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\begin{document}


\def\RunningAuthor{Veeraswami B}
\firstPage{2272}
\articleType{Original Article}
\receivedDate{23 Aug 2020}
\acceptedDate{27 Oct 2020}
\revisedDate{23 Oct 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4454}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{High Response Bio-Analytical Validation approach of Nadolol and Bendroflumethiazide by LC-MS/MS on Rat plasma}
\author{Naveen~V~M~K,  
            Veeraswami~B\textsuperscript{*}, 
            Srinivasa~Rao~G~\\[5pt]{Department of Chemistry, GITAM Institute of Science, GITAM Deemed to be University, Visakhapatnam-45, Andhra Pradesh, India}}

\begin{abstract}
A very strong responsive and straight forward the LC-MS / MS assay was developed to and witnessed for that gradation in Nadolol and Bendroflumethiazide in rat plasma. The chromatographic conditions involve isocratic mode using Waters symmetry C\ensuremath{_{18}} (150x4.6 mm, 3.5 microns) column. A 0.1 per cent Smartphone process OPA (orthophosphoric Acid) Acetonitrile and in 60:40 is employed, and therefore the detection was administered during +ve mode of electrospray ionisation by using MS. The valid method had validated in the linear range of 8-160 ng/ml Nadolol and 1-20 ng/ml bendroflumethiazide, the precise values considered to be intraday and interday within the acceptable limit. Here these drugs are extracted from the rat plasma by using the liquid-liquid extraction. And these drugs are found stable. Through freeze Thaw, sampler app, vehicle sampler and top of the bench for the future studies. Form of liquid chromatography-tandem mass spectrometry and checked in compliance with guidelines for quantification of food and drug administration Nadolol and Bendroflumethiazide plasma in rats using D\ensuremath{_{6}}-Nadolol and D\ensuremath{_{6}}-Bendroflumethiazide as internal standards utilising LC-MS incorporated with quadrupole spectrometer by using electrospray ionisation technique. The target of this analysis is to be done work out the appropriateness of this approach to Nadolol and Bendroflumethiazide Applying Nadolol and bendroflumethiazide and their internal requirements at various quantification stages and retaining different parameters such as instrument durability, precision and accuracy, sample preparation techniques, instrument synchronisation, recovery and matrix effect. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Bendroflumethiazide,\newline Bio-analytical,\newline LC-MS/MS,\newline Nadolol,\newline Rat plasma,\newline Validation
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Veeraswami~B~\\ Phone:\ +91-9493939469~\\  Email:\ veeraswamiboddu@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Nadolol was marketed, among others, under the brand Corgard, its structure is shown in Figure~\ref{f-62c40b6b8268}(A). May be medicine won't for the prevention of elevated vital sign \unskip~\citep{915901:20612416,915901:20612407}, heart pain and atrial fibrillation \unskip~\citep{915901:20612419,915901:20612404}. It's also been used to prevent migraine \unskip~\citep{915901:20612401} head-aches and complications of cirrhosis \unskip~\citep{915901:20612415,915901:20612411}. It is taken orally. Popular side dizziness has symptoms \unskip~\citep{915901:20612410}, feeling sluggish, a slow-moving feeling pulse \unskip~\citep{915901:20612398} and Reynaud syndrome \unskip~\citep{915901:20612417,915901:20612414}. Its high intake has serious side effects, coronary Bronchospasm and loss \unskip~\citep{915901:20612413}. This is used in breastfeeding, and nursing the defiance is uncertain. Everything is, it's A beta-adrenergic blocker that is non-selective \unskip~\citep{915901:20612397,915901:20612408} and acts by suppressing adrenergic \ensuremath{\beta }1 receptors \unskip~\citep{915901:20612412} the adrenergic neurons in the heart and \ensuremath{\beta }2 in blood vessels.

Nadolol is one among the well-liked \ensuremath{\beta } blockers within the control of patients with LQT for QT interval shortening and ventricular arrhythmia avoidance \unskip~\citep{915901:20612409,915901:20612418}. It's more operative than cardioselective beta-blockers \unskip~\citep{915901:20612405} such as metoprolol and propranolol within the avoidance of breakthrough Cardiac incidents \unskip~\citep{915901:20612406}. Nadolol has the benefit of daily dosing and thereby increases the weakness of the patient. It is for the person whose function of the kidney decreased, and nadolol could also provide less dose often. For many neurological disorders, such as the avoidance of migraine attacks, attention/deficit/hyperactivity disorder (ADHD), it is effective, and its use has studied as a therapy for tremor and Parkinson's disease \unskip~\citep{915901:20612420}. Still, neither is well established \unskip~\citep{915901:20612400}.

