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\begin{document}


\def\RunningAuthor{Gowtham Reddy Cheruku et al.}
\firstPage{2286}
\articleType{Original Article}
\receivedDate{06 Jul 2021}
\acceptedDate{10 Aug 2021}
\revisedDate{05 Aug 2021}
\journalVolume{2021, 12}
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\title{\textbf{UV Spectrophotometric Method Development and Validation of Esomeprazole in Bulk and Pharmaceutical Dosage Forms}}
\author{Gowtham~Reddy~Cheruku\textsuperscript{*},  
            Sai~Laasya~Mithinti, 
            Purushotham~Saidu~\\[5pt]{Department of Pharmaceutical Sciences, Shri Vishnu College of Pharmacy, Bhimavaram, West Godavari, Andhra Pradesh, India}}

\begin{abstract}
The work discusses method development and validation. An uncomplicated, accurate, and straightforward method was developed for the drug Esomeprazole in bulk as well as Pharmaceutical dosage form. NaOH was used as the solvent. The maximum wavelength (\ensuremath{\lambda} max) for Esomeprazole was found to be 305nm. 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). Esomeprazole's recovery percentage (\%) was 100.20\%, respectively. Linearity for Esomeprazole was observed between 5-25\ensuremath{\mu}g/ml, respectively. Regression equation y=0.0407x-0.0122, regression coefficient (r\ensuremath{^2}) is 0.9963 for Esomeprazole. Inter day and intraday precision were checked, \% relative standard deviation values were less than 2. The regression equations were used to derive the Limit of Detection (LOD) and Limit of Quantification (LOQ) values. LOD value was found to be 0.734 \ensuremath{\mu}g/mL and LOQ value was 2.224 \ensuremath{\mu}g/mL for Esomeprazole. The assay of the marketed formulation was performed, which was 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}Esomeprazole,\newline Sodium hydroxide (NAOH),\newline UV Spectrophotometer,\newline Sonicator,\newline Electronic Balance
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Gowtham~Reddy~Cheruku~\\ Phone:\ ~\\  Email:\ cherukugowthamreddy769@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
 Esomeprazole is a drug, which diminishes stomach acid (HCL)\unskip~\citep{1203533:23115691} and it is purchased by many people under the brand name Amaryl among others. It is used for the treatment of diseases like Gastroesophageal reflux disease (GRD), peptic ulcers, and Zollinger{\textendash}Ellison syndrome\unskip~\citep{1203533:23115689}. Potency is akin to other proton pump inhibitors (PPIs)\unskip~\citep{1203533:23115696} like Pantoprazole, Esomeprazole, Omeprazole. Mode of administration is through oral or injection into a vein (intravenous). Esomeprazole is the (\textit{S})-isomer of omeprazole. Its mechanism of action is by blocking gastric hydrogen potassium ATPase in the parietal cells of the stomach then the production of HCl will be declined\unskip~\citep{1203533:23115699}. 

In 2007, a medical utility license was granted for the drug that was patented in 1993\unskip~\citep{1203533:23115695}. Generic versions of it are available and sold over the counter in several countries\unskip~\citep{1203533:23115690}. More than 7 million prescriptions were filled for it in 2018, making it the 103rd most frequently prescribed medication in the United States\unskip~\citep{1203533:23115700,1203533:23115694}. United States residents also have access to it without a prescription\unskip~\citep{1203533:23115698}. 

To date, there are various methods to detect Esomeprazole like RP-HPLC method\unskip~\citep{1203533:23115697}, Stability indicating methods\unskip~\citep{1203533:23115692}, Planar chromatography\unskip~\citep{1203533:23115693}, High Performance Thin Layer Chromatography (HPTLC). But, there have been no studies with NaOH using the UV-Spectrophotometric method for the unmasking of Esomeprazole in pharmaceutical formulations. This paper is concerned with developing and validating a new way to quantify Esomeprazole by UV-spectroscopy in tablet preparation (Figure~\ref{f-295c5926eb89}).

UV spectrum (U.V. spectroscopy or UV/Vis) encompasses absorption and reflection spectroscopy 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. 
    
