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\def\RunningAuthor{Bhavani N L D et al.}
\firstPage{2291}
\articleType{Original Article}
\receivedDate{01 Jul 2021}
\acceptedDate{10 Aug 2021}
\revisedDate{06 Aug 2021}
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\title{\textbf{Development and Validation of UV Spectrophotometric Method of Mesalamine Using Hydrotropic Solubilizing Agents}}
\author{Bhavani~N~L~D\textsuperscript{*},  
            Gowtham~Reddy~Cheruku,  
            Bhargava~Sri~Harsha~Polina,  
            Dheeraj~Kotagiri, 
            Jnanendra~Kumar~Korukollu~\\[5pt]{Department of Pharmaceutical Analysis, Shri Vishnu College of Pharmacy, Bhimavaram, Andhra Pradesh, India}}

\begin{abstract}
The work was proposed to discuss method development and validation of the drug Mesalamine by using hydrotropic solubilizing agents. An uncomplicated, accurate, and precise method was developed for the drug Mesalamine in bulk as well as Pharmaceutical dosage form. 5M Urea was used as the hydrotropic solubilizing agent to enhance the solubility of the drug. The maximum wavelength (\ensuremath{\lambda} max) for Mesalamine was found to be 241nm. The validation was performed as per International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines for Accuracy, linearity, precision, Limit of detection (LOD) and Limit of quantification (LOQ). Percentage recovery (\%) of Mesalamine was ascertained to be between 95 to 98\%. Linearity for Mesalamine was observed between 2-10 \ensuremath{\mu}g/ml. Regression equation y=0.0571x-0.0186, regression coefficient (r\ensuremath{^2}) is 0.9996 for Mesalamine. Inter day and intraday precision were checked, \% relative standard deviation values were less than 2 for both the methods. Limit of detection (LOD) and Limit of quantification (LOQ) values were derived using regression equations. LOD value was found to be 0.55 \ensuremath{\mu}g/ml. LOQ value was found to be 1.67 \ensuremath{\mu}g/ml. The assay of the marketed formulation was performed and the results of the assay were obtained by the proposed method. The results are in between 98-102\%. So, the method developed was simple and economical that can be adopted for routine tests. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Hydrotropic Solubilization,\newline Mesalamine,\newline Citric acid,\newline UV spectrophotometer,\newline Electronic balance,\newline Sonicator
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Bhavani~N~L~D~\\ Phone:\ 9581286017~\\  Email:\ bhavani.n@svcp.edu.in
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Mesalamine is a pharmaceutical acclimated to cure severe diseases like inflammatory bowel disease (IBD), ulcerative colitis and Crohn's disease \unskip~\citep{1194132:23033554}. The primary use of this drug is treating mild to moderately severe diseases.\ensuremath{^{}}The mode of administration is through an oral or buccal cavity or rectally.\ensuremath{^{}} The formulations which are taken through the oral cavity appear to be similarly effective \unskip~\citep{1194132:23033553}. Certain formulations can trigger an allergic reaction in individuals with a sulfa or sulfonamide allergy. The mechanism of action is by direct contact with the intestine. It is the active moiety of sulfasalazine and sulfapyridine and are metabolites \unskip~\citep{1194132:23033549}. It is also the vital part or constituent of the prodrug balsalazide along with the inert carrier molecule 4-aminobenzoyl-beta-alanine \unskip~\citep{1194132:23033551}. Common side effects are throat irritation, nausea, cough, inflammation of the tissue lining the sinuses, headache, gas, burping. Mesalamine is also referred to as Mesalazine or 5-aminosalicylic acid (5-ASA). Figure~\ref{f-c71ecdd19372}

From 1987, Mesalamine was accepted for the treatment of various diseases in the United States \unskip~\citep{1194132:23033558}. Despite its generic status, it is accessible across the globe \unskip~\citep{1194132:23033554} and purchased by many individuals as a medicament under diverse brands. The drug ranked 246th in the United States in 2017, with 1.7 million prescriptions written \unskip~\citep{1194132:23033562,1194132:23033554,1194132:23033560}. 

To date, there are various methods to detect Mesalamine like RP-UHPLC method \unskip~\citep{1194132:23033559}, Spectroscopic methods \unskip~\citep{1194132:23033557}, Vireodt's method \unskip~\citep{1194132:23033550}, TLC Densitometric methods \unskip~\citep{1194132:23033555}. Hydrotropic solubilizing agents have not been studied using the UV-Spectrophotometric method to unmask Mesalamine in pharmaceutical formulations. The purpose of the current paper was to develop and validate an improved method for quantifying Mesalamine in tablet formulation using UV-Spectroscopic analysis. 

