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\def\RunningAuthor{Merugu Manasa and Vijey Aanandhi M}
\firstPage{1367}
\articleType{Original Article}
\receivedDate{23 Aug 2020}
\acceptedDate{03 Nov 2020}
\revisedDate{20 Sep 2020}
\journalVolume{2021, 12}
\journalIssue{2}
\journalDoi{ijrps.v12i2.4692}
\copyrightYear{2021}
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\title{Stability Indicating Assay Method (Siam) for Determination of Semaglutide by HPLC Using Qbd Approach}
\author{Merugu~Manasa, 
            Vijey~Aanandhi~M\textsuperscript{*}~\\[5pt]{Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai, Tamil Nadu, India}}

\begin{abstract}
An accurate, precise, robust and stability-indicating RP-HPLC method was developed for the estimation of Semaglutide using QbD approach. After conducting several trials using CCD method, one desirable method was optimized. Stationary phase selected was Kromasil C18 (250\ensuremath{\times}4.6 mm, 5 \ensuremath{\mu}m) and potassium dihydrogen orthophosphate (pH\ensuremath{_{2}}) and Methanol used as mobile phase in the ratio of 61.2: 38.8. Detection was carried out at the wavelength 230nm. Flow rate selected for separation was 0.98ml/min and the temperature of 29.15\ensuremath{^{0}}C. The retention time was found to be 2.518 at the run time of 5min. The developed method was subjected to validation as per ICH guidelines. Semaglutide linear in the concentration range of 0-9\ensuremath{\mu}g/ml and the regression coefficient was found to be 0.999. The developed method was accurate, precise and robust. When a sample solution subjected to stress conditions like  acid hydrolysis, neutral hydrolysis, photodegradation and thermal degradation, no degradation products were observed. In basic degradation studies two degradation products and oxidative conditions one degradation product was observed. From stress studies, we can observe that the degradation of Semaglutide was less than 10 in all conditions, and there is no impact of degradation products on system suitability parameters. The peak was homogenous I~all conditions, thus proving the stability-indicating nature of the method. This method can be applied for the determination of Semaglutide in the drug substance. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}RP-HPLC,\newline Semaglutide,\newline QbD,\newline CCD,\newline Desirable method
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Vijey~Aanandhi~M~\\ Phone:\ ~\\  Email:\ hodpchemistry@velsuniv.ac.in
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
SEMAGLUTIDE IUPAC name of Semaglutide is 17-\{[(1R)-3-[(2-\{2-[(\{2-[2-(\{[(5S)-5-[(2S) -2- [(2S) -2- [(2S) -2- \{2- [(2S) -2- [(2S) -2- [(2S) -2- [(2S) -2-[(2S)-2-[(2S)-2-[(2S)-2-[(2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-\{2-[(2S)-2- \{2-[(2S)-2 -amino-3- (1H-imidazol-4-yl) propanamido]-2- methyl propanamido\} -4- carboxy butanamido] acetamido\}-3-hydroxybutanamido]-3-phenylpropanamido]-3-hydroxy butanamido ]-3-hydroxypropanamido]-3-carboxy propanamido]-3-methyl butanamido]-3-hydroxy propanamido] -3- hydroxyl propanamido] -3- (4-hydroxyphenyl) propanamido] -4- methyl pentanamido]-4-carboxybutanamido]acetamido\}-4-carbamoyl butanamido] propanamido] propanamido] -5 -\{[(1S) -1 -\{[(1S) -1 -\{[(1S,2S) -1 -\{[(1S) -1- \{[(1S) -1-\{[(1S)-1-\{[(1S)-1-\{[(1S)-4-carbamimidamido-1-[(\{[(1S)-4-carbamimidamido-1-[(carboxymethyl) carbamoyl] butyl] carbamoyl\}methyl) carbamoyl]butyl]carbamoyl\}-2-methylpropyl]carbamoyl\}-3-methylbutyl] carbamoyl\}-2- (1H-indol-3-yl)ethyl]carbamoyl\}ethyl]carbamoyl\}-2-methylbutyl]carbamoyl\}-2-phenylethyl]carbamoyl\}-3-carboxypropyl] carbamoyl\} pentyl] carbamoyl\} methoxy) ethoxy]ethyl\}carbamoyl)methoxy]ethoxy\}ethyl)

