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\def\RunningAuthor{Vikrant Verma}
\firstPage{2399}
\articleType{Original Article}
\receivedDate{26.08.2019}
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\journalVolume{2020, 11}
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\title{A novel high performance liquid chromatography method development and validation of fluconazole by ezchrom software}
\author{Vikrant~Verma\textsuperscript{*}\textsuperscript{1},  
            Manish~Pathak\textsuperscript{1},  
            Amit~Kumar\textsuperscript{2}, 
            Amit~Kumar\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Pharmaceutical Chemistry, Kharvel Subharti College of Pharmacy, Swami Vivekanand Subharti University Meerut, Uttar Pradesh, India}~\\{\textsuperscript{2}Department of Pharmacognosy, Kharvel Subharti College of Pharmacy, Swami Vivekanand Subharti University Meerut, Uttar Pradesh, India}~\\{\textsuperscript{3}Department of Biotechnology, Keral Verma Subharti College of Science, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh, India}}

\begin{abstract}
The current research explains the development of a new method and its subsequent validation by a novel HPLC method in fluconazole tablets. Inertsil Octadecyl silica C18 column having dimensions of length 150 mm, diameter 4.6 mm, particle size 5 \ensuremath{\mu }m  was exploited for the method development, and the mobile phase embodies buffer pH 4 and acetonitrile in the combo of 40:60. Wavelength exploited for the segregation was 289 nm. The volume of injection was preserved at 20 \ensuremath{\mu}l. Average recovery established to be 101.571, 100.315, and 99.021, the standard deviation was established to be 0.93834, 0.986418, and 0.989741, and \% R.S.D was established to be 0.92382, 0.98331, and 0.999526. The percentage recovery was established to be within prescribed restrictions. In assay studies, an average assay was found to be 95.966, and standard deviation and \% RSD of the assay was found to be 0.6021 and 0.6274, respectively.  After carefull and systemic investigation of various research articles and review articles, it is observed that H.P.L.C method development and validation of fluconazole has been done in various pharmaceutical dosage forms, but till now a smaller amount of work is executed on dosage forms of solid nature, and hence there is huge prospective for research to be done in this area for new methodology to be developed in pharmaceutical dosage form of solid nature by means of dissimilar mobile phase combos and different strategy adopted by use of chromatography software which avoids the problems and demerits associated with present methods.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Fluconazole,\newline International Council for Harmonisation,\newline Linearity,\newline Precision,\newline Robustness
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Vikrant~Verma~\\ Phone:\ 917351096954~\\  Email:\ vijeetsingh84@rediffmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Fluconazole is 2-(2,4-difluorophenyl)-1,3- bis(1H-1,2,4-triazol-1-yl)-2-propanol, chemically an antifungal drug-containing triazole moiety that is revealed to be efficient in opposition to an extensive variety of superficial and fungal infections of systemic nature, administered both intravenous and oral administration \unskip~\citep{648611:15252063,648611:15252065}.

Fluconazole is azoles derivative inhibit the lanosine 14\ensuremath{\alpha }-demethylase, which is a fungal cytochrome P450 3A enzyme and is accountable for the transformation of lanosterol into chief sterol ergosterol, present in the cell membrane of fungi. The consequential diminution of ergosterol changes the fluidity of the membrane of the cell, and this thwarts the activity of enzymes linked with the membrane \unskip~\citep{648611:15252068}.

U.S. Food and Drug Administration has approved fluconazole for the effective treatment of certain fungal infections of systemic origin and is presently in clinical use in the United States and Europe as well as in clinical trials for unfolding its use in other applications. A gradually hike in systemic fungal infections associated with a growing population of immunosuppressed patients has increased its importance in the drug treatment of fungal infections and diseases \unskip~\citep{648611:15252052}. There are a small number of significant HPLC methods accessible for the evaluation of fluconazole in dosage forms of solid nature\ensuremath{_{}}and plasma \unskip~\citep{648611:15252057,648611:15252064,648611:15252055,648611:15252056,648611:15252062}.

The prime objective of current research is to develop a very less time taking, reproducible, precise novel and easy method and validate it according to the International Council for Harmonisation guidelines. There are so many demerits found in the current methods for the estimation of fluconazole by H.P.L.C in solid dosage form like high limit of quantitation, high limit of detection, long chromatographic run time, some methods are costly and time-consuming, some methods require complex techniques, and hence a new method is developed to avoid all the demerits in the present methods \unskip~\citep{648611:15252059,648611:15252058,648611:15252067,648611:15252053,648611:15252066}.
    
