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\def\RunningAuthor{Vijey Aanandhi M et al.}
\firstPage{6826}
\articleType{Original Article}
\receivedDate{15 Jun 2020}
\acceptedDate{03 Aug 2020}
\revisedDate{08 Jul 2020}
\journalVolume{2020, 11}
\journalIssue{4}
\journalDoi{ijrps.v11i4.3652}
\copyrightYear{2020}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{\textbf{Method Development and Validation of Fludrocortisone Acetate Tablets by Reverse Phase HPLC Method }}
\author{Vijey~Aanandhi~M\textsuperscript{*},  
            Elancheziyan~K,  
            Yamini~R,  
            Prakash~Chand~T,  
            Aysha~Jadeera~K~A, 
            Binoy~Varghese~Cheriyan~\\[5pt]{Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Sciences, Technology and Advanced Studies (VISTAS) Pallavaram, Chennai-600117, Tamil Nadu, India}}

\begin{abstract}
The purpose or intent of this current study was to establish a fast and sensitive HPLC technique for the perseverance of Fludrocortisone acetate and utilizing best frequently used HPLC technique. This method had been validated as per the ICH requirements to assure that the method consistently meets the predetermined specifications and quality attributes.Utilizing filtered and degassed pH 3.0 Phosphate buffer and Acetonitrile in the ratio 90:10 as a Mobile phase-A and pH 3.0 Phosphate buffer and Acetonitrile in the ratio 65:35 as a Mobile phase-B the established RP-HPLC technique was done. The separation was achieved by using Waters, X-Bridge Shield RP18, (150 X 4.6-mm), 3.5-\ensuremath{\mu}m column. Run time and Flow rate was set 45minutes and 1.2mL/min. Injection volume 100\ensuremath{\mu}L and wavelength was set 240nm.The correlation coefficient square for fludrocortisones acetate and Fludrocortisone Impurity was found to be 0.9991 and 0.99997. The SD and \%RSD for Fludrocortisone Impurity was found to be 0.02 and 1.48 represents method precision. Following validated parameters lies within the limit. Hence, the developed method was precise, simple, fast and accurate.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Fludrocortisone Acetate,\newline Method development,\newline Method validation,\newline Reverse Phase HPLC
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Vijey~Aanandhi~M~\\ Phone:\ ~\\  Email:\ hodpchemistry@velsuniv.ac.in
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Disorder and then postural hypotension. It is usually consumed along with hydrocortisone in case of adrenal inadequacy and medication is utilized utmost in its acetate form \unskip~\citep{869157:20178588,869157:20178581,869157:20178582,869157:20178578}. Fludrocortisone acetate is an adrenal corticosteroid and has controlled level of glucocorticoid action and has extremely maximum level of mineralocorticoid action. Though it is utilized just for its mineralocorticoid issues. Mineralocorticoid functions in the distal tubules to enhance hydrogen ion evacuation, sodium resorption, to enhance potassium evacuation and later water retention. In further secretory cells cation transfer is equally influenced, evacuation of water by sweat glands and salivary glands is also modified, though to a lower degree. At the cellular levels corticosteroids spread throughout or over cell membranes and intricate with particular or individual cytoplasmic recipient, such complexes afterwards join the cell nucleus attach to DNA and encourage arrangement of mRNA and later protein combination of different enzymes thought to be finally liable for the physiologic issues of such hormones \unskip~\citep{869157:20178586,869157:20178579}. Fludrocortisone is utilized mainly to substitute the lacking hormone aldosterone in diverse forms of adrenal inadequacy for instance classic salt squandering form of congenital adrenal hypertrophy and Addison's illness. It has been utilized in the therapy of cerebral salt squandering disorder. Because of its issues on enhancing sodium levels and hence blood volume and then used to heal postural hypotension and to heal low blood pressure \unskip~\citep{869157:20178589,869157:20178584}.