carbamoyl]-1-carboxy propyl] carbamoyl\} heptadecanoic acid \unskip~\citep{923108:20679460}. Molecular weight 4113.641 and molecular formula is C187H291N45O59. Structure was shown in Figure~\ref{f-4a465974ce93}. 

QbD (Quality by Design) \unskip~\citep{923108:20679461,923108:20679463} is a systemic approach used to produce the robust method, to increase patient safety by increasing the quality of pharmaceuticals \unskip~\citep{923108:20679465}. In present days QbD approach is taking the predominant position in pharmaceutical studies. In analytical QbD approach, five main tools are present \unskip~\citep{923108:20679464,923108:20679467}. Method goal identification, method scouting, method evaluation, method selection and risk assessment \unskip~\citep{923108:20679466}.

Literature survey \unskip~\citep{923108:20679563,923108:20679565} revealed that there are no studies on quality-based design approach of estimation of Semaglutide by HPLC method. A few HPLC studies were reported for estimation of Semaglutide \unskip~\citep{923108:20679564}. Few studies were reported on other QbD approach of other anti-diabetic drugs \unskip~\citep{923108:20679567,923108:20679566}. The pilgrimage has been made to develop a simple accurate, precise, rapid, economical, robust and stable method for determination Semaglutide by HPLC using the QbD approach according to ICH guidelines \unskip~\citep{923108:20679568}.
    
\section{Materials and Methods}
\textbf{Chemicals and Reagents}

Pure Semaglutide was procured from Spectrum pharma Pvt Ltd (Hyderabad). Hydrochloric acid AR grade (HCL) and sodium hydroxide AR grade (NaOH) were obtained from Merck India Pvt Ltd. Hydrogen Peroxide (H\ensuremath{_{2}}O\ensuremath{_{2}}) was purchased from Qauligens. Acetic acid AR grade was purchased from Fisher scientific, India and SD. Fine chem Ltd. Respectively. Ammonium acetate and ammonium formate were obtained from SD. Fine chem Ltd and Merck India Pvt Ltd. Respectively. HPLC grade Acetonitrile (ACN) and methanol (MeOH) were purchased from Fischer scientific. HPLC grade water used throughout analysis was obtained from the Merck milli-Q water purification unit.

\textbf{Equipment}

The LC system used for method development and validation. Waters carried detection with a diode array detector (model: 2996 detector 2487 separation module). The output signal was supervised and processed using Waters Empower 2 Software. Mettler Toledo balance was used to perform weighing. Received drug sample was authenticated by melting point apparatus (BUCHI), FT-IR (BRUKER ALFA), and UV-VIS spectrophotometer (Shimadzu -1800, japan). Other pieces of equipment used throughout the experimental work are hot air oven (Yorco scientific), thermostat dry air equipment Thermo scientific and pH meter (Eutech instruments pH tutor, pH meter, India).

\textbf{Drug authentication}

After procuring the API from the drug was authenticated by melting point test and scanning from 200 to 400nm in UV-VIS spectrophotometer.

\textbf{Chromatographic Conditions}

Various trials were conducted to select a mobile phase and a stationary phase. The choice of a Kromasil C18 column 0.1\%OPA: Methanol (61.2:38.8\%) was selected as mobile phase. Using the central composite design (CCD) method was optimized. CCD design summary was shown in Table~\ref{tw-787101dcf6f4}.

\textbf{Preparation of Solutions }

\textbf{Preparation of Standard Solution }

Weigh accurately about 3mg of Semaglutide and dissolved in a diluent. The solution was sonicated for 5min and make up the volume to 50ml with diluent.