\section{Materials and Methods}
\textbf{Chemicals and reagents}

Esomeprazole working standard powder was given by Metrochem API Private Limited, Hyderabad, India and was used without further purification. The Esomeprazole tablets were bought from a local pharmacy, Bhimavaram. Each tablet contains 40mg of Esomeprazole. Standard NaOH 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 Digital 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 0.1M N} \textit{\textbf{a}} \textbf{OH solution}

It was precisely weighed (4g of NaOH) and transferred into a 1000ml volumetric flask. Then a small amount of water was added to the volumetric flask and sonicated for a few minutes to dissolve the remaining NaOH completely. Later, the NaOH solution was made up to 1000ml with the required amount of distilled water. 

\textbf{Preparation of standard stock solution}

The stock solution of Esomeprazole was prepared by accurately weighing 100 mg of pure drug and was transferred into a 100ml volumetric flask. A small amount of 0.1M NaOH 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 0.1M NaOH to give a concentration of 1000\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 in the range of 5-25 \ensuremath{\mu}g/ml, where the beer's law was obeyed. The dilutions are 5 \ensuremath{\mu}g/ml, 10 \ensuremath{\mu}g/ml, 15 \ensuremath{\mu}g/ml, 20 \ensuremath{\mu}g/ml, 25 \ensuremath{\mu}g/ml respectively.

\textbf{Preparation of sample stock solution}

Ten tablets of Esomeprazole were weighed and crushed and mixed in a mortar and pestle into fine powder. Each tablet was having a content of Esomeprazole equivalent to 40mg. 100mg of powder was taken and dropped into 100ml volumetric flasks and 10ml of 0.1M NaOH 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 0.1M NaOH solution was added to make up to 100ml. A nylon filter measuring 0.45 meters in diameter was used to filter acceptable diluents of the solution. 

\textbf{Assay}

A mass of not less than 10 tablets was prepared by grinding them to a fine, similar particle 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 25\ensuremath{\mu}g/ml solution was obtained serially with the same. The absorbance was measured against the blank 0.1M NaOH. The readings were taken in triplicate.
\begin{eqnarray*}\begin{array}{l}\%assay\;=\left(\frac{mean\;test\;absorbance}{mean\;std\;absorbance}\right)\times\left(\frac{dilution\;of\;s\tan dard\;}{dilution\;of\;test}\right)\\\;\;\;\;\;\;\;\;\;\;\;\;\;\;\times\left(\frac{mean\;test\;weight}{label\;claim}\right)\times100\end{array} \end{eqnarray*}
\textbf{Method validation}

\textbf{Accuracy}

Accuracy test was performed at three different concentration levels of 80\%, 100\% 120\%, with three replicates at each percentage.

Percentage Accuracy (\%) = 
\begin{eqnarray*}\frac{observed\;concentration}{nominal\;concentration\;}\times100 \end{eqnarray*}
\textbf{Precision}

\textit{\textbf{\space }}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 with has same concentration analysed thrice in a day and \% RSD was calculated.

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

Limits of detection (LOD) and limit of quantitation (LOQ) were estimated per ICH guidelines. For this study, the standard deviation of responses was computed based on the slope of the calibration curve and the standard deviation of response.

\textbf{Linearity}

In order to construct the calibration curves, five concentrations of Esomeprazole of different potency were used (5-25 \ensuremath{\mu}g/mL). Plotting the absorbance of Esomeprazole against the concentration allows calibration curves to be constructed. Based on the least square method, linear regression analysis was performed to evaluate the linearity.
    
\section{Results and Discussion}

\bgroup
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\centering \makeatletter\IfFileExists{images/41a1c1b0-7978-4b0d-8dd6-e137bf19b889-upicture1.png}{\includegraphics{images/41a1c1b0-7978-4b0d-8dd6-e137bf19b889-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Structure of Esomeprazole }}
\label{f-295c5926eb89}
\end{figure}
\egroup

\bgroup
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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/190ebf04-b194-4c33-bcfe-8b0d5f6216a5-upicture2.png}{\includegraphics{images/190ebf04-b194-4c33-bcfe-8b0d5f6216a5-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Standard graph of Esomeprazole}}
\label{f-1993cb4f5b5d}
\end{figure}
\egroup

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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/bc32840d-54bf-4878-9dfb-4c10dc380926-upicture3.png}{\includegraphics{images/bc32840d-54bf-4878-9dfb-4c10dc380926-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Linearity graph of Esomeprazole}}
\label{f-343a64d9b578}
\end{figure}
\egroup
From the reported literature, there were few methods established for the determination of Esomeprazole in individual, combination and combination with other drugs. 