\textbf{Method Development}

Method development is the procedure or method of choosing a precise testing process to establish the constitution of a preparation. Analytical process must be utilized under GLP and GMP conditions and utilizing the customs or properties fixed out in the ICH guidelines the analytical procedure should be established. It is the method of evidence that an analytical procedure is tolerable for employ in laboratory to assess the compactness of consequent specimens. Suitable and quantified or measured instruments, Certified procedure, Change control, Dependable reference standard, Competent analysts, Integrity and Specimen choice\unskip~\citep{869157:20178583,869157:20178580,869157:20178577}.
    
\section{Materials and Methods}
\textbf{Chemicals and reagents}

Fludrocortisone Acetate tablets (Equivalent to about 2.5mg) the used pharmaceutical preparation were formulated in-house and used Fludrocortisone Acetate API with a potency 100\%. HPLC grade Acetonitrile were purchased from Merck limited and HPLC grade Methanol were purchased from Finar Limited and HPLC grade water. Analytical grade reagents were used.

\textbf{Instrumentation}

Automated Reverse Phase HPLC equipped with UV-detection chromatographic separation was achieved utilizing Waters, X-Bridge Shield RP18, (150 X 4.6-mm), 3.5-\ensuremath{\mu}m column. Run time and Flow rate was set 45minutes and 1.2mL/min. Injection volume 100\ensuremath{\mu}L and wavelength was set 240nm. For processing data Empower software were used.

The chromatographic Parameters and gradient progam are listed in Tables~\ref{tw-1e0f4630446e} and~\ref{tw-47e0fa2ef053}.

From Table~\ref{tw-af2ae0f170ef}, developed technique were met the acceptance criteria. Hence the developed method was found to be accurateTables~\ref{tw-f48b239c053e}, \ref{tw-0afc8ae346d7}, \ref{tw-3eb75aa3cb97}, \ref{tw-6f4d48c16d60}, \ref{tw-e4dcd1fa4da3} and~\ref{tw-823462d5a367} .

The correlation coefficient square (r\ensuremath{^{2}}) for Fludrocortisone Acetate and Fludrocortisone Impurity were met the acceptance criteria. The linear regression data shows that the method is linear over the entire concentration range (LOQ (10\%)-150\%) and the developed method was found to be precise and accurateFigures~\ref{f-dfe3bf29bd85}, \ref{f-2c29dcb7d017} and~\ref{f-6a2810c4dbfd}.

From Figure~\ref{f-f193f5261b6f}, developed method were met the acceptance criteria. Hence the developed method was found to be accurate and precise.

Overall average recovery for Fludrocortisone Impurity is between 80.0-120.0\%.The \%RSD for recovery of triplicate preparations at each level is NMT 10\% and hence the method is accurate

No interference was observed from diluents, placebo and all known Impurities at the retention time of Fludrocortisone Acetate peak.

The \%difference in area between initial and time points should be NMT 25.0 for Standard. The \%difference in \%Impurity between initial and time points should be NMT 25.0 for sample.

All results met the acceptance criteria. Based on above results, it is concluded that standard and sample solutions were stable up to 96 hours respectively when stored at Room temperature.

The \%difference in Peak area for Impurity between the centrifuged sample and filtered sample should be NMT 25.0.