\textbf{Preparation of buffer}

\textbf{Potassium dihydrogen orthophosphate buffer(pH\ensuremath{_{2}})}

 Accurately weighed 1.36g of potassium orthophosphate was dissolved in 900ml milli-Q water and sonicate it for degassing. Makeup the volume to 1000ml and added 1ml of Triethylamine. Using diluted orthophosphoric acid pH was adjusted to 2.

\textbf{Method Validation}

The proposed method was validated as per ICH guidelines.

 To conduct system suitability studies, six replicate samples of 6\ensuremath{\mu}g/mL solution of Semaglutide were injected into a system to calculate the retention time, area, theoretical plates, SD and \%RSD. In linearity studies conducted by injecting different concentrations range of 1.5-9\ensuremath{\mu}g/ml. The calibration curve was plotted by taking concentration on X-axis and peak area on Y-axis. The correlation coefficient (R\ensuremath{^{2}}) should be less than 1. Accuracy performed to determine percentage recovery by injecting 50\%, 100\% and 150\% concentrations of standard in triplicate. The precision of the developed method was determined by two methods like repeatability (intra-day) and intermediate precision (inter-day). \% RSD should be less than 2. Limit of detection (LOD) refers to the lowest concentration level resulting in the peak area of three times the baseline noise. Limit of Quantification (LO Q) refers to the lowest concentration level that provided a peak area with a signal to noise ratio higher than ten. Robustness referred as, the capacity of the method to remain unaffected by small or deliberate changes in chromatographic conditions like organic content in mobile phase ration (\ensuremath{\pm} 10), flow rate (\ensuremath{\pm} 10) and temperature (\ensuremath{\pm} 10).