\section{Materials and Methods}
The reference standard of standard fluconazole was provided by the Jaagruti Pharmaceuticals situated in Jeedimetla, Hyderabad, India. The pharmaceutical solid dosage form fluconazole tablets, having brand name flucozole, was acquired from native pharmacy having a claim of 150 mg within the label, manufactured by company S.B Lifesciences, Panchkula, Haryana, India. All different chemicals exploited in the study are HPLC grade. Methyl alcohol and water, each of them HPLC grade, purchased from drug company rankem situated in Gurgaon, disodium hydrogen phosphate from fisher scientific and potassium dihydrogen phosphate also from fisher scientific.

\textbf{Instrumentation and the detail of the novel developed Method}

The HPLC organization consists of Agilent model 1220 infinity emboding gradient pump with flow domain from 0.2 ml/min to10 ml/min and C- 18 column Inertsiloctadecylsilane with dimensions of (150X4.6X5\ensuremath{\mu}) was utilized. The HPLC arrangement was inbuilt with EZChrom software that has a photometer having double-beam, amid deuterium lamp used as a light source and limit of wavelength was 190 nm to 600 nm with integrated degasser \unskip~\citep{648611:15252054}. The mobile phase exploited was a combo of a buffer having pH-4 along with acetonitrile in the scale of 40:60 with flow rate preserved at 1.3 ml/minute. The utilized wavelength for the segregation was 289 nanometer. The volume of injection was 20 \ensuremath{\mu}l, and the column was preserved at ambient temperature. Purification of the mobile phase was executed by passing through the micron filter having pore size 0.45 \ensuremath{\mu }m before use.

\textbf{Diluent Preparation}

Phosphate buffer having pH 4 mixed with acetonitrile in the proportion of 40:60 was used as a diluent. Diluent was then passed through a micron filter with pore size 0.45 \ensuremath{\mu }m preliminary to utilization.

\textbf{Preparation of Blank }

Diluent was utilized as a blank solution

\textbf{Procedure for preparation of Buffer (Phosphate Buffer having pH 4)}

Accurately 5.04 gram disodium hydrogen phosphate was mixed with 3.01 gram potassium dihydrogen phosphate in an affordable amount of water so as to make a thousand ml of the solution. The pH of the solution was adjusted with the assistance of glacial acetic acid \unskip~\citep{648611:15252060}.

\textbf{Selection of suitable wavelength }

Stock solution of the standard drug Fluconazole was made by mixing twenty milligrams of the standard drug Fluconazole  with 100 ml of methyl alcohol, which provides 200 \ensuremath{\mu}g/ml of the stock solution of standard. From this solution, 1ml was then withdrawn, and then dilution was executed with the facilitate of 10 ml of the methyl alcohol to acquire twenty \ensuremath{\mu}g /ml working solution of standard drug, which was then sonicated for fifteen minutes to get rid of all of the gases which are dissolved. UltraViolet spectra was obtained amid the scale from 200 nm to 400 nm utilizing methyl alcohol as a blank.

\textbf{ Selection of suitable Mobile phase }

Stock solution of the drug fluconazole used as standard was made by adding about twenty mg of the standard drug fluconazole into 100 ml diluent which gives 200 \ensuremath{\mu}g /ml of the stock solution of standard,then1ml was withdrawn and diluted to ten ml with diluent(diverse scale of mobile phase which are utilized for trials) to give twenty \ensuremath{\mu}g /ml working solution of standard, which was then injected for selection of diverse mobile phase combinations. A diverse mobile phase combo was utilized till the parameters of system suitability was acquired.

\textbf{Method Development and validation}

\textbf{Trials acquired throughout the development of method}

Numerous trials was executed throughout the development of a method, few significant trials are given in Figures~\ref{f-07ef618213d7}, \ref{f-b97841b09e55}, \ref{f-544edeebbc66}, \ref{f-b40ba1c7c66a}, \ref{f-104a4603ee77}, \ref{f-e6c8e9778a23} and~\ref{f-b7ebf53d06d3} .

\textbf{Method Linearity and Method Range}

Stock resolution of standard fluconazole was obtained by mixing twenty mg of standard fluconazole drug in 100 ml of diluent to give 200\ensuremath{\mu}g/ml of the stock solution of standard. Linearity was established in the concentration scale of ten \ensuremath{\mu}g /ml to thirty \ensuremath{\mu}g /ml. This concentration was obtained by mixing1.5ml,1.25ml, 1ml, 0.75ml and 0.5ml of fluconazole standard stock solution with facilitate of diluent to get concentrations of 10 \ensuremath{\mu}g/ml, 15 \ensuremath{\mu}g/ml, 20 \ensuremath{\mu}g/ml, 25 \ensuremath{\mu}g/ml and 30 \ensuremath{\mu}g/ml of Fluconazole for the determination of linearity studies.

\textbf{Precision Studies}

These studies was executed in 3 dissimilar styles as intraday and interday precision executed for 3 different concentrations lower 10 \ensuremath{\mu}g/ml, middle 20 \ensuremath{\mu}g/ml and higher 30 \ensuremath{\mu}g/ml at 3 times each for single day and alternate days and repeatability studies executed for only one middle concentration at six times.