Bendroflumethiazide formerly, bendrofluazide, brand name Aprinoxmay be a thiamine diuretic won't treat hypertension, its structure is shown in Figure~\ref{f-62c40b6b8268}(B). Bendroflumethiazide may be a thiamine diuretic \unskip~\citep{915901:20612399,915901:20612403} that acts at the start of the distal convoluted tubule (DCT) by inhibiting sodium reabsorption. As a consequence of more sodium hitting the supply ducts, water is lost. Bendrofiumethiazide mark has a role to play in the treatment of minor coronary wreck, but the diuretic loop could be safer for overload reduction.  The Best use of bendroflumethiazide in hypertension \unskip~\citep{915901:20612402} at present (part of the result is due to hypertension).
    
\section{Materials and Methods}
\textbf{Natural compounds and reagents}

Acetonitrile and orthophosphoric acid, water (Level HPLC) were purchased from Worli, Mumbai, India, from Merck (India) Ltd. All APIs of Nadolol and Bendroflumethiazide as reference standards were procured from Spectrum pharma research solutions Pvt Ltd, Hyderabad. 

\textbf{Equipment}

An HPLC system (Waters alliance series e2695) associated Mass Spectrometer with QTRAP the triple quadrupole instrument 5500 (Sciex) was utilised. By the Sciex programming, activity was performed.

\textbf{Chromatographic conditions}

Chromatographic separation was administered in aristocratic mode at room temperature, by using an asymmetry C\ensuremath{_{18}} column (150x4.6 mm, 3.5 microns). A mix of Acetonitrile and 0.1\% OPA in 40: 60 as a mobile process, v / v was used at a flow of 1.0 ml /min. 10 \ensuremath{\mu }l was the infused arrangement. The running time was 15 minutes.

\textbf{Drafting of the norm and internal control samples}

\textbf{Preparation of Bendroflumethiazide parent stock}

10 mg of Bendroflumethiazide standard was weighed and dissolved reliably in 100 ml in a diluent. The Concentration of the solution is 100\ensuremath{\mu }g/ml. Take this 0.1ml of the above solution diluted to 10ml with diluent. This is often called the parent stock solution of bendroflumethiazide, and therefore the Concentration is 0.1\ensuremath{\mu}g/ml.

\textbf{Preparation of Nadolol and Bendroflumethiazide standard stock solution}

Take 0.4ml Nadolol parent stock solution and 0.4ml of Bendroflumethiazide parent stock solution into 10ml VF and made up to the mark with diluent. It's a stock solution with Nadolol concentration 320ng/ml and Bendroflumethiazide concentration 40ng/ml. Within the same way, an internal standard stock solution was prepared. 

\textbf{Preparation of traditional solution}

Normal solutions have been developed by taking 0.5 ml stock solution; 0.5 ml stock solution; 0.5 ml stock solution IS a stock solution, 0.2 ml plasma, 0.3 ml plasma Acetonitrile remaining 0.5 ml diluent in a centrifuged tube and vortexes for 15 min to combine the constituents. After centrifuging 15 min, at 5000 rpm supernatant managed solution was separated and filtered through 0.45 \ensuremath{\mu} nylon syringe filter into a vial and injected into HPLC system.

\textbf{Preparation of sample solution}

\textbf{Preparation of sample stock}

One tablet (contains 40 mg Nadolol and 5 mg Bendroflumethiazide) was weighed, note the typical weight of the tablet. The tablet was taken into a mortar and crushed into a fine powder. 13.4 mg of tablet powder was weighed accurately and Dissolved in diluents of 100 ml. From this take 0.8 ml and Diluted with diluted at 100 ml. this is often the sample stock with Nadolol concentration 320 ng/ml and Bendroflumethiazide concentration 40 ng/ml.