\begin{table*}[!htbp]
\caption{\boldmath {Design Summary of CCD} }
\label{tw-787101dcf6f4}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.178\linewidth-2\tabcolsep}p{\dimexpr.142\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(.48\linewidth-2\tabcolsep)}}{\cAlignHack File version: DX 11.0.0, Study Type: Response surface, Design Type: central composite Design, Design Model: Quadratic} & \multicolumn{3}{p{\dimexpr(.52\linewidth-2\tabcolsep)}}{\cAlignHack ATP: Robustness \mbox{}\protect\newline CQA: Retention time, Theoretical plates and Asymmetry, Runs: 21}\\
\rowcolor{kwdboxcolor}CMPs & Unit & Type & Subtype & Min. & Max.\\
\tblmidrule 
Column Temperature &
  0C &
  Numeric &
  Continuous &
  24.95 &
  35.05\\
Flow rate &
  ml/min &
  Numeric &
  Continuous &
  0.8318 &
  1.17\\
\%Org ratio &
  \% &
  Numeric &
  Continuous &
  33.27 &
  46.73\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Central composite experimental design matrix with response} }
\label{tw-2b829c48f28c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0691\linewidth-2\tabcolsep}p{\dimexpr.07550000000000001\linewidth-2\tabcolsep}p{\dimexpr.1221\linewidth-2\tabcolsep}p{\dimexpr.11679999999999998\linewidth-2\tabcolsep}p{\dimexpr.1159\linewidth-2\tabcolsep}p{\dimexpr.1605\linewidth-2\tabcolsep}p{\dimexpr.16349999999999994\linewidth-2\tabcolsep}p{\dimexpr.17660000000000004\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} &  & Factor 1 & Factor 2 & Factor 3 & Response 1 & Response 2 & Response 3\\
\rowcolor{kwdboxcolor}Std & Run & A: FR & B: MP & C: T & RT & TP & TF\\
\rowcolor{kwdboxcolor} &  & ml/min & \% & 0C & MIN & NUM & NUM\\
\tblmidrule 
6 &
  1 &
  1.1 &
  36 &
  33 &
  2.465 &
  5579.36 &
  1.4\\
16 &
  2 &
  1 &
  40 &
  30 &
  2.668 &
  6038.26 &
  1.44\\
15 &
  3 &
  1 &
  40 &
  30 &
  2.671 &
  5629.04 &
  1.55\\
3 &
  4 &
  0.9 &
  44 &
  27 &
  2.9 &
  5511.93 &
  1.44\\
7 &
  5 &
  0.9 &
  44 &
  33 &
  2.891 &
  5342.77 &
  1.37\\
2 &
  6 &
  1.1 &
  36 &
  27 &
  2.465 &
  5760.58 &
  1.39\\
20 &
  7 &
  1 &
  40 &
  30 &
  2.67 &
  5833.39 &
  1.53\\
13 &
  8 &
  1 &
  40 &
  24.9546 &
  2.655 &
  6097.28 &
  1.37\\
18 &
  9 &
  1 &
  40 &
  30 &
  2.659 &
  5666.54 &
  1.45\\
10 &
  10 &
  1.16818 &
  40 &
  30 &
  2.385 &
  5335.45 &
  1.41\\
12 &
  11 &
  1 &
  46.7272 &
  30 &
  2.633 &
  5759.36 &
  1.44\\
4 &
  12 &
  1.1 &
  44 &
  27 &
  2.447 &
  5590.03 &
  1.42\\
19 &
  13 &
  1 &
  40 &
  30 &
  2.659 &
  5534.37 &
  1.46\\
9 &
  14 &
  0.831821 &
  40 &
  30 &
  3.241 &
  6262.6 &
  1.37\\
5 &
  15 &
  0.9 &
  36 &
  33 &
  2.911 &
  5200.94 &
  1.34\\
17 &
  16 &
  1 &
  40 &
  30 &
  2.673 &
  5890.92 &
  1.4\\
1 &
  17 &
  0.9 &
  36 &
  27 &
  3.026 &
  5827.67 &
  1.42\\
11 &
  18 &
  1 &
  33.2728 &
  30 &
  2.606 &
  4978.99 &
  1.47\\
8 &
  19 &
  1.1 &
  44 &
  33 &
  2.343 &
  5450.19 &
  1.26\\
14 &
  20 &
  1 &
  40 &
  35.0454 &
  2.762 &
  6052.56 &
  1.51\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {ANOVA table for Retention time using CCD} }
\label{tw-74bb2bc62ac2}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1554\linewidth-2\tabcolsep}p{\dimexpr.1874\linewidth-2\tabcolsep}p{\dimexpr.09850000000000001\linewidth-2\tabcolsep}p{\dimexpr.17439999999999998\linewidth-2\tabcolsep}p{\dimexpr.12930000000000003\linewidth-2\tabcolsep}p{\dimexpr.12579999999999998\linewidth-2\tabcolsep}p{\dimexpr.12920000000000002\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Source & Sum of Squares & df & Mean Square & F-value & p-value & \\
\tblmidrule 
Model &
  0.9153 &
  9 &
  0.1017 &
  36.10 &
  \textless\ 0.0001 &
  Significant\\
A-FR &
  0.8703 &
  1 &
  0.8703 &
  308.90 &
  \textless\ 0.0001 &
  \\
B-MP &
  0.0042 &
  1 &
  0.0042 &
  1.50 &
  0.2481 &
  \\
C-T &
  0.0002 &
  1 &
  0.0002 &
  0.0600 &
  0.8115 &
  \\
AB &
  4.500E-06 &
  1 &
  4.500E-06 &
  0.0016 &
  0.9689 &
  \\
AC &
  0.0000 &
  1 &
  0.0000 &
  0.0177 &
  0.8967 &
  \\
BC &
  5.000E-07 &
  1 &
  5.000E-07 &
  0.0002 &
  0.9896 &
  \\
A\ensuremath{^2} &
  0.0282 &
  1 &
  0.0282 &
  10.01 &
  0.0101 &
  \\
B\ensuremath{^2} &
  0.0084 &
  1 &
  0.0084 &
  2.99 &
  0.1146 &
  \\
C\ensuremath{^2} &
  0.0008 &
  1 &
  0.0008 &
  0.2722 &
  0.6132 &
  \\
Residual &
  0.0282 &
  10 &
  0.0028 &
   &
   &
  \\
Lack of Fit &
  0.0280 &
  5 &
  0.0056 &
  147.81 &
  \textless\ 0.0001 &
  Significant\\
Pure Error &
  0.0002 &
  5 &
  0.0000 &
   &
   &
  \\
Cor Total &
  0.9435 &
  19 &
   &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Result of system suitability} }
\label{tw-ff73fd378807}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.16009999999999998\linewidth-2\tabcolsep}p{\dimexpr.23990000000000002\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & RT & Area & Asymmetry & T. Plates\\
\tblmidrule 
1 &
  2.515 &
  369765 &
  1.38 &
  4593\\
2 &
  2.518 &
