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\def\RunningAuthor{Vaishali Pardeshi et al.}
\firstPage{411}
\articleType{Original Article}
\receivedDate{20 Sep 2022}
\acceptedDate{17 Nov 2022}
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\journalVolume{2022, 13}
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\journalDoi{ijrps.v13i4.3939}
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\title{Development and Validation of Simple UV Spectrophotometric Method for the Determination of Pretomanid}
\author{Vaishali~Pardeshi\textsuperscript{*}\textsuperscript{\href{https://orcid.org/0000-0001-6757-1543}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}},  
            Tushar~Lokhande\textsuperscript{\href{https://orcid.org/0000-0002-0735-3082}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}}, 
            Ashwini~Shelke\textsuperscript{\href{https://orcid.org/0000-0001-8120-730X}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}}~\\[5pt]{Department of Pharmaceutical Chemistry, Mahatma Gandhi Vidyamandir's Pharmacy College, Nashik, Maharashtra, India}}

\begin{abstract}
In order to treat multidrug resistant TB, pretomanid, a nitroimidazooxazine antimycobacterial agent, is used with other antituberculosis medications. There is no technique for its analysis that uses spectroscopy, HPLC or HPTLC. Since a UV spectrophotometric approach for Pretomanid analysis must be developed. Utilizing a Shimadzu UV-2600, a quick, accurate, straightforward, and affordable UV spectrophotometric approach has been devised to determine Pretomanid. Solvent made of methanol to assess the bulk Pretomanid concentration. The detection process was placed at a wavelength of 321 nm. The parameters linearity, accuracy, precision, ruggedness, and robustness were taken into consideration during method validation in accordance with ICH Q2R1 criteria, as well as LOD and LOQ. It demonstrated linearity in the 10{\textendash}30(g/mL) range at a predetermined max of 321 nm, and had a strong correlation coefficient (R2-0.997) and outstanding mean recovery (99.00{\textendash}100.07\%). In terms of intraday and interday precision, Pretomanid's\% RSD was discovered to be 0.6366 and 0.666, respectively. Pretomanid identification using this approach was effective. The method's linearity, accuracy, repeatability, and reproducibility were statistically and empirically verified. The outcomes demonstrated the method's applicability for both routine examinations of protomanid bulks and industrial formulations. The suggested UV-Vis Spectrophotometric approach was verified in accordance with ICH requirements and found to be simple, accurate, precise and quick for the determination of Pretomanid.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Pretomanid,\newline UV Spectrophotometric method,\newline Process validation,\newline ICH guidelines
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Vaishali~Pardeshi~\\ Phone:\ 09156807993~\\  Email:\ vaishali.pardeshi12@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Pretomanid, a nitroimidazooxazine antimycobacterial agent, is used in conjunction with other antituberculosis medications to treat multidrug-resistant TB. \unskip~\cite{1589508:26450428} Pretomanid is (6S)-2-nitro-6-[[4-(trifluoromethoxy) phenyl] methoxy] in its chemical form. -6,7-dihydro-5H-imidazo[2,1-b] \unskip~\cite{1589508:26450425,1589508:26450424} oxazine as shown in Figure~\ref{f-0a1cd27ec1d4}\unskip~\cite{1589508:26450425,1589508:26450423}. In August 2019, the US Food and Drug Administration authorised Pretomanid. As part of a bedaquiline, pretomanid, and linezolid (BPaL) combination, this orally active medicine has been approved to treat people with pulmonary severe drug tuberculosis (TB) \unskip~\cite{1589508:26450424,1589508:26450427}. Mycolic acid production is inhibited by pretomanid. This results in faulty cell wall construction, which eventually leads to bacterial cell death. It eradicates M. tuberculosis bacteria that are reproducing and those that are not. \unskip~\cite{1589508:26450422} For its analysis, no spectroscopic, HPLC, or HPTLC methods are available. Since there is a need to establish a Pretomanid analytical technique. This project's goal was to develop a precise, specific, repeatable, and accurate UV approach for the analysis of Pretomanid. The developed method was examined for linearity, accuracy, precision, robustness, ruggedness, LOD, and LOQ in accordance with ICH guidelines. 