\textbf{Studies on recovery}

These studies was executed by spiking sample formulation with the standard drug and afterward sorting out recovery at 80 \%,100 \%, and 120 \%. An exact quantity of the standard drug was mixed with sample formulation and then estimated recovered quantity from sample formulation as percentage recovery.

\textbf{Stock solution of standard drug and working standard preparation}

The stock solution of standard drug fluconazole was obtained by dissolving 20 mg of fluconazole standard drug in 100 ml of diluent to obtain 200 \ensuremath{\mu}g /ml stock solution, from which 1ml was taken and diluted it with 10 ml of diluent to obtain working solution of standard drug.

\textbf{Sample Stock Solution}

Accurately 20 tablets was weight and then powdered, tablet powder commensurate to 20 mg fluconazole was taken in\textbf{ a }volumetric flask of 100 ml capacity, and then 60 ml of diluent was added. The solution was then filtered through micron filter of pore size 0.45 \ensuremath{\mu }m after shaking for 15 minute and sonication for about 10 minutes then the volume was finetuned with methanol to obtain 200 \ensuremath{\mu}g/ml solution, from this 1ml was acquired and diluted it with 10 ml of diluent to obtain sample solution for recovery studies.

\textbf{Procedure for preparation of recovery solution}

This solution was made for standard by mixing 0.8 ml of standard Fluconazole,1 ml of standard fluconazole, and 1.2 ml of standard drug Fluconazole and after that volume makes upto 10 ml with facilitate of the diluent.For the sample recovery solution was made by mixing 1 ml from a stock solution of a sample, and then it was diluted with10 ml of diluent to get 20 \ensuremath{\mu}g/ml. Afterward, injected the standard solution and sample solution for recovery and dissimilar recovery solutions of concentrations 80 \%, 100 \%, and 120 \% and after that calculated percentage recovery. 

\textbf{Limit of detection and quantitation }

These values are acquired by substituting the values of standard deviation and slope in the mathematical formula given below.

 L.O.D=3.3 \ensuremath{\sigma  }/S L.O.Q=10 \ensuremath{\sigma  }/S

\textbf{Assay studies }

\textbf{Stock solution of drug fluconazole used as standard and working standard preparation}

The stock solution of standard drug fluconazole was acquired by dissolving 20 mg of fluconazole standard drug in 100 ml of diluent to acquire 200 \ensuremath{\mu}g /ml stock solution of standard drug fluconazole from which 1ml was taken and diluted with 10 ml of diluent to acquire working solution of standard drug.

\textbf{Procedure for working sample preparation}

Accurately 20 tablets was weight and then powdered, tablet powder commensurate to 20 mg fluconazole was taken in\textbf{\space }a volumetric flask having a capacity of 100 ml, and then 60 ml of diluent was added. The subsequent solution was then filtered with a micron filter of pore size 0.45 \ensuremath{\mu }m after shaking for 15 minutes and sonication for about 10 minutes, and then the volume was makeup with methyl alcohol to acquire 200 \ensuremath{\mu}g/ml solution. From this solution, 1ml was taken and diluted it with 10 ml of diluent to give working sample preparation; after that, both solutions was injected.

\textbf{Specificity studies }

Specificity was analyzed by examination of dissimilar chromatograms acquired for standard drug, sample formulation, and blank.

\textbf{Robustness Studies of drug fluconazole}

Robustness parameters was studied by injecting the working standard formulation for dissimilar flow rate, dissimilar pH, and dissimilar mobile phase combo. The flow rate was altered at 0.2 ml variation, percentage of solvent in the mobile phase at 2\% variation, and pH of the Buffer at variation of 0.2.
    
\section{Results and Discussion}
\textbf{Selection of appropriate mobile phase }

The concluding mobile phase combo chosen, which exhibits parameters of system suitability, embodies Phosphate buffer having pH 4.0 and acetonitrile in the proportion of 40-60 in standard drug fluconazole. Parameters of system suitability is given in Table~\ref{tw-08e48f978298} for the final trial 7.