UV spectrum (U.V. spectroscopy or UV/Vis) encompasses absorption and reflection spectroscopy that exists in the ultraviolet and adjacent visible regions of the electromagnetic spectrum. This means it utilizes the light source in the visible and adjacent ranges. When the absorption or reflectance of chemicals is seen first-hand, it determines the perceived colour. It is during this region of the spectrum that atoms and molecules transition into electronic states. There is an interdependency between absorption and fluorescence spectroscopy because fluorescence measures transition out of the excited state to the ground state and absorption measures transition into the excited state \unskip~\citep{1194132:23033556}. 

In hydrotropy, alkali metal salts of various organic acids are dissolved in a solution with quite high concentrations of other alkali metals. The term was coined by Carl Neuberg in 1932. Although the term has been used in the literature to refer to compounds that don't form micelles, whether solids or liquids, but are capable of dissolving insoluble compounds. As the name suggests, hydrotropes are compounds capable of dissolving hydrophobic compounds in aqueous solutions by means other than micellar solubilization. It is common for hydrotropes to be composed of both a hydrophilic and hydrophobic portion (alike surfactants), but the hydrophobic portion may be too small to cause immediate self-aggregation. Hydrotropes do not have a critical concentration above which self-aggregation occurs (unlike micelle- and vesicle-forming surfactants, which have critical micelle concentrations (CMC) and critical vesicle concentrations (CVC)). The majority of hydrotropes aggrandize in a step-by-step process, gradually widening the aggregation. Hydrotropes do not seem to self-aggregate, except when a solubilate has been added. The following examples of hydrotropes may be considered: urea, tosylate, adenosine triphosphate (ATP), cumene sulfonate and xylene sulfonate. 
    
\section{Materials and Methods}
\textbf{Chemicals and reagents}

Mesalamine working standard powder was given by Lasa Supergenerics Limited, Mumbai, India and was used without any additional purification. The Mesalamine tablets were bought from a local pharmacy, Bhimavaram. Each tablet contains 400mg of Mesalamine. 

Standard urea was purchased from the laboratory of chemicals, Shri Vishnu college of pharmacy, Bhimavaram, Andhra Pradesh, India. All the chemicals used in the procedure were sorted according to their grade and listed as received. We processed all solutions with distilled water purified by reverse osmosis and filtered through a milli-Q system.

\textbf{Instrumentation}

Different instruments were used to carry out the present work, such as Electronic balance (Make-Adair Dutt), UV-Spectrophotometer (make- PG instruments, single beam UV-Visible Spectrophotometer, Model no- T60, Software- UV Win5 software v5.0.5), Sonicator (Make- Sonica ultrasonic cleaner).

\textbf{Preparation of 5M Urea solution}

Accurate weight of 30 grams of urea was transferred into a 100mL volumetric flask. Then a small amount of water was added to the volumetric flask and sonicated for a few minutes to dissolve the remaining urea completely. Later, the urea solution was made up to 100ml with the required amount of distilled water. 

\textbf{Preparation of standard stock solution}

The Standard stock solution of Mesalamine was prepared by accurately weighing 10 mg of pure drug and was transferred into a 100ml volumetric flask. A small amount of 5M urea solution was added and sonicated for a few minutes to dissolve the remaining drug completely. 

Later, the drug solution was made up to 100ml by adding the remaining amount of 5M urea solution to give a concentration of 100\ensuremath{\mu}g/ml, which was used for the further dilutions.

\textbf{Preparation of working standard solution}

From the stock solution, several dilutions were made by using distilled water in the range of 2-10 \ensuremath{\mu}g/ml, where the beer's law was obeyed. The dilutions are 2 \ensuremath{\mu}g/ml, 4 \ensuremath{\mu}g/ml, 6 \ensuremath{\mu}g/ml, 8 \ensuremath{\mu}g/ml, 10 \ensuremath{\mu}g/ml respectively.

\textbf{Preparation of sample stock solution}

Ten tablets of Mesalamine were weighed and compressed, and assorted in a mortar and pestle into filmy powder. Each tablet was having a content of Mesalamine equivalent to 400 mg. 10mg of powder was taken and dropped into 100ml volumetric flasks, and 10ml of 5M urea solution was added to the flask. The volumetric flasks were sonicated for 10 to 15 min to effect complete dissolution of the drug, and later the remaining 90ml of 5M urea solution was added to make up to 100ml. Suitable diluents of solution were filtered through a 0.45 \ensuremath{\mu}m nylon filter. 