\begin{table*}[!htbp]
\caption{\boldmath {Chromatographic Parameters} }
\label{tw-1e0f4630446e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5179\linewidth-2\tabcolsep}p{\dimexpr.4821\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Chromatographic parameters & Conditions/Specifications\\
\tblmidrule 
Mobile Phase {\textendash} A &
  pH 3.0Buffer: Acetonitrile (90:10,v/v)\\
Mobile Phase {\textendash} B &
  pH 3.0Buffer: Acetonitrile (65:35,v/v)\\
Column &
  Waters, X-Bridge{\textsuperscript{\textregistered}} Shield RP18, (150 X 4.6-mm), 3.5-\ensuremath{\mu}m \\
Flow rate &
  1.2 mL/min\\
Column Temp. &
  30\ensuremath{^\circ}C\\
Sample Temp. &
  10 \ensuremath{^\circ}C\\
Wavelength &
  240 nm\\
Injection Volume &
  100 \ensuremath{\mu}L\\
Run Time &
  45.0 minutes\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Gradient Program} }
\label{tw-47e0fa2ef053}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3525\linewidth-2\tabcolsep}p{\dimexpr.30749999999999996\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Time (min) & Mobile Phase-A (\%) & Mobile Phase-B (\%)\\
\tblmidrule 
0.00 &
  40.0 &
  60.0\\
15.00 &
  40.0 &
  60.0\\
20.00 &
  30.0 &
  70.0\\
30.00 &
  0.0 &
  100.0\\
40.00 &
  0.0 &
  100.0\\
40.10 &
  40.0 &
  60.0\\
45.00 &
  40.0 &
  60.0\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {System Precision data} }
\label{tw-af2ae0f170ef}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.2252\linewidth-2\tabcolsep}p{\dimexpr.1748\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S. No. & Injection No. & Peak Area for Fludrocortisone Acetate RS & USP Tailing & USP Plate Count\\
\tblmidrule 
1. &
  1 &
  151500 &
  1.1 &
  21354\\
2. &
  2 &
  154805 &
  1.1 &
  21172\\
3. &
  3 &
  152286 &
  1.1 &
  20984\\
4. &
  4 &
  152654 &
  1.1 &
  21231\\
5. &
  5 &
  153553 &
  1.1 &
  21452\\
6. &
  6 &
  154436 &
  1.1 &
  21012\\
Mean &
  - &
  153206 &
  - &
  -\\
STDEV &
  - &
  1284.65 &
  - &
  -\\
\%RSD &
  - &
  0.8 &
  - &
  -\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Linearity Data} }
\label{tw-f48b239c053e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{LLLLL}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(\mcWidth{1}+\mcWidth{2}+\mcWidth{3})}}{\cAlignHack Fludrocortisone Acetate} & \multicolumn{2}{p{\dimexpr(\mcWidth{4}+\mcWidth{5})}}{\cAlignHack Fludrocortisone Impurity}\\
\rowcolor{kwdboxcolor}Linearity Level (\%) & Concentration (\ensuremath{\mu}g/mL) & Peak Area for Fludrocortisone Acetate & Concentration (\ensuremath{\mu}g/mL) & Peak Area for Fludrocortisone Impurity\\
\tblmidrule 
LOQ (10\%) &
  0.05 &
  16236 &
  0.150 &
  14978\\
30 &
  0.151 &
  45752 &
  0.45 &
  45070\\
50 &
  0.252 &
  77852 &
  0.75 &
  75117\\
80 &
  0.402 &
  122749 &
  1.2 &
  120188\\
100 &
  0.503 &
  153206 &
  1.5 &
  151345\\
120 &
  0.604 &
  185421 &
  1.8 &
  180282\\
150 &
  0.755 &
  238594 &
  2.25 &
  225353\\
Correlation Coefficient Square (r2) &
  - &
  0.9991 &
  - &
  0.99997\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Method Precision Data} }
\label{tw-0afc8ae346d7}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2118\linewidth-2\tabcolsep}p{\dimexpr.2815\linewidth-2\tabcolsep}p{\dimexpr.2433\linewidth-2\tabcolsep}p{\dimexpr.26340000000000003\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S. No. & Sample Name & Peak Area for Fludrocortisone Impurity & \% Impurity\\
\tblmidrule 
1. &
  Method Precision-1 &
  150852 &
  1.513\\
2. &
  Method Precision-2 &
  151235 &
  1.476\\
3. &
  Method Precision-3 &
  150652 &
  1.495\\
4. &
  Method Precision-4 &
  151232 &
  1.458\\
5. &
  Method Precision-5 &
  152867 &
  1.505\\
6. &
  Method Precision-6 &
  153259 &
  1.512\\
Mean &
  - &
  - &
  1.49\\
STDEV &
  - &
  - &
  0.02\\
\%RSD &
  - &
  - &
  1.48\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Accuracy Data} }