  369582 &
  1.4 &
  4582\\
3 &
  2.52 &
  365226 &
  1.44 &
  4678\\
4 &
  2.523 &
  366412 &
  1.42 &
  4695\\
5 &
  2.527 &
  365262 &
  1.43 &
  4707\\
6 &
  2.53 &
  367302 &
  1.42 &
  4578\\
Mean &
  2.5 &
  367258 &
  1.4 &
  4639\\
SD &
  0.01 &
  2025.67 &
  0.02 &
  60.61\\
\%RSD &
  0.2 &
  0.6 &
  1.5 &
  1.3\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Result of linearity } }
\label{tw-3a29e0063ff9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5083\linewidth-2\tabcolsep}p{\dimexpr.4917\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Conc. (ppm) & Area\\
\tblmidrule 
0 &
  0\\
1.5 &
  91489\\
3.0 &
  183079\\
 4.5 &
  273992\\
6.0 &
  364706\\
7.5 &
  448744\\
9.0 &
  546985\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results for Accuracy} }
\label{tw-a015a9df9130}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.223\linewidth-2\tabcolsep}p{\dimexpr.277\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Value & 50\% (3\ensuremath{\mu}g/ml) & 100\% (6 \ensuremath{\mu}g/ml) & 150\% (9\ensuremath{\mu}g/ml)\\
\tblmidrule 
1 &
  543136 &
  728383 &
  905438\\
2 &
  541102 &
  728420 &
  904090\\
3 &
  546818 &
  723033 &
  906735\\
Mean &
  543685 &
  726612 &
  905421\\
SD &
  2897.3 &
  3099.6 &
  1322.6\\
\%RSD &
  0.5 &
  0.4 &
  0.1\\
\% Accuracy &
  99.5 &
  100.2 &
  99.7\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results for Precision} }
\label{tw-96bb8f8fcce8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.18480000000000008\linewidth-2\tabcolsep}p{\dimexpr.41919999999999995\linewidth-2\tabcolsep}p{\dimexpr.396\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Intra-day precision & Inter-day precision\\
\tblmidrule 
1 &
  368190 &
  345553\\
2 &
  365689 &
  341650\\
3 &
  364421 &
  344492\\
4 &
  366696 &
  340676\\
5 &
  361043 &
  339670\\
6 &
  366406 &
  337240\\
AVG &
  365408 &
  341547\\
STDEV &
  2469.9 &
  3084.3\\
\%RSD &
  0.7 &
  0.9\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of Robustness} }
\label{tw-2cdad10bc032}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.137\linewidth-2\tabcolsep}p{\dimexpr.103\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{Parameter }} & {\multirow{2}{*}{Modified condition}} & \multicolumn{2}{p{\dimexpr(.24\linewidth-2\tabcolsep)}}{\cAlignHack Area } & {\multirow{2}{*}{\% RSD }} & \multicolumn{2}{p{\dimexpr(.24\linewidth-2\tabcolsep)}}{\cAlignHack Asymmetry } & {\multirow{2}{*}{\%RSD }}\\
\rowcolor{kwdboxcolor} &  & Trial 1  & Trial 2  &  & Trial 1  & Trial 2  & \\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.137\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Flow rate}} &
  0.9 &
  332821 &
  327419 &
  1.16 &
  1.37 &
  1.4 &
  1.53\\
 &
  1.0 &
  369765 &
  369582 &
  0.04 &
  1.38 &
  1.4 &
  1.02\\
 &
  1.1 &
  383068 &
  388710 &
  1.03 &
  1.5 &
  1.49 &
  0.47\\
\multicolumn{1}{p{\dimexpr(.137\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Column temperature}} &
  250C &
  365079 &
  362042 &
  0.59 &
  1.38 &
  1.4 &
  1.02\\
 &
  300C &
  365226 &
  366412 &
  0.23 &
  1.44 &
  1.42 &
  0.99\\
 &
  350C &
  331291 &
  330928 &
  0.08 &
  1.5 &
  1.48 &
  0.95\\
\multicolumn{1}{p{\dimexpr(.137\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{\% Org ratio}} &
  36.0 &
  376493 &
  378468 &
  0.37 &
  1.42 &
  1.40 &
  1.00\\
 &
  39.9 &
  368190 &
  365689 &
  0.48 &
  1.41 &
  1.39 &
  1.01\\
 &
  42.0 &
  358893 &
  360045 &
  0.23 &
  1.38 &
  1.35 &
  1.55\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Summary of degradation study} }
\label{tw-0a81974f141f}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{LLLL}
\tbltoprule \rowcolor{kwdboxcolor}S. No. & Condition of degradation study & \% of drug degraded & The retention time of degradant\\
\tblmidrule 
1. &
  2N HCl, 8 hrs &
  4.87\% &
  -\\
2. &
  2N NaOH, 8hrs &
  5.67\% &
  -\\
3. &
  Neutral hydrolysis, 24 hrs &
  No degradation &
  -\\
4. &
  Oxidative degradation, 24 hrs &
  7.26\% &
  2.254\\
5. &
  Thermal degradation, 3 days &
  3.16\% &
  -\\
6. &
  Photodegradation, 24 hrs &
  1.77\% &
  -\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/31d5b467-4737-488a-b8c3-1aec64072f14-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/31d5b467-4737-488a-b8c3-1aec64072f14-upicture1.png}{\includegraphics{images/31d5b467-4737-488a-b8c3-1aec64072f14-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Semaglutide Structure}}
\label{f-4a465974ce93}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/50a4e770-34b6-49f5-a2b1-9fb29b2da668-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/50a4e770-34b6-49f5-a2b1-9fb29b2da668-upicture2.png}{\includegraphics{images/50a4e770-34b6-49f5-a2b1-9fb29b2da668-upicture2.png}}{}
\makeatother 
\caption{\boldmath {UV spectrum of Semaglutide}}
\label{f-a1650447faf7}
\end{figure}
\egroup
\textbf{Stress Studies}

