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\begin{document}


\def\RunningAuthor{Anuradha V et al.}
\firstPage{808}
\articleType{Original Article}
\receivedDate{09 Dec 2020}
\acceptedDate{28 Jan 2021}
\revisedDate{15 Jan 2021}
\journalVolume{2021, 12}
\journalIssue{1}
\journalDoi{ijrps.v12i1.4184}
\copyrightYear{2021}
\def\authorCount{3}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{New Validated RP-HPLC Method for Cisplatin and Topotecan in API and Vaccine Form and its Stress Studies}
\author{Subrahmanyam~Talari\textsuperscript{1,2},  
            Anuradha~V\textsuperscript{*}\textsuperscript{3}, 
            Komala~sai~prathyusha~A\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh-522510, India}~\\{\textsuperscript{2}Department of Chemistry, Sir C R Reddy College, Eluru, Andhra Pradesh-534007, India}~\\{\textsuperscript{3}Department of Chemistry, Vignan Degree College, Guntur, Andhra Pradesh-522009, India}}

\begin{abstract}
We have developed a completely unique and reliable HPLC technique for simultaneous quantification of Cisplatin and Topotecan. A chromatographic detachment was attained on a XDB C\ensuremath{_{18}} column (150x4.6mm, 3.5 \ensuremath{\mu}) using isocratic elution with a buffer containing buffer and acetonitrile with the proportion of 60:40 as a movable phase with a flow of 1 mL/min at room temperature and UV detection was carried out at 262 nm. Dissolve 1mL of triethylamine in 1 lt of HPLC grade water and filter through 0.45 \ensuremath{\mu} filter paper. This solution was used as a buffer. 10 min run time was used to separate Cisplatin and Topotecan. The analysis was achieved within 15 min over honest linearity within the concentration range from 5-75 \ensuremath{\mu}g/mL of Cisplatin and 2-30 \ensuremath{\mu}g/mL of Topotecan. By injecting the standard six times, system suitability parameters were studied and the outcomes were under the acceptable limit. Precision and recovery study results were found to be within a suitable limit. By using the above technique, the assay of the marketed formulation was performed and found to be within the limit. Degradation studies were carried out on Cisplatin and Topotecan, with a purity threshold greater than the purity angle in all conditions and within the acceptable range. The above-mentioned technique was validated according to ICH guidelines.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}HPLC,\newline Cisplatin,\newline Topotecan,\newline Development,\newline Validation
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Anuradha~V~\\ Phone:\ 9949137666~\\  Email:\ vanuradha372@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
        \textsc{DOI:}\ \href{https://doi.org/10.26452/\@journalDoi}{\textcolor{blue}{\underline{\smash{https://doi.org/10.26452/\@journalDoi}}}}\par%
        \vspace*{-11pt}\hrulefill\\{\fontsize{9.12}{10.12}\selectfont Production and Hosted by}\par{IJRPS |~\href{www.ijrps.com}{www.ijrps.com}}\par%
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        \makeatother
        
    
\section{Introduction}
Cisplatin is a chemotherapy medication \unskip~\citep{990648:21292610,990648:21292651} which is used in the treatment of a number of cancer sicknesses. These include testicular cancer \unskip~\citep{990648:21292634}, cancer of the ovaries \unskip~\citep{990648:21292639}, cancer of the cervix \unskip~\citep{990648:21292612}, cancer of the breast \unskip~\citep{990648:21292613}, cancer of the bladder \unskip~\citep{990648:21292645}, cancer of the head and neck \unskip~\citep{990648:21292648}, cancer of the oesophagus \unskip~\citep{990648:21292637}, cancer of the lungs \unskip~\citep{990648:21292646}, cancer of the mesothelioma \unskip~\citep{990648:21292636}, cancer of the brain \unskip~\citep{990648:21292611} and neuroblastoma \unskip~\citep{990648:21292650}. It is administered into a vein by injection. Bone marrow suppression \unskip~\citep{990648:21292635}, hearing problems, kidney problems and vomiting \unskip~\citep{990648:21292644} are common side effects. Other serious adverse effects include numbness, difficulty in walking, allergic reactions, problems with electrolytes \unskip~\citep{990648:21292608} and heart disease \unskip~\citep{990648:21292616}. Using this medicine during pregnancy can result in harming the baby in the womb. Cisplatin is in the anti-neoplastic platinum-based \unskip~\citep{990648:21292826} family of medications. By binding to DNA and inhibiting its replication, it works in part. 