\label{tw-3eb75aa3cb97}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.104\linewidth-2\tabcolsep}p{\dimexpr.1419\linewidth-2\tabcolsep}p{\dimexpr.12900000000000002\linewidth-2\tabcolsep}p{\dimexpr.12489999999999998\linewidth-2\tabcolsep}p{\dimexpr.1219\linewidth-2\tabcolsep}p{\dimexpr.10319999999999997\linewidth-2\tabcolsep}p{\dimexpr.10299999999999999\linewidth-2\tabcolsep}p{\dimexpr.08610000000000001\linewidth-2\tabcolsep}p{\dimexpr.086\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Accuracy Levels & Sample \# & Peak Area of Fludrocortisone Impurity & Amount Added (\ensuremath{\mu}g/mg) & Amount Recovered \mbox{}\protect\newline (\ensuremath{\mu}g/mg) & \% Recovery & Average \% Recovery & S.D & \%RSD\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.104\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{50\%}} &
  Sample 1 &
  72382 &
  0.742 &
  0.450 &
  98.9 &
  \multicolumn{1}{p{\dimexpr(.10299999999999999\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{97.3}} &
  \multicolumn{1}{p{\dimexpr(.08610000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.86}} &
  \multicolumn{1}{p{\dimexpr(.086\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{1.9}}\\
 &
  Sample 2 &
  70832 &
  0.698 &
  0.732 &
  95.3 &
   &
   &
  \\
 &
  Sample 3 &
  73135 &
  0.720 &
  0.738 &
  97.6 &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.104\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{100\%}} &
  Sample 1 &
  150218 &
  1.479 &
  1.506 &
  98.2 &
  \multicolumn{1}{p{\dimexpr(.10299999999999999\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{98.9}} &
  \multicolumn{1}{p{\dimexpr(.08610000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{0.97}} &
  \multicolumn{1}{p{\dimexpr(.086\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{0.99}}\\
 &
  Sample 2 &
  152457 &
  1.501 &
  1.524 &
  98.5 &
   &
   &
  \\
 &
  Sample 3 &
  153632 &
  1.513 &
  1.512 &
  100.0 &
   &
   &
  \\
\multicolumn{1}{p{\dimexpr(.104\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{150\%}} &
  Sample 1 &
  216083 &
  2.128 &
  2.256 &
  94.3 &
  \multicolumn{1}{p{\dimexpr(.10299999999999999\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{96.5}} &
  \multicolumn{1}{p{\dimexpr(.08610000000000001\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.51}} &
  \multicolumn{1}{p{\dimexpr(.086\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{2.6}}\\
 &
  Sample 2 &
  218753 &
  2.154 &
  2.244 &
  96.0 &
   &
   &
  \\
 &
  Sample 3 &
  226789 &
  2.233 &
  2.250 &
  99.3 &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Solution Stability of Fludrocortisone Acetate at Room Temperature} }
\label{tw-6f4d48c16d60}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2928\linewidth-2\tabcolsep}p{\dimexpr.4144\linewidth-2\tabcolsep}p{\dimexpr.2928\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Time Interval & Fludrocortisone Acetate Peak Area & \% Difference in Area\\
\tblmidrule 
Initial &
  105924 &
  N/A\\
12 Hours &
  103568 &
  2.22\\
24 Hours &
  102559 &
  3.18\\
48 Hours &
  101068 &
  4.58\\
72 Hours &
  100956 &
  4.69\\
96 Hours &
  100582 &
  5.04\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Solution Stability of Fludrocortisone Impurity at Room Temperature} }
\label{tw-e4dcd1fa4da3}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.24410000000000004\linewidth-2\tabcolsep}p{\dimexpr.4676\linewidth-2\tabcolsep}p{\dimexpr.2883\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Time Interval & Fludrocortisone Impurity Peak Area & \% Difference in Area\\
\tblmidrule 
Initial &
  152532 &
  N/A\\
12 Hours &
  150980 &
  1.02\\
24 Hours &
  150264 &
  1.49\\
48 Hours &
  150004 &
  1.66\\
72 Hours &
  149925 &
  1.71\\
96 Hours &
  149856 &
  1.75\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Filter Study Data of Fludrocortisone Impurity} }
\label{tw-823462d5a367}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5682000000000001\linewidth-2\tabcolsep}p{\dimexpr.2221\linewidth-2\tabcolsep}p{\dimexpr.2097\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Fludrocortisone Impurity}\\
\rowcolor{kwdboxcolor}Sample & Peak Area & \% Difference\\
\tblmidrule 
Centrifuged/Unfiltered &
  152532 &
  N/A\\
0.45\ensuremath{\mu}m Nylon/2mL discard &
  153252 &
  0.12\\
0.45\ensuremath{\mu}m Nylon/4mL discard &
  152117 &
  0.27\\
0.45\ensuremath{\mu}m Nylon/6mL discard &
  152029 &
  0.33\\
0.45\ensuremath{\mu}m Nylon/8mL discard &
  151863 &
  0.44\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Preparation of Buffer}