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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/f4db5247-ff90-4356-9f8a-8d2861acfe1b-upicture2.png}{\includegraphics{images/f4db5247-ff90-4356-9f8a-8d2861acfe1b-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Trial executed with D.M.S.O-methanol (30-70)}}
\label{f-b97841b09e55}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/2d16d5f1-ad2b-4e9b-ad38-647f0c63ac05-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/2d16d5f1-ad2b-4e9b-ad38-647f0c63ac05-upicture3.png}{\includegraphics{images/2d16d5f1-ad2b-4e9b-ad38-647f0c63ac05-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Trial executed with Water-Methanol (20-80)}}
\label{f-544edeebbc66}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/71e3514a-db9f-429a-80a1-837efb5909bf-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/71e3514a-db9f-429a-80a1-837efb5909bf-upicture4.png}{\includegraphics{images/71e3514a-db9f-429a-80a1-837efb5909bf-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Trial executed with phosphate buffer having pH 4.0-methanol (60-40)}}
\label{f-b40ba1c7c66a}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/28aa285f-8b74-4ad5-aa50-1b37377d893c-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/28aa285f-8b74-4ad5-aa50-1b37377d893c-upicture5.png}{\includegraphics{images/28aa285f-8b74-4ad5-aa50-1b37377d893c-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Trial executed with phosphate buffer having pH 5.5-acetonitrile (30-70)}}
\label{f-104a4603ee77}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/c6811d7d-8425-4c24-8ecb-8769988bb6c7-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c6811d7d-8425-4c24-8ecb-8769988bb6c7-upicture6.png}{\includegraphics{images/c6811d7d-8425-4c24-8ecb-8769988bb6c7-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Trial executed with phosphate buffer having pH6.5-Acetonitrile (80-20)}}
\label{f-e6c8e9778a23}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/9a7300fa-7d77-417f-9101-37577e5948af-upicture7.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/9a7300fa-7d77-417f-9101-37577e5948af-upicture7.png}{\includegraphics{images/9a7300fa-7d77-417f-9101-37577e5948af-upicture7.png}}{}
\makeatother 
\caption{\boldmath {Trial executed with phosphate buffer having pH 4.0-Methanol (30-70)}}
\label{f-b7ebf53d06d3}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/69cb41b5-7ff3-4183-8dcd-442109e60f0e-upicture8.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/69cb41b5-7ff3-4183-8dcd-442109e60f0e-upicture8.png}{\includegraphics{images/69cb41b5-7ff3-4183-8dcd-442109e60f0e-upicture8.png}}{}
\makeatother 
\caption{\boldmath {Linearity graph of fluconazole}}
\label{f-d06c82ec7009}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/8cb30bde-fa76-4ec6-b90a-743f3f2e5fe0-upicture9.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/8cb30bde-fa76-4ec6-b90a-743f3f2e5fe0-upicture9.png}{\includegraphics{images/8cb30bde-fa76-4ec6-b90a-743f3f2e5fe0-upicture9.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram for linearity of drug Fluconazole}}
\label{f-8e037cbf34f5}
\end{figure}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Parameters for system suitability acquired for the final trial} }
\label{tw-08e48f978298}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.1778\linewidth-2\tabcolsep}p{\dimexpr.2416\linewidth-2\tabcolsep}p{\dimexpr.1806\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Retention time* & Height  & Area & Theoretical plate & Assymetry\\
\tblmidrule 
1.657 &
  493.141 &
  3423.24 &
  7740 &
  1.228\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*In minutes}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Linearity Data } }
\label{tw-05693fa39717}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.20083333333333332\linewidth-2\tabcolsep}p{\dimexpr.4071333333333333\linewidth-2\tabcolsep}p{\dimexpr.3920333333333333\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Concentration* & Area\\
\tblmidrule 
1 &
  10 &
  2436.791\\
2 &
  15 &
  2967.695\\
3 &
  20 &
  3576.669\\
4 &
  25 &
  4112.87\\
5 &
  30 &
  4701.921\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*Concentration in \ensuremath{\mu}g/ml}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Intraday Precision (10 \ensuremath{\mu}g/ml)} }
\label{tw-eb1b311c1e67}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.43010000000000005\linewidth-2\tabcolsep}p{\dimexpr.5699\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Standard & Area\\
\tblmidrule 
1 &
  2324.4\\
2 &
  2335.658\\
3 &
  2328.802\\
Average &
  2329.62\\
Standard deviation &
  5.673401449\\
\%RSD &
  0.243533342\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Intraday Precision (20 \ensuremath{\mu}g/ml)} }
\label{tw-0c6a7730e8f7}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.47580000000000005\linewidth-2\tabcolsep}p{\dimexpr.52419999999999995\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Standard & Area\\
\tblmidrule 
1 &
  3467.69\\
2 &
  3456.368\\
3 &
  3460.882\\
Average &
  3461.6466\\
Standard deviation &
  5.6996\\
\%RSD &
  0.1646\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Intraday Precision (30 \ensuremath{\mu}g /ml)} }
\label{tw-a6407498b7ac}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.4758\linewidth-2\tabcolsep}p{\dimexpr.5242\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Standard  & Area \\
\tblmidrule 
1 &
  4590.793\\
2 &
  4566.973\\
3 &
  4583.848\\
Average &
  4580.538\\
Standard deviation &
  12.25010918\\
\%RSD &
  0.267438218\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Interday Precision (10 \ensuremath{\mu}g/ml)} }