\bgroup
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\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/07aefa18-c27b-420f-b65f-f9939cb651da-upicture1.png}{\includegraphics{images/07aefa18-c27b-420f-b65f-f9939cb651da-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Structure (A) Nadolol and (B) Bendroflumethiazide Structures}}
\label{f-62c40b6b8268}
\end{figure*}
\egroup
\textbf{Preparation of sample solution}

For sample preparation takes 0.2 ml plasma, 0.5 ml sample stock, 0.3 ml acetonitrile and 0.5 ml IS, 0.5 ml diluent were taken into a centrifuge tube and vortexed for 15 min to precipitate all the proteins. Centrifuge it for 15 min at 5000 rpm and collect the supernatant solution into a vial and inject it into an HPLC system. 

\textbf{Method validation}

\textbf{Selectivity}

Selectivity was undertaken by investigating the rodent of plasma tests from six-point six, distinct rodents to check for impedance yeah, at the maintenance season in analyses.

\textbf{Impact Matrix}

Effect of a matrix for Nadolol and Bendroflumethiazide it was calculated by comparing the height Ratio of Region within plasma samples from six separate drug-free blank plasma samples, and tidy reconstitution samples were collected from the post. At MQC stages, trials were carried out in triplicate with six separate plasma parcels with a satisfactory accuracy of 15 per cent.

\textbf{Accuracy and precision}

It was dictated that within monitoring checks (n=6) at a lower quantification limit (LLOQ), low-quality monitoring (LQC), medium quality control (MQC), top quality control (HQC) standards were repeatedly examined. The half CV should be less than 15 per cent and precision should within15 per cent apart from LLQC where it should be within 20percent. 

\textbf{Recovering }

Extracting the productivity in Nadolol and Bendroflumethiazide are determined by an examination of six reproduce at each internal Concentration in power. The share Recovery was measured by matching the height areas of the removed standards with the height areas of the removed standards. 

\textbf{Carryover }

The analyte held by the chromatographic framework during the infusion of an example that shows up in ensuing clear or obscure examples.

\textbf{Dilution integrity}

Dilution integrity ought to be shown by spiking the grid with an analyte focus over the ULOQC and weakening this example with a clear framework. 

\textbf{Stability}

Stability of stock solution was carried out by looking at Analyses global reaction inside the stability evaluation for the worldwide test reaction arranged through the current product structure. At the LQC and HQC concentration levels, plasma stability tests were carried out using six copies at each dose. If the shift is less than 15 per cent as per US FDA guidance, Analyze was deemed stable. At room temperature, the steadiness of spiked rodent plasma experiments is placed aside; it was calculated for twenty-four hrs. Twenty four hours.

The safety of spiked rat plasma deposited in the autosampler at 2-8\ensuremath{^{o}}C was measured for twenty-four hours. The durability of the autosampler was tested by looking at the correctly infused extract plasma reports, with the samples re-injected at 2-8\ensuremath{^{o}}C for twenty-four hours after storage in the autosampler at 2-8\ensuremath{^\circ}C twenty-four hours. The reproducibility of reinjection was tested by looking at collected plasma tests that were injected promptly, with the samples that were reinvested in the wake of putting away in the autosampler for 24 hours at 2-8\ensuremath{^{o}}C.

The durability of the cold thaw was led by looking at the steadiness tests that they had been solidified at -30\ensuremath{^{o}}C except for defrosted multiple occasions, with newly sharp spikes internal samples for monitoring. Six LQC and HQC aliquots per focus the levels have been utilised for the freeze defrost soundness assessment. The Concentration obtained after 24 hours were for long-term stability assessment contrasted and beginning fixation.
    
\section{Results and Discussion}
Lionisation of electrospray having Maximum reaction about reaction air pressure, a mode for chemical lionisation selected in this method. The optimisation of an instrument to offer sensitivity and signal stability during in fusen of analyte within the Continuous-flow to electrospray at all polarities, the ion source worked at the flow 10 \ensuremath{\mu}l/min standard chromatogram of Nadolol and Bendroflumethiazide gives more response in positive particle mode in comparison with negative particle model shown in Figure~\ref{f-610d2be767c9}.