To conduct acid hydrolysis, base hydrolysis and neutral hydrolysis, 1ml of stock solution added 1ml of 2N HCl, 1ml of stock solution and 1ml of 2N NaOH solution and 3mg of Semaglutide was weighed and dissolved in 1ml in individual volumetric flask respectively. Three solutions were shaken in Radley apparatus 70\text{\textordmasculine}C for 1 hr then neutralized and diluted to 10ml. In oxidative degradation, to 1ml of stock solution, a 1ml of 20\% H\ensuremath{_{2}}O\ensuremath{_{2}} solution was added and kept in the dark area at room temperature for 24 hrs and diluted to 10ml. In thermal degradation 3mg of Semaglutide was kept in a petri dish and kept in a hot air oven at 70\text{\textordmasculine}C for 24hrs. Sampling was done at multiple time points and dissolved in the diluent to produce 10ml. In photodegradation 3mg of Semaglutide was uniformly spread in the petri dish and exposed to sunlight for 24hrs. Sampling was done at multiple time points and dissolved in the diluent to produce 10ml. 
    
\section{Results and Discussion}
\textbf{Drug authentication}

After procuring the API from the drug was authenticated by melting point test and scanning from 200 to 400nm in UV-VIS spectrophotometer. The melting point was observed between 187-189\ensuremath{^{0}}C. From UV spectrum maximum absorption point found at 230nm in methanol. The UV spectrum was shown in Figure~\ref{f-a1650447faf7}.