\bgroup
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\centering \makeatletter\IfFileExists{images/6a52e404-d91b-4dae-afa6-ca123d6ad7b2-upicture1.png}{\includegraphics{images/6a52e404-d91b-4dae-afa6-ca123d6ad7b2-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Pretomanid}}
\label{f-0a1cd27ec1d4}
\end{figure}
\egroup
It is branded as Pretomanid tablets 200mg and is available in tablet form. Methanol, DMSO (Dimethyl Sulfoxide), and ethanol are all solubalising solvents for it. Pretomanid is a prodrug that is activated by the Ddn nitroreductase enzyme in the body, resulting in a variety of active metabolites that are responsible for the drug's additional therapeutic effects, including nitric oxide induction. The reduced cofactor F420 is necessary for the pretomanid-activating nitroreductase enzyme, which is deazaflavin-dependent. The enzyme glucose-6-phosphate dehydrogenase reduces F420. \unskip~\cite{1589508:26450426} The pretomanid's imidazole ring is reduced at the C-3 position to produce the metabolites, one of which is a des-nitro derivative. Increased amounts of nitric oxide result from the synthesis of this derivative, which has bactericidal properties under anaerobic circumstances due to its function as a bacterial respiratory toxin.\unskip~\cite{1589508:26450419} Bactericidal activity against anaerobes has been linked to a reduction in antibiotic treatment duration. In a pharmacokinetic investigation of healthy people, the elimination half-life was shown to be 16.9-17.4 hours. A half-life of 18 hours is reported in an FDA briefing paper. \unskip~\cite{1589508:26450421}
    
\section{Materials and Methods}
Pretomanid was received as a gift sample from Mylan, Hyderabad. Analytical grade Ethyl acetate, methanol and n-hexane were bought from Qualigen (India) Ltd., Mumbai, India. 

All other chemicals come from S.D. Fine Chemical Ltd. in Worli, India, and are of analytical quality. And for all of the experimental work, volumetric glassware of class A grade was employed. 

The UV spectrophotometric method was used on a SCHIMADZU-2600 Double Beam UV-Visible Spectrophotometer with two 10 mm matched quartz cells.

\textbf{Solvent Selection }

Various solvents such as methanol, ethanol, toluene, petroleum ether, acetonitrile, chloroform, n-hexane and acetone were tried for the estimation of Pretomanid. Methanol was selected as the solvent for the analysis of Pretomanid due to its easy availability, better solubility and cost-effectiveness, 

\textbf{Standard Stock Solution Preparation }

Pretomanid was precisely weighed at 5 mg and then added to the 50 ml volumetric flask. Prepared up to 50 ml with the same substance after being dissolved in a minimum amount of methanol to achieve a concentration of 100 g/ml (100 ppm). After being diluted with methanol, 1 ml of the stock solution was added to a 10 ml volumetric flask. 10 g/ ml of the dilution was found to be present.

\textbf{Determination of Maximum Absorption Wavelength}

Using methanol as a blank, a 10 \ensuremath{\mu}g/ ml concentration solution was scanned between 200 and 400 nm, \ensuremath{\lambda }max was determined to be 321 nm from the UV spectra and was chosen as the analytical wavelength. Figure~\ref{f-09ddbca32fae} depicted the UV spectrum of Pretomanid. 


\bgroup
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\centering \makeatletter\IfFileExists{images/caa7a079-c643-415a-8648-49eff9745a44-upicture2.png}{\includegraphics{images/caa7a079-c643-415a-8648-49eff9745a44-upicture2.png}}{}
\makeatother 
\caption{\boldmath {UV spectra showing Absorption Maxima (\ensuremath{\lambda } max) of Pretomanid}}
\label{f-09ddbca32fae}
\end{figure}
\egroup
\textbf{Preparation of Calibration Graph }

In this procedure, a series of eight 10 ml volumetric flasks (10 - 24 \ensuremath{\mu}g/ml) were filled to capacity with methanol using aliquots of Pretomanid stock solution containing 100 \ensuremath{\mu}g /ml and ranging in size from 1.2 to 2.4 ml. 

At 321 nm, the absorbance of various concentrations of liquids was measured in comparison to a blank. By graphing concentration vs absorbance, the calibration curve was created. At 321 nm, it was discovered that the sample was linear with a concentration range of 10 - 30\ensuremath{\mu}g/ ml.

\textbf{Validation of Developed UV Spectrophotometric Method}

\textbf{Linearity }

Between concentration and absorbance, a calibration graph was drawn. Pretomanid was linear at 321 nm, with a concentration range of 10 to 24 g/ml. The assay's limit of detection (LOD) and limit of quantitation (LOQ) were also determined \unskip~\cite{1589508:26450420}.