Topotecan (trade name Hycamtin) is a chemotherapeutic agent who is an inhibitor of topoisomerase \unskip~\citep{990648:21292640}. It is a synthetic, water-soluble analogue of camptothecin \unskip~\citep{990648:21292641}, a natural chemical compound. It is used to treat ovarian cancer, lung cancer and other types of cancer in the form of the hydrochloride salt. Experiments with neuroblastoma, brainstem glioma, Ewing's sarcoma \unskip~\citep{990648:21292642} and Angelman's syndrome \unskip~\citep{990648:21292632} have been underway since 2016. Topotecan is also utilized to handle non-small cell lung cancer, colorectal cancer \unskip~\citep{990648:21292609}, breast cancer, non-Hodgkin lymphoma, Endometrial cancer \unskip~\citep{990648:21292643} and Oligodendroglioma \unskip~\citep{990648:21292633}. Figure~\ref{f-00de193e00c8} represents the chemical representation of Cisplatin and Topotecan. 

\textbf{Experiment}

\textbf{Chemicals and Reagents}

Acetonitrile and Orthophosphoric acid, triethylamine, water (HPLC grade) were acquired from Merck (India) Ltd, Worli, Mumbai, India. The reference standards of Cisplatin and Topotecan APIs were taken from Spectrum Pharma Research Solutions Pvt. Ltd., Hyderabad.


\begin{table*}[!htbp]
\caption{\boldmath { Optimized Chromatographic Conditions} }
\label{tw-fc092ced24c5}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.473\linewidth-2\tabcolsep}p{\dimexpr.527\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Parameter  & Proposed method\\
\tblmidrule 
Stationary Phase &
  XDB C18 (150x4.6 mm, 3.5 \ensuremath{\mu})\\
Mobile Phase &
  0.1\% Triethylamine: Acetonitrile (60:40)\\
Injection Volume &
  10 \ensuremath{\mu}l\\
Flow Rate &
  1.0 mL/min\\
Column Temperature &
  25\ensuremath{^\circ}C\\
Wave Length &
  262 nm\\
Run Time &
  10.0 min\\
Retention time of Cisplatin &
  4.358 min\\
Retention time of Topotecan &
  7.744 min\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of System Suitability} }
\label{tw-5702b059733a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3715\linewidth-2\tabcolsep}p{\dimexpr.3217\linewidth-2\tabcolsep}p{\dimexpr.3068\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Parameter & Cisplatin & Topotecan\\
\tblmidrule 
Theoretical plate count &
  6261 &
  11234\\
Tailing factor &
  1.06 &
  1.14\\
Resolution &
  - &
  13.21\\
Retention time &
  4.358 &
  7.744\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Linearity} }
\label{tw-2ec8d1791c1d}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10040000000000001\linewidth-2\tabcolsep}p{\dimexpr.29959999999999997\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{S. No}} & \multicolumn{2}{p{\dimexpr(.49959999999999994\linewidth-2\tabcolsep)}}{Cisplatin} & \multicolumn{2}{p{\dimexpr(.40\linewidth-2\tabcolsep)}}{Topotecan}\\
\rowcolor{kwdboxcolor} & Concentration (\ensuremath{\mu}g/mL) & Area & Concentration (\ensuremath{\mu}g/mL) & Area\\
\tblmidrule 
1 &
  5 &
  345271 &
  2 &
  182053\\
2 &
  12.5 &
  932015 &
  5 &
  615013\\
3 &
  25 &
  1802501 &
  10 &
  1152468\\
4 &
  50 &
  3520164 &
  20 &
  2154785\\
5 &
  62.5 &
  4265021 &
  25 &
  2785436\\
6 &
  75 &
  5159206 &
  30 &
  3205684\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Method Precision} }
\label{tw-6eafc688852e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.19730000000000004\linewidth-2\tabcolsep}p{\dimexpr.4254\linewidth-2\tabcolsep}p{\dimexpr.3773\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S. No. & Area of Cisplatin & Area of Topotecan\\
\tblmidrule 
1 &
  3562417 &
  2145012\\
2 &
  3501269 &
  2150234\\
3 &
  3542815 &
  2145025\\
4 &
  3546251 &
  2145873\\
5 &
  3536215 &
  2102125\\
6 &
  3524864 &
  2153641\\
Mean &
  3535639 &
  2140318\\
Std. dev &
  20875.344 &
  19024.101\\
\% RSD &
  0.59 &
  0.89\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Intermediate Precision} }
\label{tw-de2f57121f94}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2832\linewidth-2\tabcolsep}p{\dimexpr.2313\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.2355\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Area of Cisplatin & Relative standard Deviation & Area of Topotecan & Relative standard Deviation\\
\tblmidrule 
3562012 &
  \multicolumn{1}{p{\dimexpr(.2313\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{0.42}} &
  2102568 &
  \multicolumn{1}{p{\dimexpr(.2355\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{0.85}}\\
3521365 &
   &
  2145278 &
  \\
3547215 &
   &
  2135624 &
  \\
3535626 &
   &
  2135265 &
  \\
3528692 &
   &
  2155869 &
  \\
3548576 &
   &
  2143206 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Accuracy} }
\label{tw-000570dd4f95}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.13689999999999996\linewidth-2\tabcolsep}p{\dimexpr.2721\linewidth-2\tabcolsep}p{\dimexpr.191\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Accuracy & Amount of Cisplatin & \% Recovery & Amount of Topotecan & \% Recovery\\
\tblmidrule 
50 &
  25 &
  99.9 &
  10 &
  98.2\\
100 &
  50 &
  99.9 &
  20 &
  99.5\\
150 &
  75 &
  99.7 &
  30 &
  99.8\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Robustness} }
\label{tw-8382e1ccccaf}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3383\linewidth-2\tabcolsep}p{\dimexpr.3258\linewidth-2\tabcolsep}p{\dimexpr.3359\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Parameter & \% RSD of Cisplatin & \% RSD of Topotecan\\
\tblmidrule 
Flow (0.8 mL/min) &
  0.57 &
  0.38\\
Flow (1.2 mL/min) &
  0.64 &
  0.87\\
Organic phase (36:64) &
  0.21 &
  0.42\\
Organic phase (44:56) &
  0.69 &
  0.56\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Forced Degradation} }
\label{tw-9224c0e38bec}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.46699999999999996\linewidth-2\tabcolsep}p{\dimexpr.193\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{Stress Parameter}} & \multicolumn{2}{p{\dimexpr(.533\linewidth-2\tabcolsep)}}{\% of Degradation}\\
\rowcolor{kwdboxcolor} & Cisplatin & Topotecan\\
\tblmidrule 
Acid degradation (1N HCl) &
  14.6 &
  14.9\\
Alkali degradation (1N NaOH) &
  14.1 &
  15.3\\
Peroxide degradation (30\% Peroxide) &
  15.2 &
  16.6\\
Reduction degradation (30\% sodium bisulphate) &
  13.9 &
  12.4\\
Thermal (sample, 70\ensuremath{^\circ}C, 6 Hrs) &
  13.4 &
  11.5\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Equipment}