\textbf{Assay}

A mass of not less than 10 tablets was prepared by grinding them to a fine, similar fragment size powder using a mortar and pestle. A compound with a weight equal to the 10 mg tablet was carried out quantitatively by weighing into a volumetric flask and transferring it to a 100 mL volumetric flask based on the weight estimate. Approximately 100 ml of solvent was added, the solution was sonicated for 10 min, then diluted till 10\ensuremath{\mu}g/ml solution was obtained serially with the same. The absorbance was measured against the blank 5M urea solution: water. The readings were taken in triplicate.

\% assay = (mean test absorbance/mean std absorbance) * (dilution of standard /dilution of test)

* (mean test weight/label claim) * 100. LOD and LOQ were within limits. The results indicate a satisfactory method for the drug Mesalamine.

\textbf{Method validation}

\textbf{Accuracy}

The accuracy test was performed at three different concentration levels of 80\%, 100\% 120\%, with three replicates at each percentage.
\begin{eqnarray*}\begin{array}{l}Percentage\;Accuracy\;(\%)=\\observed\;concentration/nominal\;\\concentration\times100\end{array} \end{eqnarray*}
\textbf{Precision}

It was demonstrated by interday and intraday studies. In interday, the solution of the same concentration was analysed twice for two consecutive days. In intraday, the solution has the same concentration analysed thrice in a day and \% RSD was calculated.

\textbf{Detection and quantitation}\textbf{ of} \textbf{ limits (sensitivity)}

Limits of detection (LOD) and limit of quantitation (LOQ) were estimated as per ICH guidelines. The method used in this project is based on a standard deviation of response and the slope of the calibration curve. 

\textbf{Linearity}

There were five different concentrations of Mesalamine in the calibration curves, ranging from 2 to 10 g/ml. Mesalamine absorbance was plotted against concentration to construct calibration curves. Using the least-squares method, linear regression analysis was performed to determine the linearity.
    
\section{Results and Discussion}
From the reported literature, there were few methods established for the determination of Mesalamine in individual, combination and combination with other drugs. 