\begin{table*}[!htbp]
\caption{\boldmath {Precision and Accuracy of Nadolol} }
\label{tw-e5e4424fd448}
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  \cAlignHack 8 &
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  \cAlignHack 80 &
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  \cAlignHack 8.236 &
  \cAlignHack 40.111 &
  \cAlignHack 80.163 &
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  \cAlignHack 40.169 &
  \cAlignHack 80.157 &
  \cAlignHack 120.258\\
\cAlignHack QC sample-3 &
  \cAlignHack 8.225 &
  \cAlignHack 40.118 &
  \cAlignHack 80.146 &
  \cAlignHack 120.247\\
\cAlignHack QC sample-4 &
  \cAlignHack 8.204 &
  \cAlignHack 40.172 &
  \cAlignHack 80.129 &
  \cAlignHack 120.266\\
\cAlignHack QC sample-5 &
  \cAlignHack 8.213 &
  \cAlignHack 40.139 &
  \cAlignHack 80.155 &
  \cAlignHack 120.137\\
\cAlignHack QC sample-6 &
  \cAlignHack 8.247 &
  \cAlignHack 40.158 &
  \cAlignHack 80.194 &
  \cAlignHack 120.174\\
\cAlignHack Mean &
  \cAlignHack 8.223 &
  \cAlignHack 40.145 &
  \cAlignHack 80.157 &
  \cAlignHack 120.221\\
\cAlignHack SD &
  \cAlignHack 0.01596 &
  \cAlignHack 0.02605 &
  \cAlignHack 0.02151 &
  \cAlignHack 0.05284\\
\cAlignHack \%CV &
  \cAlignHack 0.19 &
  \cAlignHack 0.06 &
  \cAlignHack 0.03 &
  \cAlignHack 0.04\\
\cAlignHack Accuracy &
  \cAlignHack 99.84 &
  \cAlignHack 99.93 &
  \cAlignHack 100.25 &
  \cAlignHack 99.89\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Precision and Accuracy of Bendroflumethiazide} }
\label{tw-e63e8b25ad29}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.23650000000000006\linewidth-2\tabcolsep}p{\dimexpr.16349999999999994\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Qc Name & \cAlignHack LLQC & \cAlignHack LQC & \cAlignHack MQC & \cAlignHack HQC\\
\tblmidrule 
\cAlignHack Conc. (ng/ml) &
  \cAlignHack 1 &
  \cAlignHack 5 &
  \cAlignHack 10 &
  \cAlignHack 15\\
\cAlignHack QC Sample-1 &
  \cAlignHack 1.056 &
  \cAlignHack 5.107 &
  \cAlignHack 10.126 &
  \cAlignHack 15.174\\
\cAlignHack QC Sample-2 &
  \cAlignHack 1.047 &
  \cAlignHack 5.128 &
  \cAlignHack 10.134 &
  \cAlignHack 15.128\\
\cAlignHack QC Sample-3 &
  \cAlignHack 1.029 &
  \cAlignHack 5.139 &
  \cAlignHack 10.117 &
  \cAlignHack 15.139\\
\cAlignHack QC Sample-4 &
  \cAlignHack 1.022 &
  \cAlignHack 5.121 &
  \cAlignHack 10.120 &
  \cAlignHack 15.147\\
\cAlignHack QC Sample-5 &
  \cAlignHack 1.034 &
  \cAlignHack 5.142 &
  \cAlignHack 10.151 &
  \cAlignHack 15.134\\
\cAlignHack QC Sample-6 &
  \cAlignHack 1.018 &
  \cAlignHack 5.127 &
  \cAlignHack 10.148 &
  \cAlignHack 15.124\\
\cAlignHack Mean &
  \cAlignHack 1.034 &
  \cAlignHack 5.127 &
  \cAlignHack 10.133 &
  \cAlignHack 15.141\\
\cAlignHack Stddev &
  \cAlignHack 0.01468 &
  \cAlignHack 0.01269 &
  \cAlignHack 0.01431 &
  \cAlignHack 0.01809\\
\cAlignHack \%CV &
  \cAlignHack 1.42 &
  \cAlignHack 0.25 &
  \cAlignHack 0.14 &
  \cAlignHack 0.12\\
\cAlignHack Accuracy &
  \cAlignHack 99.78 &
  \cAlignHack 100.13 &
  \cAlignHack 100.06 &
  \cAlignHack 100.27\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/31fabc62-24b8-41ce-8b77-37a12a01d517-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/31fabc62-24b8-41ce-8b77-37a12a01d517-upicture2.png}{\includegraphics{images/31fabc62-24b8-41ce-8b77-37a12a01d517-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of Standard}}
\label{f-610d2be767c9}
\end{figure}
\egroup
\textbf{Specificity}

Interfering peaks weren't observed at Nadolol, Bendroflumethiazide, Nadolol-D\ensuremath{_{6}}, and Bendroflumethiazide-D\ensuremath{_{6}} retention times within the chromatogram of blank rat plasma shown in Figure~\ref{f-22d8707da861}. This proved specificity of the tactic to research Nadolol and Bendroflumethiazide simultaneously.