\label{tw-6057463d1279}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.42575\linewidth-2\tabcolsep}p{\dimexpr.57425\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Standard & Area\\
\tblmidrule 
1 &
  2317.623\\
2 &
  2295.277\\
3 &
  2306.229\\
Average &
  2306.376\\
Standard deviation &
  11.17373\\
\%RSD &
  0.484471\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Interday Precision (20 \ensuremath{\mu}g/ml)} }
\label{tw-915ba49788ff}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.4792\linewidth-2\tabcolsep}p{\dimexpr.5208\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Standard & Area\\
\tblmidrule 
1 &
  3476.815\\
2 &
  3497.355\\
3 &
  3474.376\\
Average &
  3482.849\\
Standard deviation &
  12.6219\\
\%RSD &
  0.362402\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Interday Precision (30 \ensuremath{\mu}g/ml)} }
\label{tw-2c12b33a8a26}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.4844\linewidth-2\tabcolsep}p{\dimexpr.5156\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Standard & Area\\
\tblmidrule 
1 &
  4585.015\\
2 &
  4608.77\\
3 &
  4578.073\\
Average &
  4590.619\\
Standard deviation &
  16.0976\\
\%RSD &
  0.350663\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Repeatability examination performed at 20 \ensuremath{\mu}g/ml} }
\label{tw-f973440d38ef}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.4706\linewidth-2\tabcolsep}p{\dimexpr.5294\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Standard & Area\\
\tblmidrule 
1 &
  3469.981\\
2 &
  3483.65\\
3 &
  3495.139\\
4 &
  3485.97\\
5 &
  3508.867\\
6 &
  3490.403\\
Average &
  3489.001667\\
Standard deviation &
  12.91417178\\
\%RSD &
  0.370139456\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Area of Sample and area of Standard along with spiked and net area} }
\label{tw-bf2199b6d68c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1695\linewidth-2\tabcolsep}p{\dimexpr.25540000000000003\linewidth-2\tabcolsep}p{\dimexpr.17509999999999998\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Spiking of standard & Area of sample spike with standard & Area of sample & Net area of standard & Area of standard\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.1695\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{80\%}} &
  3256.222 &
  2333.024 &
  923.198 &
  1139.535\\
 &
  3252.145 &
  2333.024 &
  919.121 &
  1139.535\\
 &
  3268.573 &
  2333.024 &
  935.549 &
  1139.535\\
\multicolumn{1}{p{\dimexpr(.1695\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{100\%}} &
  3483.841 &
  2333.024 &
  1150.817 &
  1139.535\\
 &
  3463.255 &
  2333.024 &
  1130.231 &
  1139.535\\
 &
  3481.372 &
  2333.024 &
  1148.348 &
  1139.535\\
\multicolumn{1}{p{\dimexpr(.1695\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{120\%}} &
  3699.043 &
  2333.024 &
  1366.019 &
  1139.535\\
 &
  3689.807 &
  2333.024 &
  1356.783 &
  1139.535\\
 &
  3672.39 &
  2333.024 &
  1339.366 &
  1139.535\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Percentage Recovery, Average, Standard Deviation, and Percentage relative standard deviation } }
\label{tw-6244250916f7}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.12690000000000001\linewidth-2\tabcolsep}p{\dimexpr.14879999999999999\linewidth-2\tabcolsep}p{\dimexpr.1668\linewidth-2\tabcolsep}p{\dimexpr.14070000000000002\linewidth-2\tabcolsep}p{\dimexpr.1463\linewidth-2\tabcolsep}p{\dimexpr.1512\linewidth-2\tabcolsep}p{\dimexpr.1193\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Sample amount added \mbox{}\protect\newline (\ensuremath{\mu}g/ml) & \cAlignHack Standard \mbox{}\protect\newline amount \mbox{}\protect\newline added \mbox{}\protect\newline ( \ensuremath{\mu}g \mbox{}\protect\newline /ml) & \cAlignHack Standard \mbox{}\protect\newline amount \mbox{}\protect\newline recovered \mbox{}\protect\newline ( \ensuremath{\mu}g \mbox{}\protect\newline /ml) & \%Recovery & \cAlignHack Average & Standard Deviation & \%R.S.D\\
\tblmidrule 
10 &
  8 &
  8.1015 &
  101.269 &
  101.571 &
  0.93834 &
  0.92382\\
10 &
  8 &
  8.0657 &
  100.821 &
   &
   &
  \\
10 &
  8 &
  8.2099 &
  102.623 &
   &
   &
  \\
10 &
  10 &
  10.0990 &
  100.990 &
  100.315 &
  0.98641 &
  0.98331\\
10 &
  10 &
  9.9183 &
  99.183 &
   &
   &
  \\
10 &
  10 &
  10.0773 &
  100.773 &
   &
   &
  \\
10 &
  12 &
  11.9875 &
  99.895 &
  99.021 &
  0.98974 &
  0.99952\\
10 &
  12 &
  11.9064 &
  99.220 &
   &
   &
  \\
10 &
  12 &
  11.7536 &
  97.946 &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Area of Samples, percentage assay and area of standard along with average assay, standard Deviation and percentage relative standard deviation of assay} }
\label{tw-61c7fa1c9a7f}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.12254999999999999\linewidth-2\tabcolsep}p{\dimexpr.32065\linewidth-2\tabcolsep}p{\dimexpr.29155\linewidth-2\tabcolsep}p{\dimexpr.26525\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Area of Samples & \% Assay & Area of standard\\
\tblmidrule 
1. &
  3432.55 &
  95.96926568 &
  \multicolumn{1}{p{\dimexpr(.26525\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{3576.718}}\\
2. &
  3453.959 &
  96.56783118 &
  \\
3. &
  3410.886 &
  95.36357074 &
  \\
 &
  Average assay &
  95.9668892 &
  \\
 &
  Standard deviation &
  0.602133738 &
  \\
 &
  \% RSD of assay &
  0.62743905 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Label claim of a tablet, percentage assay, average of percentage assay, standard deviation and percentage relative standard deviation along with the result } }