It was concluded that there was no method reported for the estimation of the drug (Esomeprazole) individually using NaOH as a solvent, which promoted the pursuit of the present work. The scope and objective of the current work is to develop a method, to validate a simple, sensitive and rapid analytical method for the estimation of the drug Esomeprazole by using UV spectroscopy. 


\begin{table*}[!htbp]
\caption{\boldmath {Accuracy of Esomeprazole} }
\label{tw-9da7dda36606}
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\tbltoprule \rowcolor{kwdboxcolor}Concentration \mbox{}\protect\newline  & \multicolumn{2}{p{\dimexpr(.3253\linewidth-2\tabcolsep)}}{\cAlignHack Amount added (\ensuremath{\mu }g/ml) } & Amount  \mbox{}\protect\newline Found (\ensuremath{\mu }g/ml) & \% Recovery\\
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\multicolumn{1}{p{\dimexpr(.2183\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{80\%}} &
  20 &
  25 &
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  20 &
  25 &
  19.49 &
  97.46\\
 &
  20 &
  25 &
  20.06 &
  100.28\\
\multicolumn{1}{p{\dimexpr(.2183\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{100\%}} &
  25 &
  25 &
  24.90 &
  99.59\\
 &
  25 &
  25 &
  25.68 &
  102.73\\
 &
  25 &
  25 &
  25.44 &
  101.75\\
\multicolumn{1}{p{\dimexpr(.2183\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{120\%}} &
  30 &
  25 &
  29.93 &
  99.78\\
 &
  30 &
  25 &
  29.82 &
  99.94\\
 &
  30 &
  25 &
  30.47 &
  101.58\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Inter day precision of Esomeprazole} }
\label{tw-995f323ad44b}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.41919999999999995\linewidth-2\tabcolsep}p{\dimexpr.274\linewidth-2\tabcolsep}p{\dimexpr.3068\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Concentration(\ensuremath{\mu}g/ml) & Day 1 & Day 2\\
\tblmidrule 
15 &
   0.5972  &
  0.596\\
15 &
  0.6123 &
  0.6139\\
15 &
  0.6146 &
  0.6135\\
15 &
  0.6137 &
  0.6182\\
15 &
  0.6233 &
  0.6202\\
Average &
  0.6122 &
  0.6124\\
SD &
  0.0094 &
  0.0096\\
\%RSD &
  1.541 &
  1.564\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Average \% RSD: 1.553}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Intraday precision of Esomeprazole} }
\label{tw-fd55a99626a9}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2377\linewidth-2\tabcolsep}p{\dimexpr.2457\linewidth-2\tabcolsep}p{\dimexpr.25219999999999995\linewidth-2\tabcolsep}p{\dimexpr.2644\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Concentration (\ensuremath{\mu}g/ml) & Absorbance 1 log (Io/I)  & Absorbance 2 log (Io/I)  & Absorbance 3  log (Io/I)\\
\tblmidrule 
15 &
  \cAlignHack 0.5972 &
  \cAlignHack 0.5992 &
  \cAlignHack 0.5974\\
15 &
  \cAlignHack 0.6123 &
  \cAlignHack 0.613 &
  \cAlignHack 0.6129\\
15 &
  \cAlignHack 0.6146 &
  \cAlignHack 0.6166 &
  \cAlignHack 0.6132\\
15 &
  \cAlignHack 0.6137 &
  \cAlignHack 0.6138 &
  \cAlignHack 0.6145\\
15 &
  \cAlignHack 0.6233 &
  \cAlignHack 0.6226 &
  \cAlignHack 0.6225\\
Average &
  \cAlignHack 0.6122 &
  \cAlignHack 0.6130 &
  \cAlignHack 0.6121\\
SD &
  \cAlignHack 0.0094 &
  \cAlignHack 0.008605 &
  \cAlignHack 0.009109\\
\%RSD &
  \cAlignHack 1.541 &
  \cAlignHack 1.403 &
  \cAlignHack 1.488\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Average \% RSD: 1.478}
\end{tablenotes}
\end{threeparttable}

\end{table*}
The analysis was carried out at 305 nm for Esomeprazole, respectively. The correlation coefficient (r\ensuremath{^{2}}) was found to be remarkable. The drug esomeprazole showed linearity between 5-25\ensuremath{\mu}g/ml, respectively. The method was validated by Accuracy, precision, LOD and LOQ, Linearity. The precision and Accuracy of the enhanced procedure was studied. The \%RSD values for precision and Accuracy were observed to be within the admissible limit, which revealed that the developed method was precise. LOD and LOQ were within the acceptable range. The results indicate a satisfactory method for the drug Esomeprazole (Figure~\ref{f-1993cb4f5b5d}). 