\textbf{Precision }

The precision of the developed method was determined by intra-day and inter-day variation studies using only three concentrations of Pretomanid (10,14,18 ppm) 9 times. Intra-day studies and inter-day studies were determined by analyzing three sample solutions of concentrations 10,14,18. The mean, standard deviation and \% RSD were determined \unskip~\cite{1589508:26450420}

\textbf{Accuracy (Recovery Studies)}

In this investigation, a blend of pure Pretomanid and common excipients was used. Calculations were made using the label claim and the typical final product weight. The admixture was diluted using the same method as before to achieve three concentrations: 80\%, 100\%, and 120\% of the reference solution. By incorporating a known quantity of the sample solution into the standard stock solution, the investigation was conducted \unskip~\cite{1589508:26450420}.

\textbf{LOD and} \textbf{LOQ }

The LOD and LOQ were estimated from formulas 3.3\ensuremath{\sigma  }/S and 10\ensuremath{\sigma  }/S, respectively. Where, \ensuremath{\sigma  } = the standard deviation of y-intercepts of regression lines S = the slope of the calibration curve \unskip~\cite{1589508:26450420}.

\textbf{Ruggedness}

Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions. The method's ruggedness was established by the determination of Pretomanid by change the analyst. The data were then subjected to statistical analysis and the results are expressed in mean, standard deviation and \% RSD. \unskip~\cite{1589508:26450420}

\textbf{Robustness}

To determine the robustness of the method, absorbance was taken at wavelength change applied as \ensuremath{\pm}2 nm. The data were subjected to statistical analysis and the findings are shown as mean, standard deviation, and \%RSD. \unskip~\cite{1589508:26450420}
    
\section{Result and Discussion}
\textbf{Development and Validation of Method}

Pretomanid is mainly insoluble in water but readily soluble in organic solvents like methanol and DMSO. Methanol utilised as the diluent resulted in a more favourable UV analysis result. The 321 nm maximum absorption wavelength (max) was predetermined.

\textbf{Method Validation}

\textbf{Linearity }

The calibration curve revealed linearity in the 10-24 g/ml (Table~\ref{tw-711e988b8daa}) range with a regression value of 0.997. The equation y = 0.0192+0.038 was obtained using linear regression of absorbance on concentration and has a correlation value (r) of 0.9974 (Figure~\ref{f-93c899c851a9}). The maximum wavelength, or detecting wavelength, is 321 nm.


\bgroup
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\makeatother 
\caption{\boldmath {Calibration Curve of Pretomanid}}
\label{f-93c899c851a9}
\end{figure}
\egroup

\begin{table}[!htbp]
\caption{\boldmath {Calibration Parameters} }
\label{tw-711e988b8daa}
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\end{table}
\textbf{Accuracy (Recovery Studies)}

The percent RSD and percentage recovery were computed. The mean percentage recovery and percent RSD were determined to be within acceptable ranges, both less than 2, indicating that the current study work is correct in its Pretomanid technique development. Calculations were made to determine the mean, standard deviation, and percentage relative standard deviation (\%RSD). Table~\ref{tw-0092f474f52c} presented the outcomes.