\label{tw-a3575d20e9ae}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.138\linewidth-2\tabcolsep}p{\dimexpr.142\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Serial no & Label claim & Result &  \%Assay & Average \% assay & \%Standard \mbox{}\protect\newline Deviation & \%RSD\\
\tblmidrule 
1 &
  150 &
  143.9538 &
  95.9692 &
  \multicolumn{1}{p{\dimexpr(.14\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{95.9668}} &
  \multicolumn{1}{p{\dimexpr(.14\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{0.6021}} &
  \multicolumn{1}{p{\dimexpr(.16\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{0.6274}}\\
2 &
  150 &
  144.8517 &
  96.5678 &
   &
   &
  \\
3 &
  150 &
  143.0453 &
  95.3635 &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Increase and decrease in Flow Rate} }
\label{tw-b97380470d2e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2897\linewidth-2\tabcolsep}p{\dimexpr.2501\linewidth-2\tabcolsep}p{\dimexpr.2413\linewidth-2\tabcolsep}p{\dimexpr.2189\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.53980000000000004\linewidth-2\tabcolsep)}}{\cAlignHack Flow Rate of 1.5ml/min} & \multicolumn{2}{p{\dimexpr(.46019999999999996\linewidth-2\tabcolsep)}}{\cAlignHack Flow Rate of 1.1ml/min}\\
\rowcolor{kwdboxcolor}S.no & Area & S.no & Area\\
\tblmidrule 
1 &
  3434.003 &
  1 &
  3486.739\\
2 &
  3451.865 &
  2 &
  3499.579\\
3 &
  3433.858 &
  3 &
  3493.494\\
Average &
  3439.909 &
  Average &
  3493.271\\
Standard deviation &
  10.35474 &
  Standard deviation &
  6.422913\\
\%RSD &
  0.301018 &
  \%RSD &
  0.183865\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Effect of variation in the ratio of Mobile Phase} }
\label{tw-e860fe8f1889}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.28800000000000004\linewidth-2\tabcolsep}p{\dimexpr.21199999999999996\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.50\linewidth-2\tabcolsep)}}{\cAlignHack At Mobile Phase (42:58)} & \multicolumn{2}{p{\dimexpr(.50\linewidth-2\tabcolsep)}}{\cAlignHack  At Mobile Phase (38:62)}\\
\rowcolor{kwdboxcolor}S.no & Area & S.no & Area\\
\tblmidrule 
1 &
  3409.068 &
  \multicolumn{1}{p{\dimexpr(.28800000000000004\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{1 \mbox{}\protect\newline 2 \mbox{}\protect\newline 3 \mbox{}\protect\newline Average \mbox{}\protect\newline Standard deviation \mbox{}\protect\newline \%RSD}} &
  \multicolumn{1}{p{\dimexpr(.21199999999999996\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{3512.125 \mbox{}\protect\newline 3522.16 \mbox{}\protect\newline 3496.091 \mbox{}\protect\newline 3510.125 \mbox{}\protect\newline 13.14904 \mbox{}\protect\newline 0.374603}}\\
2 &
  3422.375 &
   &
  \\
 3 &
  3393.491 &
   &
  \\
Average &
  3408.311 &
   &
  \\
Standard deviation &
  14.45686 &
   &
  \\
\%RSD &
  0.424165 &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Effect of the increase and decrease in pH } }
\label{tw-f88c790c5bd8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3054\linewidth-2\tabcolsep}p{\dimexpr.2396\linewidth-2\tabcolsep}p{\dimexpr.2448\linewidth-2\tabcolsep}p{\dimexpr.2102\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.545\linewidth-2\tabcolsep)}}{\cAlignHack  At increase pH value by 0.2} & \multicolumn{2}{p{\dimexpr(.455\linewidth-2\tabcolsep)}}{\cAlignHack At decrease pH value by 0.2}\\
\rowcolor{kwdboxcolor}S.no & Area & S.no & Area\\
\tblmidrule 
1 &
  3474.519 &
  1 &
  3445.274\\
2 &
  3490.516 &
  2 &
  3427.228\\
3 &
  3483.64 &
  3 &
  3438.356\\
Average &
  3482.892 &
  Average &
  3436.953\\
Standard deviation &
  8.024712 &
  Standard deviation &
  9.104479\\
\%RSD &
  0.230404 &
  \%RSD &
  0.2649\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/787d963b-3d4b-47d6-9b4e-6ba63fc0ae91-upicture10.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/787d963b-3d4b-47d6-9b4e-6ba63fc0ae91-upicture10.png}{\includegraphics{images/787d963b-3d4b-47d6-9b4e-6ba63fc0ae91-upicture10.png}}{}
\makeatother 
\caption{\boldmath {Recovery chromatogram acquired for the sample }}
\label{f-5f99372c5ea8}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/cd2f3761-30e0-4550-aa1d-5c1dc2866a62-upicture11.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/cd2f3761-30e0-4550-aa1d-5c1dc2866a62-upicture11.png}{\includegraphics{images/cd2f3761-30e0-4550-aa1d-5c1dc2866a62-upicture11.png}}{}
\makeatother 
\caption{\boldmath {Recovery chromatogram acquired for the standard }}
\label{f-deb722e6ae88}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/8703ca8d-c733-48b0-a98a-04c73dc4236a-upicture12.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/8703ca8d-c733-48b0-a98a-04c73dc4236a-upicture12.png}{\includegraphics{images/8703ca8d-c733-48b0-a98a-04c73dc4236a-upicture12.png}}{}
\makeatother 
\caption{\boldmath {Sample formulation chromatogram acquired for the first determination }}
\label{f-46a547f57aad}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/6dcdcc86-47e6-41a4-8875-e28b33d73b0a-upicture13.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/6dcdcc86-47e6-41a4-8875-e28b33d73b0a-upicture13.png}{\includegraphics{images/6dcdcc86-47e6-41a4-8875-e28b33d73b0a-upicture13.png}}{}
\makeatother 
\caption{\boldmath {Sample formulation chromatogram acquired for the second determination }}
\label{f-f980de5096d9}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/a5355dbd-8d91-42c5-ac0f-2f509d4d7435-upicture14.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/a5355dbd-8d91-42c5-ac0f-2f509d4d7435-upicture14.png}{\includegraphics{images/a5355dbd-8d91-42c5-ac0f-2f509d4d7435-upicture14.png}}{}
\makeatother 
\caption{\boldmath {Sample formulation chromatogram acquired for third determination}}
\label{f-a7e2727c87ad}
\end{figure}
\egroup