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\caption{\boldmath {Structure of Mesalamine}}
\label{f-c71ecdd19372}
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\caption{\boldmath {Standard graph of Mesalamine}}
\label{f-ee232d6d045f}
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\caption{\boldmath {Linearity graph of Mesalamine}}
\label{f-23fe79f30c54}
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\caption{\boldmath {Accuracy of Mesalamine} }
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  \cAlignHack 95.69\%\\
 &
  \cAlignHack 8 &
  \cAlignHack 10 &
  \cAlignHack 17.47 &
  \cAlignHack 97.06\%\\
 &
  \cAlignHack 8 &
  \cAlignHack 10 &
  \cAlignHack 17.52 &
  \cAlignHack 97.35\%\\
\multicolumn{1}{p{\dimexpr(.1933\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 100\%}} &
  \cAlignHack 10 &
  \cAlignHack 10 &
  \cAlignHack 19.25 &
  \cAlignHack 96.28\%\\
 &
  \cAlignHack 10 &
  \cAlignHack 10 &
  \cAlignHack 19.30 &
  \cAlignHack 96.54\%\\
 &
  \cAlignHack 10 &
  \cAlignHack 10 &
  \cAlignHack 19.30 &
  \cAlignHack 96.54\%\\
\multicolumn{1}{p{\dimexpr(.1933\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\cAlignHack 120\%}} &
  \cAlignHack 12 &
  \cAlignHack 10 &
  \cAlignHack 21.14 &
  \cAlignHack 96.13\%\\
 &
  \cAlignHack 12 &
  \cAlignHack 10 &
  \cAlignHack 21.20 &
  \cAlignHack 96.37\%\\
 &
  \cAlignHack 12 &
  \cAlignHack 10 &
  \cAlignHack 21.18 &
  \cAlignHack 96.29\%\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Interday precision of Mesalamine} }
\label{tw-13fd742f2e64}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.38599999999999994\linewidth-2\tabcolsep}p{\dimexpr.27189999999999998\linewidth-2\tabcolsep}p{\dimexpr.3421\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Concentration (\ensuremath{\mu}g/ml) & \cAlignHack Day 1 & \cAlignHack Day 2\\
\tblmidrule 
\cAlignHack 6 &
  \cAlignHack 0.342 &
  \cAlignHack 0.335\\
\cAlignHack 6 &
  \cAlignHack 0.331 &
  \cAlignHack 0.347\\
\cAlignHack 6 &
  \cAlignHack 0.334 &
  \cAlignHack 0.345\\
\cAlignHack 6 &
  \cAlignHack 0.333 &
  \cAlignHack 0.334\\
\cAlignHack 6 &
  \cAlignHack 0.343 &
  \cAlignHack 0.337\\
\cAlignHack 6 &
  \cAlignHack 0.337 &
  \cAlignHack 0.330\\
\cAlignHack Average &
  \cAlignHack 0.336 &
  \cAlignHack 0.338\\
\cAlignHack SD &
  \cAlignHack 0.00449 &
  \cAlignHack 0.00605\\
\cAlignHack \% RSD &
  \cAlignHack 1.3071 &
  \cAlignHack 1.7751\\
\cAlignHack Average \% RSD &
  \multicolumn{2}{p{\dimexpr(.614\linewidth-2\tabcolsep)}}{\cAlignHack 1.5411}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Intraday precision of Mesalamine} }
\label{tw-162b6bb3bdff}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2293\linewidth-2\tabcolsep}p{\dimexpr.26240000000000002\linewidth-2\tabcolsep}p{\dimexpr.2541\linewidth-2\tabcolsep}p{\dimexpr.2542\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Concentration (\ensuremath{\mu}g/ml) & \cAlignHack Absorbance 1 \mbox{}\protect\newline log (I\ensuremath{_{o}}/I)  & \cAlignHack Absorbance 2 \mbox{}\protect\newline log (I\ensuremath{_{o}}/I)  & \cAlignHack Absorbance 3 \mbox{}\protect\newline log (I\ensuremath{_{o}}/I) \\
\tblmidrule 
\cAlignHack 6 &
  \cAlignHack 0.336 &
  \cAlignHack 0.342 &
  \cAlignHack 0.345\\
\cAlignHack 6 &
  \cAlignHack 0.322 &
  \cAlignHack 0.331 &
  \cAlignHack 0.338\\
\cAlignHack 6 &
  \cAlignHack 0.329 &
  \cAlignHack 0.334 &
  \cAlignHack 0.335\\
\cAlignHack 6 &
  \cAlignHack 0.327 &
  \cAlignHack 0.333 &
  \cAlignHack 0.342\\
\cAlignHack 6 &
  \cAlignHack 0.331 &
  \cAlignHack 0.343 &
  \cAlignHack 0.341\\
\cAlignHack 6 &
  \cAlignHack 0.330 &
  \cAlignHack 0.337 &
  \cAlignHack 0.346\\
\cAlignHack Average &
  \cAlignHack 0.329 &
  \cAlignHack 0.336 &
  \cAlignHack 0.341\\
\cAlignHack SD &
  \cAlignHack 0.00422 &
  \cAlignHack 1.3071 &
  \cAlignHack 0.0038\\
\cAlignHack \% RSD &
  \cAlignHack 1.2762 &
  \cAlignHack 1.3071 &
  \cAlignHack 1.1140\\
\cAlignHack Average \% RSD &
  \multicolumn{3}{p{\dimexpr(.7707000000000001\linewidth-2\tabcolsep)}}{\cAlignHack 1.2324}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Linearity of Mesalamine} }
\label{tw-e8bc207e140a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.471\linewidth-2\tabcolsep}p{\dimexpr.529\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Concentration (\ensuremath{\mu}g/ml) & \cAlignHack Absorbance log (I\ensuremath{_{o}}/I) \\
\tblmidrule 
\cAlignHack 2 &
  \cAlignHack 0.100\\
\cAlignHack 4 &
  \cAlignHack 0.206\\
\cAlignHack 6 &
  \cAlignHack 0.321\\
\cAlignHack 8 &
  \cAlignHack 0.438\\
\cAlignHack 10 &
  \cAlignHack 0.555\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Assay of the formulation} }
\label{tw-f37e07940497}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.20759999999999998\linewidth-2\tabcolsep}p{\dimexpr.3881\linewidth-2\tabcolsep}p{\dimexpr.4043\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \multicolumn{2}{p{\dimexpr(.7924000000000001\linewidth-2\tabcolsep)}}{\cAlignHack The absorbance of sample solution}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack Concentration & \cAlignHack  Absorbance log (I\ensuremath{_{o}}/I) \\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 10\ensuremath{\mu}g/ml &
  \cAlignHack 0.563\\
\cAlignHack 2 &
  \cAlignHack 10\ensuremath{\mu}g/ml &
  \cAlignHack 0.565\\
\cAlignHack 3 &
  \cAlignHack 10\ensuremath{\mu}g/ml &
  \cAlignHack 0.563\\
\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Average: 0.564}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
It was concluded that there was no method reported for the estimation of the drug (Mesalamine) individually using Hydrotropic solubilizing agents and 5M urea solution as a solvent, which promoted the pursuit of the present work. The scope and objective of the present work is to develop a method and to validate a simple, sensitive and rapid analytical method for the estimation of the drug Mesalamine by using UV spectroscopy.