\textbf{Phosphate Buffer pH 3.0}

The buffer was prepared by dissolving 2.07 g of sodium di-hydrogen phosphate monohydrate in 1000 mL of water. Mixed well and then the buffer adjusted to pH 3.0\ensuremath{\pm}0.05 with diluted phosphoric acid, then the solution filtered through 0.45 \ensuremath{\mu}m membrane filter and sonicated the buffer solution to degas.

\textbf{Mobile Phase -A}

900mL of pH 3.0 Phosphate buffer and 100mL of Acetonitrile into a suitable container and then sonicated to degas.

\textbf{Mobile Phase-B}

650 mL of pH 3.0 Phosphate buffer and 350 mL of Acetonitrile into a suitable container and then sonicated to degas.

\textbf{Preparation of Diluent}

Used Mobile Phase-B as a diluent.

\textbf{Procedure}

\textbf{Standard Stock Preparation}

50.23mg of Fludrocortisone Acetate RS was weighed and transferred into a 100mL volumetric flask. To that 3/4th volume of diluent was added. Sonicated to dissolve, diluted to volume with diluent and mixed well.

\textbf{Intermediate Stock Preparation}

Pipetted out 6mL of Fludrocortisone Acetate Standard Stock solution into 100mL volumetric flask. Diluted to volume with diluent and mixed well.

\textbf{Standard Preparation}

Pipetted out 2.5mL of Fludrocortisone Acetate Intermediate Stock solution into 50mL volumetric flask. Diluted to volume with diluent and mixed well.

\textbf{Preparation of Sample Solution}

Randomly selected the twenty-five (25) tablets and recorded the weight of tablets. Transferred the tablets into a 25mLof volumetric flask. (Equivalent to about 2.5 mg of Fludrocortisone Acetate) added about 15mL of diluent and sonicated the flask until the tablets dispersed. Then spiked the 0.75mL of Fludrocortisone Impurity stock preparation into the same sample solution. Further sonicated to 15 minutes with frequent intermittent shake. After the sonication, diluted to volume with diluent and mixed well. Kept the flask on bench top for about 10 minutes and allowed to settle down. Centrifuged the sample for about 5 minutes and collected the supernatant. Filtered the clear aliquot through 0.45-\ensuremath{\mu}m Nylon syringe filter and collected the filtrate after discarded the first 4mL of filtrate. 