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\fixFloatSize{images/a5a212d3-ae3f-452a-bfc5-b30d8bda3342-upicture15.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/a5a212d3-ae3f-452a-bfc5-b30d8bda3342-upicture15.png}{\includegraphics{images/a5a212d3-ae3f-452a-bfc5-b30d8bda3342-upicture15.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram acquired for the standard drug}}
\label{f-eca9a9bceb2e}
\end{figure}
\egroup

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\fixFloatSize{images/5198b0f0-9661-4f22-9bc4-0d53aaa1806b-upicture16.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/5198b0f0-9661-4f22-9bc4-0d53aaa1806b-upicture16.png}{\includegraphics{images/5198b0f0-9661-4f22-9bc4-0d53aaa1806b-upicture16.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram for method specificity acquired for sample}}
\label{f-a2e657d1d6ab}
\end{figure}
\egroup

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\fixFloatSize{images/00840507-e34f-4f3d-b140-0553ce10c8de-upicture17.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/00840507-e34f-4f3d-b140-0553ce10c8de-upicture17.png}{\includegraphics{images/00840507-e34f-4f3d-b140-0553ce10c8de-upicture17.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram for method specificity acquired for blank }}
\label{f-630505c128c1}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/e5a9f478-adfd-48d4-82a9-2a4850b1c169-upicture18.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/e5a9f478-adfd-48d4-82a9-2a4850b1c169-upicture18.png}{\includegraphics{images/e5a9f478-adfd-48d4-82a9-2a4850b1c169-upicture18.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram for method specificity acquired for standard}}
\label{f-fafb5bd444c4}
\end{figure}
\egroup
\textbf{Linearity studies of drug fluconazole }

Linearity graph and linearity chromatogram for linearity are given in Figure~\ref{f-d06c82ec7009}, and Figure~\ref{f-8e037cbf34f5}, respectively, and Table~\ref{tw-05693fa39717} shows data of linearity, which gives a relationship between concentration vs. Area. The new developed method be established to demonstrate linearity in the range of 10 \ensuremath{\mu}g /ml to 30 \ensuremath{\mu}g /ml, respectively.\textbf{\space }

\textbf{Precision examination of the Fluconazole }

Precision examination was executed, and the method was established to be precise in all precision parameters with percentage relative standard deviation conforming to the prescribed limits. Intraday precision data is given in Tables~\ref{tw-eb1b311c1e67} and~\ref{tw-0c6a7730e8f7} and Table~\ref{tw-a6407498b7ac}.