\bgroup
\fixFloatSize{images/f6162484-c1b6-4530-a5e6-e59d965df627-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/f6162484-c1b6-4530-a5e6-e59d965df627-upicture3.png}{\includegraphics{images/f6162484-c1b6-4530-a5e6-e59d965df627-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of Blank}}
\label{f-22d8707da861}
\end{figure}
\egroup
\textbf{Matrix effect}


\begin{table*}[!htbp]
\caption{\boldmath {Stability results of nadolol} }
\label{tw-c09c56d9200b}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2365\linewidth-2\tabcolsep}p{\dimexpr.15200000000000003\linewidth-2\tabcolsep}p{\dimexpr.26459999999999997\linewidth-2\tabcolsep}p{\dimexpr.225\linewidth-2\tabcolsep}p{\dimexpr.1219\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.3885\linewidth-2\tabcolsep)}}{\cAlignHack Stability experiment spiked plasma} & \cAlignHack Spiked plasma conc. (n=6, ng/ml) & \cAlignHack Conc. measured (n=6, ng/ml) & \cAlignHack \% CV\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.2365\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Benchtop stability }} &
  \cAlignHack LQC &
  \cAlignHack 40 &
  \cAlignHack 40.126 &
  \cAlignHack 1.1\\
 &
  \cAlignHack MQC &
  \cAlignHack 80 &
  \cAlignHack 80.324 &
  \cAlignHack 0.47\\
 &
  \cAlignHack HQC &
  \cAlignHack 120 &
  \cAlignHack 120.041 &
  \cAlignHack 0.23\\
\multicolumn{1}{p{\dimexpr(.2365\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Autosampler stability }} &
  \cAlignHack LQC &
  \cAlignHack 40 &
  \cAlignHack 40.325 &
  \cAlignHack 0.24\\
 &
  \cAlignHack MQC &
  \cAlignHack 80 &
  \cAlignHack 80.187 &
  \cAlignHack 0.52\\
 &
  \cAlignHack HQC &
  \cAlignHack 120 &
  \cAlignHack 120.259 &
  \cAlignHack 0.29\\
\multicolumn{1}{p{\dimexpr(.2365\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Long term (Day 28) stability}} &
  \cAlignHack LQC &
  \cAlignHack 40 &
  \cAlignHack 40.375 &
  \cAlignHack 0.26\\
 &
  \cAlignHack MQC &
  \cAlignHack 80 &
  \cAlignHack 80.421 &
  \cAlignHack 0.14\\
 &
  \cAlignHack HQC &
  \cAlignHack 120 &
  \cAlignHack 120.305 &
  \cAlignHack 0.07\\
\multicolumn{1}{p{\dimexpr(.2365\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Wet extract stability}} &
  \cAlignHack LQC &
  \cAlignHack 40 &
  \cAlignHack 40.825 &
  \cAlignHack 0.57\\
 &
  \cAlignHack MQC &
  \cAlignHack 80 &
  \cAlignHack 80.327 &
  \cAlignHack 0.28\\
 &
  \cAlignHack HQC &
  \cAlignHack 120 &
  \cAlignHack 120.159 &
  \cAlignHack 0.14\\
\multicolumn{1}{p{\dimexpr(.2365\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Dry extract stability}} &
  \cAlignHack LQC &
  \cAlignHack 40 &
  \cAlignHack 40.284 &
  \cAlignHack 1.55\\
 &
  \cAlignHack MQC &
  \cAlignHack 80 &
  \cAlignHack 80.307 &
  \cAlignHack 0.52\\
 &
  \cAlignHack HQC &
  \cAlignHack 120 &
  \cAlignHack 120.172 &
  \cAlignHack 0.17\\
\multicolumn{1}{p{\dimexpr(.2365\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Freeze-thaw stability }} &
  \cAlignHack LQC &
  \cAlignHack 40 &
  \cAlignHack 40.296 &
  \cAlignHack 0.70\\
 &
  \cAlignHack MQC &
  \cAlignHack 80 &
  \cAlignHack 80.421 &
  \cAlignHack 0.36\\
 &
  \cAlignHack HQC &
  \cAlignHack 120 &
  \cAlignHack 120.317 &
  \cAlignHack 0.19\\
\multicolumn{1}{p{\dimexpr(.2365\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Short term stability}} &
  \cAlignHack LQC &
  \cAlignHack 40 &
  \cAlignHack 40.329 &
  \cAlignHack 1.11\\
 &
  \cAlignHack MQC &
  \cAlignHack 80 &
  \cAlignHack 80.127 &
  \cAlignHack 0.31\\
 &
  \cAlignHack HQC &
  \cAlignHack 120 &
  \cAlignHack 120.478 &
  \cAlignHack 0.35\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Stability results of bendroflumethiazide} }
\label{tw-baad97f61da8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2415\linewidth-2\tabcolsep}p{\dimexpr.18009999999999998\linewidth-2\tabcolsep}p{\dimexpr.2598\linewidth-2\tabcolsep}p{\dimexpr.20819999999999997\linewidth-2\tabcolsep}p{\dimexpr.1104\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.4216\linewidth-2\tabcolsep)}}{\cAlignHack Stability experiment spiked plasma} & \cAlignHack Spiked plasma conc. (n=6, ng/ml) & \cAlignHack Conc. measured (n=6, ng/ml)  & \cAlignHack \% CV\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.2415\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Benchtop stability }} &