An HPLC system (Waters alliance e2695 model) consisting of a quaternary pump, PDA detector-2998 was used. Data processing was performed with Empower 2.0 software. 

\textbf{Chromatographic Conditions}

A chromatographic detachment was achieved in isocratic mode at room temperature using XDB column of C\ensuremath{_{18}} (150x4.6 mm, 3.5 \ensuremath{\mu}). A mixture of acetonitrile and 0.1 percent triethylamine (TEA) in 40:60 v/v at a stream of 1 mL/min was used as a movable phase. The injection volume was 10 \ensuremath{\mu}l and the run time was 10.0 min.

\textbf{Preparation of Buffer}

1 mL of triethylamine is dissolved in 1 litre of HPLC grade water and filtered through 0.45 \ensuremath{\mu} filter paper. 

\textbf{Diluent}

The movable phase was used as diluent.

\textbf{Standard Preparation }

Carefully weigh and transfer 50 mg of cisplatin and 20 mg of topotecan in a volumetric flask of capacity 100 mL and add the app. 70 mL of diluents, sonicated to melt it for 30 min. and add diluents up to mark. Further, dilute 5 mL of the above solution to 50 mL with diluents. 

\textbf{Sample Preparation }

Carefully weigh and transfer weight similar to 50 mg of cisplatin injection powder and 20 mg of topotecan injection powder in a flask of 100 mL and add 70 mL of diluent. Sonicated to melt and dilute up to the mark with diluent. Take 5 mL of the above solution and dilute it to 50 mL and filtered through a 0.45 \ensuremath{\mu} nylon syringe filter. 

\textbf{Method Validation}

\textbf{System Suitability}

In order to confirm the system performance, system suitability parameters were measured. The parameters, including USP plate count, USP tailing and \%RSD, were calculated and found to be within the limit. 

\textbf{Specificity}

Specificity is the capacity to evaluate the analyte unambiguously the analyte in the presence of other constituents that may be expected to be combined in the sample and standard solution. It is checked by examining the chromatograms of blank samples and samples spiked with cisplatin and topotecan.