\textbf{Intraday Precision}

Standard area, average of area, standard deviation and percentage relative standard deviation at  10, 20 and 30 \ensuremath{\mu}g/ml designed for intraday precision studies are given in Tables~\ref{tw-eb1b311c1e67} and~\ref{tw-0c6a7730e8f7} and Table~\ref{tw-a6407498b7ac}.

\textbf{Interday Precision}

Standard area and its average, standard deviation and percentage relative standard deviation at 10 ,20 and 30 \ensuremath{\mu}g/ml concentration for interday precision studies are given in Tables~\ref{tw-6057463d1279} and~\ref{tw-915ba49788ff} and Table~\ref{tw-2c12b33a8a26}.

\textbf{Repeatability }\textbf{examination} \textbf{\space }

The results of repeatability examination are presented in Table~\ref{tw-f973440d38ef}, consisting of a standard area and its average, standard deviation, and percentage relative standard deviation performed at middle concentration  20 \ensuremath{\mu}g/ml. The method was established to be precise.

\textbf{Recovery Studies}

Recovery studies was executed with average recovery established as 101.571, 100.315, and 99.021, and standard deviation established as 0.93834, 0.986418, and 0.989741, and the percentage relative standard deviation was established as 0.92382, 0.98331 and 0.999526. Percentage recovery was established to be within prescribed limits. Recovery chromatogram acquired for the sample is given in Figure~\ref{f-5f99372c5ea8}, and the recovery chromatogram acquired for the standard is given in Figure~\ref{f-deb722e6ae88}, and results are given in Table~\ref{tw-bf2199b6d68c} and Table~\ref{tw-6244250916f7}. 

\textbf{Limit of detection and quantitation }

The limit of detection and quantitation was established to be 0.0080 and 0.0051. 

\textbf{Assay studies }

The results of the assay studies presented in Table~\ref{tw-61c7fa1c9a7f} which gives Area of Samples, percentage assay and area of standard along with average assay, percentage relative standard deviation and standard deviation of assay and Table~\ref{tw-a3575d20e9ae} which shows claim of label of tablet, percentage assay and its average, standard deviation and percentage relative standard deviation along with result and Figures~\ref{f-46a547f57aad}, \ref{f-f980de5096d9} and~\ref{f-a7e2727c87ad} shows sample formulation chromatogram acquired for all three determination of sample and Figure~\ref{f-eca9a9bceb2e} shows Chromatogram acquired for the drug used as standard. \textbf{\space }

\textbf{Specificity }

To analyze whether the method is specific or not, three individual chromatograms are acquired for the standard drug, sample formulation, and the blank, and there was no intrusion from impurities and excipients observed the new method which is developed. Chromatograms for standard drug, sample formulation, and blank are given in Figures~\ref{f-a2e657d1d6ab}, \ref{f-630505c128c1} and~\ref{f-fafb5bd444c4}.

\textbf{Robustness results of fluconazole }

A detailed robustness analysis was executed, and the results was found satisfactory, which given the evidence that the method which was developed is robust. Data regarding the variation of flow rate by increasing or decreasing by 0.2 are given in Table~\ref{tw-b97380470d2e} . Table~\ref{tw-e860fe8f1889}  shows the outcome of change in the composition of the mobile phase by 2\%, and Table~\ref{tw-f88c790c5bd8} shows the effect of alteration in pH.
    
\section{Conclusions}
A novel easy, specific, less time-wasting method was developed for the drug fluconazole. This method can be efficiently used in pharmaceutical industries. Several trials was executed with the help of various combinations of mobile phase till the parameters of system suitability are met. There was no interference found in the developed method from the excepients and degradation products. The developed method was observed precise in all dimensions of precision examination. Final selection of mobile phase from which the method was developed embodies phosphate buffer having pH 4 and acetonitrile in the combination of 40:60. This developed method is reproducible, giving rapid results. 

The percentage relative standard deviation of the various validation parameters was found to be within the prescribed, defined limits. Insignificant dissimilarity in the pH of the mobile phase, rate of flow, and the constitution of the mobile phase has not effected the execution of the method. 
\section*{Acknowledgement}The authors extend their appreciation to the Dean of kharvel subharti college of pharmacy for providing research facilities for the research work.
    

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