-31d8-429f-b9bd-2e21ea41e93d-upicture8.png}}{}
\makeatother 
\caption{\boldmath {Representative example of LLOQ chromatogram of Tenofovir}}
\label{f-9cd945339db9}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/ffda6afd-7234-4856-ae28-b09740d21a89-upicture9.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/ffda6afd-7234-4856-ae28-b09740d21a89-upicture9.png}{\includegraphics{images/ffda6afd-7234-4856-ae28-b09740d21a89-upicture9.png}}{}
\makeatother 
\caption{\boldmath {Representative example of LQC chromatogram of Tenofovir}}
\label{f-815160ff1c6f}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/e5a857c0-8431-4ec9-8a03-4e40d50bc248-upicture10.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/e5a857c0-8431-4ec9-8a03-4e40d50bc248-upicture10.png}{\includegraphics{images/e5a857c0-8431-4ec9-8a03-4e40d50bc248-upicture10.png}}{}
\makeatother 
\caption{\boldmath {Representative example of MQC chromatogram of Tenofovir}}
\label{f-ade3977bde53}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/1411b351-ca62-46f8-8a58-7b84e3ff9dfc-upicture11.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/1411b351-ca62-46f8-8a58-7b84e3ff9dfc-upicture11.png}{\includegraphics{images/1411b351-ca62-46f8-8a58-7b84e3ff9dfc-upicture11.png}}{}
\makeatother 
\caption{\boldmath {Representative example of HQC chromatogram of Tenofovir}}
\label{f-36d028822e83}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/458a283c-b7b5-4635-8494-35f67fd7a2ec-upicture12.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/458a283c-b7b5-4635-8494-35f67fd7a2ec-upicture12.png}{\includegraphics{images/458a283c-b7b5-4635-8494-35f67fd7a2ec-upicture12.png}}{}
\makeatother 
\caption{\boldmath {Representative example of the blank chromatogram of Pioglitazone}}
\label{f-2cecff447447}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/ab1187ee-2f16-4ced-86fc-51595c345ad9-upicture13.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/ab1187ee-2f16-4ced-86fc-51595c345ad9-upicture13.png}{\includegraphics{images/ab1187ee-2f16-4ced-86fc-51595c345ad9-upicture13.png}}{}
\makeatother 
\caption{\boldmath {Representative example of LLOQ chromatogram of pioglitazone}}
\label{f-5255adcfa38a}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/cd31baf7-a62f-4b81-b625-54ac5a30f09f-upicture14.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/cd31baf7-a62f-4b81-b625-54ac5a30f09f-upicture14.png}{\includegraphics{images/cd31baf7-a62f-4b81-b625-54ac5a30f09f-upicture14.png}}{}
\makeatother 
\caption{\boldmath {Representative example of LQC chromatogram for Pioglitazone}}
\label{f-4c7ddaa28a6e}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/775354e1-a43b-4f1a-b0c7-10c5fbbdb4ff-upicture15.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/775354e1-a43b-4f1a-b0c7-10c5fbbdb4ff-upicture15.png}{\includegraphics{images/775354e1-a43b-4f1a-b0c7-10c5fbbdb4ff-upicture15.png}}{}
\makeatother 
\caption{\boldmath {Representative example of MQC chromatogram for Pioglitazone}}
\label{f-e28366d2cdbd}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/cd87c54a-77a3-4d21-9818-96439c9edc5e-upicture16.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/cd87c54a-77a3-4d21-9818-96439c9edc5e-upicture16.png}{\includegraphics{images/cd87c54a-77a3-4d21-9818-96439c9edc5e-upicture16.png}}{}
\makeatother 
\caption{\boldmath {Representative example of HQC chromatogram for Pioglitazone}}
\label{f-c5ec51478ef2}
\end{figure}
\egroup
\textbf{Construction of calibration curve}

To obtain the linear response, five samples were taken on an alternate day by using similar plasma and stock solution. Each one of these contains nine concentration levels which differed from one another. These are named as inter and intra-day data results in stability and reproducible on alternate days. The peak area is calculated from extracted ion to the internal standard. The main purpose of an experiment is to assess the least level. Form the nominal concentration of the analyte calibration curve was constructed. Calibration curve of Tenofovir Disoproxil Fumarate is shown in Figure~\ref{f-1effbdc803a7} and pioglitazone HCL is shown in Figure~\ref{f-258d766a768c}.