\textbf{Accuracy}

Accuracy is the closeness of the test findings gained by the technique to the real value. It is assessed by the recovery studies will be evaluated at three different concentration levels. In each level, a minimum of three injections was administered at each level and quantity of the drug present, percentage recovery and the associated standard deviations were calculated. 

\textbf{Precision}

The precision of the analytical technique is the degree of agreement among independent test results. It was studied by analysis of multiple sampling of a homogeneous sample. The precision of the present method was assessed in terms of repeatability, intra-day and inter-day variations. It was checked by analyzing the samples at different time intervals of the same day as well as on different days. 

\textbf{Linearity}

The linearity of an analytical method is its ability to obtain results in a defined range directly proportional to the concentration of the analyte in the sample within a definite range. The six series of standard solutions were selected for assessing linearity range, six series of standard solutions were selected. Using peak area versus concentration of the standard solution, the calibration curve was plotted and the regression equations were measured. The least-squares method was used to calculate the slope, intercept and correlation coefficient. 

\textbf{Stress Degradation}

Stress Degradation should not have any interference between the peaks of forced degradation preparations obtained for the chromatogram. Stress degradation studies were conducted in accordance with ICH guidelines Q1 (A) R2. The peaks of degradation should be well spaced and the resolution between the peaks should be at least 1.0. The peak purity of the principle peaks will pass only when there is separation. Forced degradation studies were performed to obtain the degradation of about 20 per cent by different types of stress conditions. 

\textbf{Robustness}

An analytical procedure's robustness is a measure of its ability to remain unchanged by small but intentional differences in method parameters and gives an indication of its reliability during normal use. Robustness study was carried out by injecting a standard solution into the HPLC system and modifying chromatographic settings such as flow rate (\ensuremath{\pm}0.2 mL/min), organic content in the mobile phase (\ensuremath{\pm}10\%). By establishing the effect of the modified parameters, the separation factor, retention time and peak asymmetry were estimated.


\bgroup
\fixFloatSize{images/ac731be6-9d70-45a6-a208-7177c8a1858a-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/ac731be6-9d70-45a6-a208-7177c8a1858a-upicture1.png}{\includegraphics{images/ac731be6-9d70-45a6-a208-7177c8a1858a-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Structural representations of (A) Cisplatin and (B) Topotecan}}
\label{f-00de193e00c8}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/aae80c07-ef99-438d-a2da-30bbf155fedb-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/aae80c07-ef99-438d-a2da-30bbf155fedb-upicture2.png}{\includegraphics{images/aae80c07-ef99-438d-a2da-30bbf155fedb-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of System Suitability}}
\label{f-20f795a0b5f2}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/5334cf4d-b96b-45c6-ab28-06279ffccc8d-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/5334cf4d-b96b-45c6-ab28-06279ffccc8d-upicture3.png}{\includegraphics{images/5334cf4d-b96b-45c6-ab28-06279ffccc8d-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Chromatogram of Blank}}
\label{f-219997f6e1ef}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/c2a3c839-a4a4-4684-b618-7ad7962ea30a-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c2a3c839-a4a4-4684-b618-7ad7962ea30a-upicture4.png}{\includegraphics{images/c2a3c839-a4a4-4684-b618-7ad7962ea30a-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Calibration plots of (A) Cisplatin and (B) Topotecan}}
\label{f-f06d259192b1}
\end{figure}
\egroup

    
\section{Results and Discussion}
The present study was designed to develop a simple, accurate and rapid analytical RP-HPLC method, that can be used for the simultaneous estimation of cisplatin and topotecan in bulk and pharmaceutical dosage form for the analysis of the assay method. In order to provide good assay performance, the chromatographic conditions were optimized. To optimize mobile phase, various combinations were tried for cisplatin and topotecan. 

The final working mobile phase is (0.1\%) triethylamine and acetonitrile in the composition of (60:40 v/v) Mobile phase for each drug were selected on the basis of its polarity. In order to obtain sufficient sensitivity for the two APIs in smaller proportions (cisplatin and topotecan) detection was carried out in several wavelengths. 

Finally, the 262 nm wavelength was selected, where the two drugs showed good absorption. The flow rate was 1.0 mL/min. For Cisplatin and Topotecan, the retention time was 4.358 min, 7.744 min, respectively. The proposed method is validated by all the outcomes within limits in accordance with the ICH guidelines. The detection was carried out with a total runtime of 10.0 min. Below Table~\ref{tw-fc092ced24c5}  represents the optimized chromatographic method.