\bgroup
\fixFloatSize{images/20e8ca52-675e-48f3-ae31-15e31ba76f9e-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/20e8ca52-675e-48f3-ae31-15e31ba76f9e-upicture1.png}{\includegraphics{images/20e8ca52-675e-48f3-ae31-15e31ba76f9e-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of Fludrocortisone Acetate Standard Injection}}
\label{f-dfe3bf29bd85}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/ed4e0ba6-82d3-4144-b430-ab7af844d7ea-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/ed4e0ba6-82d3-4144-b430-ab7af844d7ea-upicture2.png}{\includegraphics{images/ed4e0ba6-82d3-4144-b430-ab7af844d7ea-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Linearity Graph for Fludrocortisone Acetate}}
\label{f-2c29dcb7d017}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/3926d75a-3845-426d-a31d-fb73d808cd0c-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/3926d75a-3845-426d-a31d-fb73d808cd0c-upicture3.png}{\includegraphics{images/3926d75a-3845-426d-a31d-fb73d808cd0c-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Linearity Graph for Fludrocortisone Impurity}}
\label{f-6a2810c4dbfd}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/bb3d2aee-ad24-461c-9677-8738d949984d-upicture4.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/bb3d2aee-ad24-461c-9677-8738d949984d-upicture4.png}{\includegraphics{images/bb3d2aee-ad24-461c-9677-8738d949984d-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of Fludrocortisone Impurity Sample Injection}}
\label{f-f193f5261b6f}
\end{figure*}
\egroup
\textbf{Preparation of Placebo Solution}

Weighed about 2498.12mgof Placebo into 25mL of volumetric flask and added 15mL of diluent and sonicated for 15 minutes with intermittent shaking. Diluted to volume with diluent and mixed well. Centrifuged the sample for about 5 minutes and collected the supernatant. Filtered the clear aliquot through 0.45-\ensuremath{\mu}m Nylon syringe filter and collected the filtrate after discarded the first 4mL of filtrate.

\textbf{Linearity Procedure}

\textbf{Standard Stock Preparation} \textbf{\space }

50.35mg of Fludrocortisone Acetate RS was weighed and transferred into a 100mL volumetric flask. To that 3/4th volume of diluent was added. Sonicated to dissolve, diluted to volume with diluent and mixed well.

\textbf{Intermediate Stock Preparation}

Pipetted out 5mL of Fludrocortisone Acetate Standard Stock solution into 250mL volumetric flask. Diluted to volume with diluent and mixed well.

\textbf{Standard Preparation}

Pipetted out 5mL of Fludrocortisone Acetate Intermediate Stock solution into 100mL volumetric flask. Diluted to volume with diluent and mixed well.

\textbf{Fludrocortisone Impurity Stock Preparation}

2.52mg of Fludrocortisone Impurity was weighed and transferred into a 50mL volumetric flask added 35mL of diluent and sonicated to dissolve. After sonication diluted to volume with diluent and mixed well.

\textbf{Fludrocortisone Impurity Preparation}

Pipetted out 3mL of Fludrocortisone Impurity stock solution into 100mL volumetric flask. Diluted to volume with diluent and mixed well.

\textbf{Initialization of Instrument}

By running mobile phase continuously stabilization of chromatographic system and saturation of column is done and baseline graph developed.
    
\section{Results and Discussion}
The \%difference in peak area for Impurity between the centrifuged sample and filtered sample was calculated and found less than 25.0. Based on filter study data, it is concluded that samples should be filtered through 0.45\ensuremath{\mu}m Nylon filter after discarding first 4mL of filtrate. 

The purpose or intent of current study was to establish a fast and sensitive HPLC technique for the perseverance of Fludrocortisone acetate and Fludrocortisone Impurity utilizing best frequently used HPLC technique. The method has been validated as per the guidelines given by ICH requirements\textbf{\space }to assure that the method consistently meets the predetermined specifications and quality attributes. The system suitability parameter i.e., correlation coefficient square for fludrocortisones acetate and fludrocortisones impurity was found to be 0.9991 and 0.99997. The SD and \%RSD for Fludrocortisone Impurity was found to be 0.02 and 1.48 represents method precision. Following validated parameters lies within the limit. Hence the developed method was precise, simple, fast and accurate.
    
\section{Conclusions}
The developed method was validated as per the ICH guidelines instructions. From the determined values it is concluded that the established technique was simple, fast, precise and accurate. 
    
\section{Acknowledgements}
The authors are thankful to Novitium Labs Private Limited, Chennai for providing sample for research work. The authors are thankful for Vels Institute of Sciences, Technology and Advanced Studies (VISTAS), Chennai, Tamil Nadu for providing all the facilities to make this work success.

\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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\end{document}