  \cAlignHack LQC &
  \cAlignHack 5 &
  \cAlignHack 5.017 &
  \cAlignHack 3.12\\
 &
  \cAlignHack MQC &
  \cAlignHack 10 &
  \cAlignHack 10.247 &
  \cAlignHack 0.86\\
 &
  \cAlignHack HQC &
  \cAlignHack 15 &
  \cAlignHack 15.326 &
  \cAlignHack 0.86\\
\multicolumn{1}{p{\dimexpr(.2415\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Autosampler stability }} &
  \cAlignHack LQC &
  \cAlignHack 5 &
  \cAlignHack 5.365 &
  \cAlignHack 3.87\\
 &
  \cAlignHack MQC &
  \cAlignHack 10 &
  \cAlignHack 10.249 &
  \cAlignHack 1.69\\
 &
  \cAlignHack HQC &
  \cAlignHack 15 &
  \cAlignHack 15.028 &
  \cAlignHack 0.99\\
\multicolumn{1}{p{\dimexpr(.2415\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Long term (Day 28) stability}} &
  \cAlignHack LQC &
  \cAlignHack 5 &
  \cAlignHack 5.268 &
  \cAlignHack 0.34\\
 &
  \cAlignHack MQC &
  \cAlignHack 10 &
  \cAlignHack 10.248 &
  \cAlignHack 2.37\\
 &
  \cAlignHack HQC &
  \cAlignHack 15 &
  \cAlignHack 15.327 &
  \cAlignHack 0.13\\
\multicolumn{1}{p{\dimexpr(.2415\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Wet extract stability}} &
  \cAlignHack LQC &
  \cAlignHack 5 &
  \cAlignHack 5.147 &
  \cAlignHack 2.71\\
 &
  \cAlignHack MQC &
  \cAlignHack 10 &
  \cAlignHack 10.469 &
  \cAlignHack 1.8\\
 &
  \cAlignHack HQC &
  \cAlignHack 15 &
  \cAlignHack 15.007 &
  \cAlignHack 0.74\\
\multicolumn{1}{p{\dimexpr(.2415\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Dry extract stability}} &
  \cAlignHack LQC &
  \cAlignHack 5 &
  \cAlignHack 5.629 &
  \cAlignHack 3.07\\
 &
  \cAlignHack MQC &
  \cAlignHack 10 &
  \cAlignHack 10.487 &
  \cAlignHack 2.21\\
 &
  \cAlignHack HQC &
  \cAlignHack 15 &
  \cAlignHack 15.326 &
  \cAlignHack 0.64\\
\multicolumn{1}{p{\dimexpr(.2415\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Freeze-thaw stability }} &
  \cAlignHack LQC &
  \cAlignHack 5 &
  \cAlignHack 5.471 &
  \cAlignHack 3.73\\
 &
  \cAlignHack MQC &
  \cAlignHack 10 &
  \cAlignHack 10.482 &
  \cAlignHack 1.55\\
 &
  \cAlignHack HQC &
  \cAlignHack 15 &
  \cAlignHack 15.326 &
  \cAlignHack 0.88\\
\multicolumn{1}{p{\dimexpr(.2415\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack Short term stability}} &
  \cAlignHack LQC &
  \cAlignHack 5 &
  \cAlignHack 5.289 &
  \cAlignHack 3.24\\
 &
  \cAlignHack MQC &
  \cAlignHack 10 &
  \cAlignHack 10.582 &
  \cAlignHack 0.61\\
 &
  \cAlignHack HQC &
  \cAlignHack 15 &
  \cAlignHack 15.327 &
  \cAlignHack 1.22\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Pharmacokinetic parameters of Nadolol and Bendroflumethiazide} }
\label{tw-ae6af48c4c1e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.36940000000000005\linewidth-2\tabcolsep}p{\dimexpr.29059999999999995\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Pharmacokinetic parameters & \cAlignHack Nadolol & \cAlignHack Bendroflumethiazide\\
\tblmidrule 
\cAlignHack AUC0-t &
  \cAlignHack 1216 ng h/ml &
  \cAlignHack 118 ng h/ml\\
\cAlignHack Cmax &
  \cAlignHack 60 ng/ml &
  \cAlignHack 7.7 ng/ml\\
\cAlignHack AUC0-\ensuremath{\infty } &
  \cAlignHack 1241 ng h/ml &
  \cAlignHack 122 ng h/ml\\
\cAlignHack T1/2 &
  \cAlignHack 24 Hr &
  \cAlignHack 8 Hr\\
\cAlignHack tmax &
  \cAlignHack 24 Hr &
  \cAlignHack 8 Hr\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
In matrix effect, six many rat plasma at LQC and HQC of Nadolol was 99.1, and 99.9 and bendroflumethiazide were 99.8, 99.3\%. \% CV of both drugs at LQC level were 1.67, 6.93 and HQC level is 0.39, 1.72 respectively. It shows that the matrix effect on the ionisation of the analytes is below the acceptable range, and results are shown in Tables~\ref{tw-e5e4424fd448} and~\ref{tw-e63e8b25ad29}. 