The analysis was carried out at 241nm for Mesalamine, respectively. The correlation coefficient (r\ensuremath{^{2}}) was found to be remarkable. The drug Mesalamine showed linearity between 2-10\ensuremath{\mu}g/ml respectively. The method was validated by Accuracy, precision, LOD and LOQ and Linearity. The precision and Accuracy of the enhanced procedure was studied. The \%RSD values for precision and accuracy were found to be within the acceptable limit, which revealed that the developed method was precise. Figure~\ref{f-ee232d6d045f}

\textbf{Validation}

The method was validated with respect to parameters including accuracy and precision, linearity, LOD and LOQ.

\textbf{Accuracy}

Accuracy test was performed at three different concentration levels of 80\%,100\%,120\%, i.e., 8, 10, 12\ensuremath{\mu }g/ml solutions for UV with three replicates at each level in which the amount of sample was kept constant, i.e., 10 \ensuremath{\mu }g/ml in UV. The percentage recovery is calculated for all the 9 readings were found to be 96.47\%. The results are shown in Table~\ref{tw-e04aa41131bf}.

\textbf{Precision (Reproducibility)}

A study of intra-day and inter-day variation was conducted to determine the precision of the method. As part of the inter-day variation experiment, the same concentration solutions (6\ensuremath{\mu }g/ml) were prepared and analyzed twice on consecutive days, and their absorbance measurements were taken. The intra-day variation study included the preparation of five separate solutions at the same concentration (6*g/ml) and its analysis three times in the day (morning, afternoon, and evening). The \% RSD of inter-day precision was found to be 1.5411. The \% RSD of intraday precision was found to be 1.2324. The results are viewed in Table~\ref{tw-13fd742f2e64} and Table~\ref{tw-162b6bb3bdff}.

\textbf{Linearity}

Using the Absorbance of Mesalamine and its concentration as a calibration curve, a linear calibration curve for Mesalamine was calculated and constructed. The correlation coefficient of regression \textit{r}\ensuremath{^{2}} = 0.9996 over a concentration range (2 to 10\ensuremath{\mu}g/ml), the representative linear regression equation for Mesalamine is \textit{Y} = 0.0571x-0.0186 as shown in Figure~\ref{f-23fe79f30c54}, and the corresponding results given in Table~\ref{tw-e8bc207e140a}.

\textbf{Limits of detection and quantification}

In accordance with ICH guidelines, LOD and LOQ may be determined. A slope of the calibration curve and standard deviation of response are used in this project to measure the results of the measurement. The limit of detection (LOD) was found to be 0.55\ensuremath{\mu}g/ml. The limit of quantification (LOQ) was found to be 1.67\ensuremath{\mu}g/ml.

\textbf{Estimation of the main drug in bulk and tablet dosage form (Assay)}

Using the same experimental conditions and dose forms of the bulk drug and the tablet dosage form, three solutions of Mesalamine were made and analyzed. An average concentration of 101.7\% was observed. The results are viewed in Table~\ref{tw-f37e07940497}.
    
\section{Conclusion}
In accordance with the ICH Guidelines, a rapid, precise, user-friendly and reproducible ultraviolet spectrophotometer was developed to estimate mesalamine in bulk and in its tablet pharmaceutical dosage forms. To maximize the use of our method, we developed the Limit of detection (LOD) and Limit of Quantification (LOQ) measurements. Consequently, this method appears to be valid, and the results obtained by this method are fairly trustworthy. For estimating Mesalamine, this method can be used by industries and academic institutions. 
\section*{Acknowledgement}The authors are expressing sincere thanks and appreciation to Shri Vishnu College of Pharmacy for funding this research work to publish in the journal. The authors extend thanks to the Management of Shri Vishnu College of Pharmacy, Bhimavaram, for their cooperation in the present 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.
    

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