\begin{table*}[!htbp]
\caption{\boldmath {Recovery study} }
\label{tw-0092f474f52c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1393\linewidth-2\tabcolsep}p{\dimexpr.18759999999999998\linewidth-2\tabcolsep}p{\dimexpr.19740000000000002\linewidth-2\tabcolsep}p{\dimexpr.1476\linewidth-2\tabcolsep}p{\dimexpr.15849999999999998\linewidth-2\tabcolsep}p{\dimexpr.1696\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Drug & \cAlignHack Initial amount \mbox{}\protect\newline Standard drug (100ppm) \% & \cAlignHack Amount added (200ppm) & \cAlignHack Amount found & \cAlignHack Recovery (\%) & \cAlignHack \% RSD (n = 3)\\
\tblmidrule 
\cAlignHack  &
  \cAlignHack 80\%(10ppm) &
  \cAlignHack 8ppm &
  \cAlignHack 18.06 &
  \cAlignHack 99.4 &
  \cAlignHack 0.3\\
\cAlignHack Pretomanid &
  \cAlignHack 100\%(10ppm) &
  \cAlignHack 10ppm &
  \cAlignHack 19.9 &
  \cAlignHack 99.7 &
  \cAlignHack 0.4\\
\cAlignHack  &
  \cAlignHack 120\%(10ppm) &
  \cAlignHack 12ppm &
  \cAlignHack 21.6 &
  \cAlignHack 98.4 &
  \cAlignHack 0.2\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Precision} }
\label{tw-5cd14c977cc2}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1157\linewidth-2\tabcolsep}p{\dimexpr.1126\linewidth-2\tabcolsep}p{\dimexpr.18922500000000007\linewidth-2\tabcolsep}p{\dimexpr.09388750000000002\linewidth-2\tabcolsep}p{\dimexpr.14368750000000007\linewidth-2\tabcolsep}p{\dimexpr.12910000000000007\linewidth-2\tabcolsep}p{\dimexpr.08670000000000003\linewidth-2\tabcolsep}p{\dimexpr.12910000000000004\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Drug & \cAlignHack  & \multicolumn{3}{p{\dimexpr(.42680000000000014\linewidth-2\tabcolsep)}}{\cAlignHack Intra {\textendash} Day} & \multicolumn{3}{p{\dimexpr(.34490000000000016\linewidth-2\tabcolsep)}}{\cAlignHack Inter {\textendash} Day}\\
\rowcolor{kwdboxcolor} & \cAlignHack Conc. (\ensuremath{\mu}g/ml) & \cAlignHack Absorbance measured \mbox{}\protect\newline Mean \ensuremath{\pm} SD \%  & \cAlignHack RSD & \cAlignHack Average  \mbox{}\protect\newline Potency \% & \cAlignHack Mean \ensuremath{\pm} SD  & \cAlignHack RSD & \cAlignHack Average  \mbox{}\protect\newline Potency \%\\
\tblmidrule 
 &
  \cAlignHack 14 &
  \cAlignHack 0.319\ensuremath{\pm} 0.004 &
  \cAlignHack 0.14 &
  \cAlignHack 104 &
  \cAlignHack 0.315\ensuremath{\pm}0.002 &
  \cAlignHack 0.7 &
  \cAlignHack 103.4\\
Pretomanid &
  \cAlignHack 18 &
  \cAlignHack 0.384\ensuremath{\pm} 0.004 &
  \cAlignHack 1.07 &
  \cAlignHack 100.1 &
  \cAlignHack 0.394\ensuremath{\pm}0.003 &
  \cAlignHack 0.6 &
  \cAlignHack 100.6\\
 &
  \cAlignHack 22 &
  \cAlignHack 0.464\ensuremath{\pm} 0.003 &
  \cAlignHack 0.7 &
  \cAlignHack 100.8 &
  \cAlignHack 0.474\ensuremath{\pm}0.003 &
  \cAlignHack 0.7 &
  \cAlignHack 102\\
 &
  \cAlignHack  &
  \cAlignHack Mean RSD &
  \cAlignHack 0.63666 &
  \cAlignHack  &
  \cAlignHack  &
  \cAlignHack 0.666 &
  \cAlignHack \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {LOD and LOQ} }
\label{tw-3faf0dd82f51}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3238\linewidth-2\tabcolsep}p{\dimexpr.3258\linewidth-2\tabcolsep}p{\dimexpr.3504\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Drug & \cAlignHack LOD & \cAlignHack LOQ\\
\tblmidrule 
\cAlignHack Pretomanid &
  \cAlignHack 0.037ppm &
  \cAlignHack 0.113ppm\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Ruggedness} }
\label{tw-19a3b276c9e9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.15115714285714285\linewidth-2\tabcolsep}p{\dimexpr.13455714285714285\linewidth-2\tabcolsep}p{\dimexpr.1428571428571429\linewidth-2\tabcolsep}p{\dimexpr.13577142857142862\linewidth-2\tabcolsep}p{\dimexpr.14520000000000005\linewidth-2\tabcolsep}p{\dimexpr.14640000000000002\linewidth-2\tabcolsep}p{\dimexpr.14405714285714291\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Drug & \multicolumn{3}{p{\dimexpr(.4131857142857144\linewidth-2\tabcolsep)}}{\cAlignHack Analyst-1} & \multicolumn{3}{p{\dimexpr(.435657142857143\linewidth-2\tabcolsep)}}{\cAlignHack Analyst-2}\\
\rowcolor{kwdboxcolor}\cAlignHack Concentration (ppm) & \cAlignHack Mean \mbox{}\protect\newline Absorbance & \cAlignHack \% Amount found & \cAlignHack \% RSD \mbox{}\protect\newline (n=3) & \cAlignHack Mean \mbox{}\protect\newline Absorbance & \cAlignHack \% Amount found & \cAlignHack \% RSD \mbox{}\protect\newline (n=3)\\
\tblmidrule 
\cAlignHack 14 &
  \cAlignHack 0.314 &
  \cAlignHack 102 &
  \cAlignHack 0.3 &
  \cAlignHack 0.307 &
  \cAlignHack 100 &
  \cAlignHack 0.3\\
\cAlignHack 18 &
  \cAlignHack 0.386 &
  \cAlignHack 100.6 &
  \cAlignHack 0.4 &
  \cAlignHack 0.380 &
  \cAlignHack 98.9 &
  \cAlignHack 0.5\\
\cAlignHack 22 &
  \cAlignHack 0.467 &
  \cAlignHack 101.5 &
  \cAlignHack 0.2 &
  \cAlignHack 0.458 &
  \cAlignHack 99.4 &
  \cAlignHack 0.3\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Robustness data by taking absorbance at 321\ensuremath{\pm}2nm} }
\label{tw-23301f172880}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.09905714285714284\linewidth-2\tabcolsep}p{\dimexpr.2553571428571429\linewidth-2\tabcolsep}p{\dimexpr.1002571428571429\linewidth-2\tabcolsep}p{\dimexpr.10372857142857146\linewidth-2\tabcolsep}p{\dimexpr.15588571428571434\linewidth-2\tabcolsep}p{\dimexpr.1428571428571429\linewidth-2\tabcolsep}p{\dimexpr.1428571428571429\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Sr. No. & \cAlignHack Concentration (ppm) & \cAlignHack 319nm & \cAlignHack 321nm & \cAlignHack 323nm & \cAlignHack \% Amount & \cAlignHack \% RSD\\
\tblmidrule 
\cAlignHack 1. &
  \cAlignHack 14 &
  \cAlignHack 0.315 &
  \cAlignHack 0.315 &
  \cAlignHack 0.314 &
  \cAlignHack 102 &
  \cAlignHack 0.15\\
\cAlignHack 2. &
  \cAlignHack 18 &
  \cAlignHack 0.389 &
  \cAlignHack 0.389 &
  \cAlignHack 0.386 &
  \cAlignHack 101.2 &
  \cAlignHack 0.36\\
\cAlignHack 3. &
  \cAlignHack 22 &
  \cAlignHack 0.466 &
  \cAlignHack 0.467 &
  \cAlignHack 0.467 &
  \cAlignHack 101.4 &
  \cAlignHack 0.10\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Precision}