\textbf{Precision and accuracy}

The accuracy and precision calculated by pooling all individual assay results of the assay various internal control samples. The accuracy results of nadolol in quality control samples 98.7-99.9 and bendroflumethiazide in quality control samples 99.1-99.9. The half of the CV of Nadolol and Bendroflumethiazide is \textless\ 5\% altogether internal control samples. Based on the reported data shown in Table~\ref{tw-e5e4424fd448} for Nadolol \& Table~\ref{tw-e63e8b25ad29} for bendroflumethiazide supports the strategy is reliable and precise.


\bgroup
\fixFloatSize{images/b296e6ac-cf38-44d5-9096-a86c2173b77c-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/b296e6ac-cf38-44d5-9096-a86c2173b77c-upicture4.png}{\includegraphics{images/b296e6ac-cf38-44d5-9096-a86c2173b77c-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Recovery plot of Nadolol}}
\label{f-0a8de48073c6}
\end{figure}
\egroup
\textbf{Recovery}

The recoveries for Nadolol (98.79\%-100.68\%) and Bendroflumethiazide (99.61\%-100.37\%) At the LQC, MQC, HQC, and levels and \%CV ranged from 0.18-0.62 for nadolol and 0.81-2.56 for bendroflumethiazide. The results demonstrated that the bioanalytical method owns good extraction efficiency. This also showed that the recovery wasn't hooked into Concentration.  

\textbf{Ruggedness}

The per cent recoveries and per cent CV of Nadolol and Bendroflumethiazide determined with two different analysts and on two different columns were within acceptable criteria in HQC, LQC, MQC and LLQC samples. The per cent recoveries ranged from 99.84-100.72\% for nadolol and 99.34\%-99.95\% for bendroflumethiazide. The \%CV values ranged from 0.11-0.38 for nadolol and 0.53-8.23 for bendroflumethiazide. The results proved the method is ruggedness. 