\textbf{Validation}

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

\textbf{Accuracy}

Accuracy test was performed at three concentration levels of 80\%,100\%,120\%, i.e., 24, 30.0, 36.0\ensuremath{\mu }g/ml solutions for UV with three replicates at each level in which the amount of sample was kept constant, i.e., 30 \ensuremath{\mu }g/ml in UV. The percentage recovery is calculated for all the 9 readings were found to be 100.20\%. The results were presented in Table~\ref{tw-9da7dda36606}.


\begin{table}[!htbp]
\caption{\boldmath {Linearity of Esomeprazole} }
\label{tw-779e9bf0ad55}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{LL}
\tbltoprule \rowcolor{kwdboxcolor}Concentration (\ensuremath{\mu}g/ml) & Absorbance  log (Io/I) \\
\tblmidrule 
5 &
  0.1702\\
10 &
  0.4086\\
15 &
  0.6136\\
20 &
  0.8165\\
25 &
  0.9844\\
\tblbottomrule 
\end{tabulary}\par 
\end{table}

\begin{table}[!htbp]
\caption{\boldmath {Assay of the Formulation} }
\label{tw-ed9b76c4b028}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{LLL}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(\mcWidth{1}+\mcWidth{2}+\mcWidth{3})}}{\cAlignHack Absorbance for sample solution}\\
\rowcolor{kwdboxcolor}S.NO & Concentration & Absorbance log (Io/I) \\
\tblmidrule 
1 &
  25\ensuremath{\mu}g/ml &
  0.9543\\
2 &
  25\ensuremath{\mu}g/ml &
  0.9542\\
3 &
  25\ensuremath{\mu}g/ml &
  0.9543\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{ Average: 0.9544}
\end{tablenotes}
\end{threeparttable}

\end{table}
\textbf{Precision (Reproducibility)}

Precision for the method was calculated by evaluating intra-day and inter-day variations. In the inter-day variation study, the solutions of the same concentration (15\ensuremath{\mu }g/ml) were prepared and analyzed twice for two consecutive days, and the absorbance was recorded. In the intra-day variation study, five discrete solutions of the same concentration (15 \ensuremath{\mu }g/ml) were prepared and analyzed thrice in a day (morning, afternoon, and evening). The \% RSD of inter-day precision was found to be 1.553. The \% RSD of intraday precision was found to be 1.478. The results are viewed in Tables~\ref{tw-995f323ad44b} and~\ref{tw-fd55a99626a9}.

\textbf{Linearity}

Plotting the absorbance of Esomeprazole versus the concentration of the drug yielded the linearity of the calibration curve. The correlation coefficient of regression \textit{r}\ensuremath{^{2}} = 0.9963 over a concentration range (5 to 25 \ensuremath{\mu}g/ml), the representative linear regression equation for Esomeprazole is \textit{Y} = 0.0407x-0.0122 as shown in the Figure~\ref{f-343a64d9b578} and the corresponding results are given in Table~\ref{tw-779e9bf0ad55}.

\textbf{Limits of detection and quantification }

The LOD AND LOQ can be determined by the method as per ICH guidelines. We base our method on the standard deviation of response and slope of the calibration curve in this study. The limit of detection was observed to be 0.7338 \ensuremath{\mu}g/ml. The limit of quantification was observed to be 2.224 \ensuremath{\mu}g/ml. 

\textbf{Dosage estimation for the main drug in bulk and tablet form (Assay)}

\textit{\textbf{\space }}Esomeprazole solutions were prepared using bulk drug and tablet dosage forms and analyzed at the same conditions with 100.5\% accuracy. The results are viewed in Table~\ref{tw-ed9b76c4b028}.
    
\section{Conclusions}
 A rapid, precise, user friendly and reproducible UV-Spectrophotometric Method for estimation of Esomeprazole in bulk and its tablet pharmaceutical dosage forms was developed and validated as per ICH Guidelines. Additionally, the LOQ and LOD measurements were developed as part of this method's further development. The validity of the method and results obtained by this method are relatively reliable, which indicates the validity of the method. It can be applied both in the private and academic sectors to estimate Esomeprazole.
\section*{Acknowledgement} The authors express 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{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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