\bgroup
\fixFloatSize{images/11940f2c-fb53-4411-9741-1c79c8316025-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/11940f2c-fb53-4411-9741-1c79c8316025-upicture3.png}{\includegraphics{images/11940f2c-fb53-4411-9741-1c79c8316025-upicture3.png}}{}
\makeatother 
\caption{\boldmath {2Dcontour plots of retention time as a function of FR, Column temperature and organic ratio}}
\label{f-1b39eb863bcd}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/1a33cc58-46c8-42df-b78c-af776d4380d5-upicture4.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/1a33cc58-46c8-42df-b78c-af776d4380d5-upicture4.png}{\includegraphics{images/1a33cc58-46c8-42df-b78c-af776d4380d5-upicture4.png}}{}
\makeatother 
\caption{\boldmath {3D contour plots of retention time as a function of FR (a), Column temperature (b)and organic ratio(c) }}
\label{f-998625646499}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/6eafa0bb-1998-453c-b132-04aa507de548-upicture5.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/6eafa0bb-1998-453c-b132-04aa507de548-upicture5.png}{\includegraphics{images/6eafa0bb-1998-453c-b132-04aa507de548-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Overall desirability of the final method }}
\label{f-8152a9d1ee23}
\end{figure*}
\egroup
\textbf{Method Development }

Various trials were conducted to select the mobile phase and the stationary phase. The choice of Kromasil C18 column acetonitrile was selected and finalized as the organic modifier for further optimization study. Using the central composite design (CCD) method was optimized. The factors viz; \% Organic concentration(33.3-46.7\% Org \%), flow rate (0.83-1.17ml/min), Column temperature (ranging from 24.95\ensuremath{^{0}}C and 35.05\ensuremath{^{0}}C) were taken and counter and a 3D surface plot showing the effect of each parameter on Retention Time, Theoretical plates and Asymmetry (CQA) were generated. Results and ANOVA studies were shown in Tables~\ref{tw-2b829c48f28c} and~\ref{tw-74bb2bc62ac2}, respectively. A desirability function applied to the optimized conditions to predict retention time, asymmetry and theoretical plates.

2D contour plot was developed as a function of 1\% organic concentration, pH and buffer strength. To understand the results, 2D contour plots and 3D plot were generated from data using Design Expert{\textsuperscript{\textregistered}} software (shown in Figures~\ref{f-1b39eb863bcd} and~\ref{f-998625646499}). 


\bgroup
\fixFloatSize{images/4bc062b0-e591-48c5-a079-9294b63745a6-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/4bc062b0-e591-48c5-a079-9294b63745a6-upicture6.png}{\includegraphics{images/4bc062b0-e591-48c5-a079-9294b63745a6-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Optimized chromatogram of Semaglutide}}
\label{f-2019958c8363}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/c982762e-7665-4c77-881f-9171b857a1b0-upicture7.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c982762e-7665-4c77-881f-9171b857a1b0-upicture7.png}{\includegraphics{images/c982762e-7665-4c77-881f-9171b857a1b0-upicture7.png}}{}
\makeatother 
\caption{\boldmath {Linearity plot of Semaglutide }}
\label{f-d9fd9a9ea578}
\end{figure}
\egroup
To get an optimum set of conditions, composite desirability was applied based on the specified goals and limits of each response. If the response on the desirability scale is on 1, it is a thoroughly desirable response, and response is on 0 it is an undesirable response. Responses based on specified goals and boundaries for retention time, area, and asymmetry obtained desirability composite was 1. Shown in Figure~\ref{f-8152a9d1ee23}.