\textbf{Autosampler Carryover }

Peak area response of Nadolol and Bendroflumethiazide, Nadolol-D\ensuremath{_{6}}, Bendroflumethiazide-D\ensuremath{_{6}} wasn't observed within the blank rat plasma samples after successive injections of LLQC and ULQC at the retention times of Nadolol and Bendroflumethiazide, Nadolol-D\ensuremath{_{6}}, Bendroflumethiazide-D\ensuremath{_{6}}. Therefore, this method doesn't exhibit autosampler carryover.  

\textbf{Stability}

In solution stability analysis, Nadolol and Bendroflumethiazide solutions were prepared with diluents and put in storage in a refrigerator at 2-8\ensuremath{^{o}}C. New product options have been correlated with prepared stock solution earlier 24Hrs. The half the shift the nadolol and bendroflumethiazide the stock solutions were steady at 1.26 per cent and 0.79 per cent, respectively up to 24 Hrs when stored in 2-8\ensuremath{^\circ}C. Benchtop autosampler and stabilities LQC and HQC levels were observed at room temperature Nadolol, and bendroflumethiazide was stable in plasma for twenty-four Hrs in an autosampler at 20\ensuremath{^\circ}C. From this, it has been verified that at LQC and HQC levels plasma samples correlated with frequent freezing and thawing Nadolol and Bendroflumethiazide their stability has not affected From future stability it had been clear that Nadolol and Bendroflumethiazide were stable up to 24 Hrs at a storage temperature of -30\ensuremath{^\circ}C. The general stability results of Nadolol and Bendroflumethiazide were tabulated in Tables~\ref{tw-c09c56d9200b} and~\ref{tw-baad97f61da8}, respectively.

\textbf{Pharmacokinetic study}

The technique has been approved successfully to measure the Concentration of Nadolol life, as measured by the 0.693 / Kel quotient) and bendroflumethiazide in six many different rats, after oral administration of Nadolol and Bendroflumethiazide sam


\bgroup
\fixFloatSize{images/72af17c8-e641-4e48-b2a5-e4aecec5f954-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/72af17c8-e641-4e48-b2a5-e4aecec5f954-upicture5.png}{\includegraphics{images/72af17c8-e641-4e48-b2a5-e4aecec5f954-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Recovery plot of Bendroflumethiazide (B)}}
\label{f-a54e26c4ec32}
\end{figure}
\egroup
ple as an oral dose under fasting condition. After injecting the drug samples into a rat body collect the samples at different time intervals like 1, 2, 4, 6, 8, 12, 16, 20, 24, 28 and 32 hrs from the rat body. Then according to test technique sample is ready and injected into the chromatographic framework and record the values. The evaluated pharmacokinetic parameters were C\ensuremath{_{max}} (greatest watched tranquilise focus measured using trapezoidal principle), t\ensuremath{_{max}} (time to observed most extreme medication focus), K\ensuremath{_{el}} (evident first request terminal rate constant determined from the semi-log map of a plasma concentration versus time curves of nadolol shown in Figure~\ref{f-0a8de48073c6} and Bendroflumethiazide are shown in Figure~\ref{f-a54e26c4ec32} using the tactic of the smallest amount regression) and t1/2 (terminal half The ratio of test/reference for C\ensuremath{_{max}}, AUC\ensuremath{_{0-12}} and AUC were 88.54 and 94.32 respectively and located to be within the suitable limit of 80\%-125\%. The further tabulated pharmacokinetic boundaries of Nadolol and Bendroflumethiazide are shown in Table~\ref{tw-ae6af48c4c1e}.
    
\section{Conclusions}
For the primary time, higher sensitive HPLC-ESI-LCMS/MS method was created and approved for the determination of Nadolol and Bendroflumethiazide in rat plasma. Here the described approach is a rugged, fast, reproducible bioanalytical method. A simple and efficient method was developed and may be utilised in pharmacokinetic studies and to see the investigated analyte in body fluids.
    
\section{Acknowledgement}
The author is thankful to GITAM Deemed to be University for providing necessary facilities to carry out my research work.

\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.
    

\bibliographystyle{pharmascope_apa-custom}

\bibliography{\jobname}
\end{document}