The assay's repeatability (intraday) and intermediate precision (inter-day) were quantified and expressed as a percentage of RSD. For this, the concentration solutions of 14 \ensuremath{\mu }g/mL, 18 \ensuremath{\mu }g/mL, and 22 \ensuremath{\mu }g/mL were tested three times a day, and the RSD\% was computed. (Table~\ref{tw-5cd14c977cc2}) In terms of intraday and interday precision, Pretomanid's\% RSD was discovered to be 0.6366 and 0.666, respectively. Table~\ref{tw-5cd14c977cc2} displayed the outcomes for intra-day and inter-day. 

\textbf{LOD and LOQ}

Limit of Detection (LOD) and Limit of Quantitation (LOQ) estimates were used to determine the suggested method's sensitivity. The ICH indicates that LOD (DL, the detection limit) can be calculated as LOD = 3.3\ensuremath{\sigma  } / S, and the limit of quantification (which they call QL, the quantitation limit) LOQ = 10\ensuremath{\sigma  } / S. LOD and LOQ values for Pretomanid is given in Table~\ref{tw-3faf0dd82f51}. Here \ensuremath{\sigma  } is the standard deviation of the response and S is the slope of the calibration curve. S is estimated from the slope of the calibration curve for the LOD and LOQ.

\textbf{Ruggedness}

Table~\ref{tw-19a3b276c9e9} shows the ruggedness results

\textbf{Robustness}

The method's robustness is demonstrated by a slight planned change in the analytical wavelength. The wavelength change applied as \ensuremath{\pm}2 nm. The \% RSD was found to be 0.15, 0.36, and 0.10 for variables shown in Table~\ref{tw-23301f172880}. 
    
\section{\textbf{Conclusion}}
The suggested UV-Vis Spectrophotometric approach was found to be simple, precise, and accurate for the estimation of Pretomanid, and it was verified according to ICH requirements. As a result, this approach may be utilised for regular Pretomanid analysis without interfering with commonly used excipients in quality control laboratories, whether in bulk or in dosage formulations.
\section*{Acknowledgement}The authors are thankful to Mylan Lab, Hyderabad, India for providing a gift sample. Also thankful to the Principal Mahatma Gandhi Vidyamandir's Pharmacy College Panchavati Nashik for providing facilities for carrying out this 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. 
    

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