\textbf{Optimization of Chromatographic Conditions }

Initial trials were conducted to optimize the method according to the central composite design (CCD) method. HPLC studies were carried out using a Kromasil C18 (250\ensuremath{\times}4.6 mm, 5 \ensuremath{\mu }m) and 0.1\%OPA: Methanol (61.2:38.8\%) as mobile phase. At wavelength detection of 230nm. The flow rate was selected as 0.98ml/min at 29.15\ensuremath{^{0}}C column temperature. The retention time was found to be 2.518min. The optimized chromatogram is shown in Figure~\ref{f-2019958c8363}. 

\textbf{Method Validation}

The optimized method was subjected to method validation as per ICH guidelines. The method was validated to demonstrate that it is suitable for its intended purpose by the standard procedure to evaluate adequate validation characteristics.

\textbf{System Suitability }

After injecting six replicate samples of 6\ensuremath{\mu}g/mL solution of Semaglutide into the system. Different parameters like retention time, area, theoretical plates, SD and \%RSD were calculated. All the parameters were within limits as per guidelines. Results were shown in Table~\ref{tw-ff73fd378807}. 

\textbf{Linearity}

The regression equation obtained was Y=60407x+0. r\ensuremath{^{2 }} (Correlation co-efficient) was found to be 0.999. Results were shown in Figure~\ref{f-d9fd9a9ea578} and Table~\ref{tw-3a29e0063ff9}.

\textbf{Accuracy}

The accuracy of Semaglutide showed good recovery at each level and mean peak area and standard deviation are given in Table~\ref{tw-a015a9df9130}.

\textbf{Precision}

The \% RSD of intraday and inter-day precision were found be 0.7 and 0.9, respectively. The results were within limits as per the guidelines and shown in the Table~\ref{tw-96bb8f8fcce8}.

\textbf{Limit of Detection (LOD) and Limit of Quantitation (LO Q) }

LOD and LOQ were calculated according to the signal to noise ratio method. The LOD and LOQ obtained were 0.019 and 0.056 \ensuremath{\mu}g /mL, respectively.

\textbf{Robustness}

Robustness studies were performed by altering the parameters like column temperature, flow rate and \% organic concentration. The \% RSD was calculated and found that the results within limits. Results were shown in Table~\ref{tw-2cdad10bc032}.

\textbf{Stress Studies}

Stress degradation studies have been performed for the drug by using various stress conditions. No degradation products were found in case of acid hydrolysis, neutral hydrolysis, photodegradation and thermal degradation. Two significant degradation products were found in the case of 2N base degradation, and one degradation product was found in 10\% H2O2. The results were showing satisfactory, that is \% degradation should be less than 10\% and should not affect system suitability parameters of Semaglutide. Results were shown in Table~\ref{tw-0a81974f141f}. 
    
\section{Conclusions}
A simple analytical and robust HPLC method was developed for the determination of Semaglutide by using the QbD approach using Design Expert{\textsuperscript{\textregistered}} software. Results which were obtained from the validation of the developed analytical method were within the limit as per ICH guidelines. Validated stability-indicating HPLC method for Semaglutide was developed, which is capable of separating drug substance from the degradation products. 
\section*{Acknowledgement}The authors wish to thank the management of Vels University for supporting this work. The authors wish to thank Spectrum labs for providing facilities to conduct this work. The authors wish to acknowledge the Hetero labs for providing the samples for their research. They would also like to thank colleagues for their support to complete research work. 

\textbf{Conflict Of Interest}

The authors have no conflict of interests to disclose other than what has been acknowledged above.

\textbf{Funding Support}

The authors declare that they have no funding support for this study.
    

\bibliographystyle{pharmascope_apa-custom}

\bibliography{\jobname}
\end{document}