\textbf{System Suitability}

The System Suitability was performed by injecting a standard solution containing 50 \ensuremath{\mu}g/mL of cisplatin and 20 \ensuremath{\mu}g/mL of topotecan in six replicates. The results indicate that the system suitability parameter is within the limit. Table~\ref{tw-5702b059733a} represents the system suitability results and Figure~\ref{f-20f795a0b5f2} represents the standard chromatogram.

\textbf{Specificity}

There was no interference from blank at the retention time of cisplatin and topotecan. Below Figure~\ref{f-219997f6e1ef} represents the blank chromatogram.

\textbf{Linearity}

By plotting a calibration curve of the peak area against its respective concentration, linearity was determined. From this calibration curve, it was found that the curve was linear in the range of 5-75 \ensuremath{\mu}g/mL of cisplatin and 2-30 \ensuremath{\mu}g/mL of topotecan. 

The regression equations for calibration curve of cisplatin was Y= 68450.45x + 40082.06 (R\ensuremath{^{2}}-0.9997) and Y= 68450.45 + 40082.06 (R\ensuremath{^{2}}) for topotecan respectively. Linearity results were shown in Table~\ref{tw-2ec8d1791c1d}, calibration plots of cisplatin and topotecan were shown in Figure~\ref{f-f06d259192b1}. 

\textbf{Precision}

In terms of intraday and intermediate precision variations, the accuracy of this method was assessed. Six repeated analyses of the sample solution of cisplatin and topotecan on the same day under the same experimental conditions were used to determine the intraday studies. 

In the same laboratory, the intermediate precision of the method was carried out by studying the analysis with different analysts and with different instruments. As percentage RSD values were found to be {\textless}2 per cent, the method is highly precise. 

At each added concentration, good recoveries of the drug were obtained, indicating that the method was exact. Method precision results were shown in the below Table~\ref{tw-6eafc688852e} and Table~\ref{tw-de2f57121f94} represents intermediate precision results. 

\textbf{Intermediate Precision (Ruggedness)}

\textbf{Accuracy}

By calculating the recovery experiments at three levels, the accuracy of the method was achieved (50\%, 100\% and 150\%). APIs were prepared with a concentration of 25, 50, 75 \ensuremath{\mu}g/mL of cisplatin and 10, 20, 30 \ensuremath{\mu}g/mL of topotecan. For each spike level, the test solution was injected three times and the assay was performed as per the test method. The recovery outcomes were close to 100\% and also the RSD values were less than \ensuremath{\pm}2\% as well. The recovery percentage, mean and relative standard deviations have been calculated. Recovery values showed within the desired range. The method was accurate. Accuracy results were given in Table~\ref{tw-000570dd4f95}.

\textbf{Robustness}

Robustness of the chromatographic technique was determined by changing the flow rate and movable phase composition. \% RSD was found to be within the acceptable limit. Robustness results were shown in Table~\ref{tw-8382e1ccccaf}.

\textbf{Forced Degradation}

The proposed method can be used for release and stability studies for effective evaluations and can be considered as stability preferable technique. The forced degradation study carried out according to the ICH requirements includes acid, base, oxidation, reduction, thermal degradation. From the chromatograms, it is evident that the selected drugs were stable under the applied stress conditions though degraded peaks were observed. Results of forced degradation were given in Table~\ref{tw-9224c0e38bec}.
    
\section{Conclusions}
In this study, a novel, rapid, economical, sensitive and easily available HPLC method was evolved for the simultaneous estimation of cisplatin and topotecan in API and pharmaceutical dosage form. The main advantages of this method are no HPLC methods were reported. Other merits are shorter run time, low price, accessibility, sensitivity, reliability and reproducibility. These properties are important when a more number of samples are to be analyzed. The validation of all the parameters like linearity, accuracy, specificity, robustness, method precision were done and found to be within the acceptable limit. The relative standard deviation values for all the parameters were found to be less than 2\%, which indicates the validity of the method and the results gained by this technique are in fair agreement. So the proposed method could be easily applied for the routine analysis and the pharmaceutical formulations of cisplatin and topotecan in quality control laboratories without any preliminary separation. 

\textbf{Acknowledgement}

The authors are grateful and show their acknowledgment to Acharya Nagarjuna University, Guntur, AP, India, and Sir C R Reddy college, Eluru, AP, India and Shree Icon Pharmaceutical Laboratories for providing all the required facilities to carry out this research work successfully. 

\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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