\textbf{MRM transition optimization}

To provide the spectra, MRM tracks the precursor ion and their multiple product ions that are quantifiable. They form different collision ions with each transition from parent ion to daughter ion. Each transition of collision energy is optimized by MRM transitions. Precursor ion is known as parent ion. Selective transmission of precursor ions (MS1) is passed through Q1 by m/z separation and are fragmented with collision energy to form product ions (MS2). 

Product ions are known daughter ions. The m/z values of Tenofovir Disoproxil Fumarate are 520.13 \ensuremath{\rightarrow}270.12 and Pioglitazone HCL is 357.07 \ensuremath{\rightarrow}134.17. Table~\ref{tw-0e6d7da10ca5} shows the MRM transitions. Figure~\ref{f-a9dc5df594ad} \& Figure~\ref{f-be95af910ed7} shows the product ion mass spectra of Tenofovir disoproxil fumarate and pioglitazone. 

\textbf{Linearity}

It is defined as the relationship between analyte signals and analyte concentration in calibration samples. By using 1$\times $ linear regression calibration curves are generated by using analyst 1.5 software. The linearity ranges differ from one ion source to others. In ESI source, the linearity is lost in high concentrations and at lower concentrations, linearity is maintained. Table~\ref{tw-a245d2615327} and Table~\ref{tw-5d53153fa4eb} show the back-calculated standards. 

\textbf{Accuracy and precision}

Inter and intra batch precision and accuracy values are shown below. Precision is expressed as a standard deviation. In a multistep preparation procedure, the results obtained show the acceptable accuracies. Accuracy refers to the closeness of true value and for measurement of uncertainty. The coefficients of variation are less than 15\% for each analyte. The precision and accuracy analyzed by LQC, MQC and HQC are shown in Table~\ref{tw-f52a3ebe4f74}. Table~\ref{tw-428d3ebb701f} shows the intra and inter run precision and accuracy. Figure~\ref{f-2f3dc937ffdd}, Figure~\ref{f-9cd945339db9}, Figure~\ref{f-815160ff1c6f}, Figure~\ref{f-ade3977bde53} \& Figure~\ref{f-36d028822e83} shows the chromatogram of Tenofovir Disoproxil Fumarate and Figure~\ref{f-2cecff447447}, Figure~\ref{f-5255adcfa38a}, Figure~\ref{f-4c7ddaa28a6e}, Figure~\ref{f-e28366d2cdbd} \& Figure~\ref{f-c5ec51478ef2} shows the chromatogram of Pioglitazone HCL.

\textbf{Recovery}

The recovery is related to the separation approach inside variability stages\textbf{. }By comparing with the detector response, the recovery of an analyte is defined as the added amount of analyte to the extracted from the matrix. The average recovery rate of Tenofovir and pioglitazone is 93.9\% and 99.85\%. LQC, MQC and HQC values were also calculated as shown in Table~\ref{tw-676927fddfd3} \& Table~\ref{tw-dee7deb3387f}.

\textbf{Stability}

Freezing and thawing quality control samples are validated by three cycles and the obtained values are shown in Table~\ref{tw-e00763a6fa52}, which are within the range of inter-day precision and accuracy samples result in no instability. In autosampler, the quality control sample is kept for 24 hours at 4\ensuremath{^{0}}C and the values obtained are within the range of inter-day QC samples leads to the deduction that no instability is found during the runtime. In Bench-top stability quality control sample are kept for 4 hours at room temperature and the values obtained are within the range of inter-day QC samples leads to the deduction that no instability is found during the runtime.
    
\section{Conclusion }
This method describes a simple, rapid, sensitive, specific, LC-ESI-MS/MS assay of Tenofovir and Pioglitazone for simultaneous detection and accurate measurement in human plasma using Metoprolol as an internal standard and to extract the analytes the precipitation technique was used. Linearity is fixed at 5 to 5000ng/mL. The m/z values of tenofovir were 520.13\ensuremath{\rightarrow}270.12 and for pioglitazone 357.07\ensuremath{\rightarrow}134.17. They calculated the Intra-day and inter-day precision and accuracy values at n=6. The average recovery rate of tenofovir and pioglitazone is 93.9\%and 99.85\%. No instability was found during the runtime. 
\section*{Acknowledgement}The author is thankful to Dr N.Saritha for providing gift sample of drugs.

\textbf{Funding Support}

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

\textbf{Conflict of Interest}

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

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