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\def\RunningAuthor{Venkateswara Rao A et al.}
\firstPage{603}
\articleType{Original Article}
\receivedDate{15 Nov 2020}
\acceptedDate{19 Dec 2020}
\revisedDate{18 Dec 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4002}
\copyrightYear{2020}
\def\authorCount{4}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{A specified and accurate RP-HPLC method stimulation and estimation in bulk pharmaceutical dosage forms}
\author{Venkateswara~Rao~A\textsuperscript{*}\textsuperscript{1},  
            Sireesha\textsuperscript{1},  
            Malini~Evangeline~Rose\textsuperscript{2}, 
            Sivasankari~S\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Microbiology, Konaseema Institute of Medical Sciences Research Foundation, Amalapuram, Andhra Pradesh, India}~\\{\textsuperscript{2}Department of Microbiology, Meenakshi Academy of Higher Education and Research, Chennai, Tamilnadu, India}}

\begin{abstract}
A specified, accurate and exact RP-HPLC technique is established for the approximation of Hydrochlorothiazide and Losartan Potassium in pharmaceutical dosage form. The novel method was developed by make use of waters HPLC System on a Zodiac C18   column. The Mixture of 0.01\% Ortho Phosporic Acid: Acetonitrile 70:30 v/v portable segment is used at a flow rate of 1.5 ml/min. The vaccination capacity selected about 20 \ensuremath{\mu}l. The wavelength recognition is approved out at 220 nm. The preservation period of Hydro chlorthiazide and Losartan were originated to be 3.1 min and 7.2 min correspondingly. The established RP- HPLCtechniqueis authenticated as each ICH strategies for the estimation of together the drugs in bulk in addition to in pharmaceutical dosage forms. The established technique is authenticated with regard to Accuracy, Precision, Linearity, Robustness and Ruggedness. Hence, the projected technique is effectively used by simultaneous determination of Hydrochlorothiazide and Losartan Potassium. It is habitually utilized for the medication for Hypertension, congestive heart failure, symptomatic Edema, diabetes Insipidus, renal tubular acidosis. The Thiazide-Type Diuretics (counting Hydrochlorothiazide) would lesquerella powerful over the Thiazide-Like Diuretics (Chlortalidone and Indapamide) for decreasing those danger for heart Attack, Stroke, and heart disappointment in Persons with high bar. HCTZ will be likewise here and there used to prevent Osteopenia Also to medicine of Hypoparathyroidism, hypercalciuria. Thiazides need aid likewise utilized in the medicine about osteoporosis.  
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}RP-HPLC,\newline Losartan Potassium (LOS),\newline Hydrochlorothiazide (HCTZ)
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Venkateswara~Rao~A~\\ Phone:\ 8328643646~\\  Email:\ venkateswararao46@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
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\section{Introduction}
Hydrochlorothiazide \unskip~\citep{969564:21099173,969564:21099169,969564:21099167}  will be a diuretic solution regularly utilized will treat high bar. To high bar it may be recognized Concerning illustration First-Line Treatment, despite chlortalidone may be viable for a comparative rate for unfriendly impacts. HCTZ may be joined with different Circulatory strain solution as An solitary Pill on increment viability. Those novel agenize tie focused Also specific of the Aii Subtype 1 Receptor, Subsequently blocking Aii-Induced receptor. An animated metabolite - E3174, Contributes antihypertensive impact. Hence, available point from claiming Our fill in may be on create An Specific, Precise, Accurate, Linear, Simple, fast and cosset powerful Stability Indicating HPLC Method \unskip~\citep{969564:21099171,969564:21099168}. The scope of our work also extends to validation for the development methods. It abatements mineral bone passing Eventually Tom's perusing pushing calcium maintenance in the Kidney, Furthermore Eventually Tom's perusing straightforwardly empowering osteoblast separation Also bone mineral arrangement \unskip~\citep{969564:21099172}. It might be given with other antihypertensive operators for mix Preparation, for example, Previously, LOS/HCTZ. Losartan Potassium is a orally Active, Nonpeptide Atii receptor adversary. Elbasvir is an inhibitor of non-structural 5A (NS5A) hepatitis C virus (HCV), which is important for viral RNA replication and virion assembly. Elbasvir is a material with 882,035 gm/mol1 molecular formula C49H55N9O7 \unskip~\citep{969564:21099170}. It's methanol-soluble and water-insoluble. Grazoprevir is a straightforwardly second-generation antiviral drug used in combined therapy to manage persistent hepatitis C, a liver condition triggered by hepatitis C infection (HCV). Grazoprevir is a chemical formula C38H50N6O9S 766, 903 gm/mol2 material. Grazoprevir is very highly soluble in water in methanol and acetonitrile. Elbasvir, when used in conjunction with grazoprevir as a combination agent Zapatier3,4, is suggested for use with or without in adult hepatitis C (HCV) genotypes 1 or 4. Literature survey showed that no official or recorded methods were accessible for estimating both drugs in combination. The main objective of this research is to establish a reliable, specific, responsive, selective, reproducible and rapid analytical technique for simultaneous estimation of Grazoprevir, Elbasvir in bulk ant tablet dosage type.
    
\section{Materials and Methods}
\textbf{Chemicals}

Zorbax Cn, Hypersil Silica, Acetonitrile (HPLC Grade), Triethylamine Buffer, Sodium Acetate, Dil. Sodium Hydroxide, Hydrochlorothiazide, Losartan Potassium, Dis.Water (Distilled Grade).

\textbf{Equipments}

Waters HPLC With Empower {\textendash} 2 Software, Zodiac C18(250 X 4.6mm, 5\ensuremath{\mu}m) Column. The mixture of 0.01\% OPA: Acetonitrile 70:30V/V at a flow rate 1.5ml/min are used as a mobile phase.The injection volume was 20\ensuremath{\mu}l. The run time was 10 min.

\textbf{Preparation of Standard Stock Solution}

The standard stock solution was prepared by adding 20 mg of HCTZ and 80 mg of Losartan Potassium were accurately weighed and were taken into 100 Ml Volumetric Flask. 70 ml of diluent were added and was sonicated to dissolve. It was cooled to room temperature and the volume is ade upto 100ml by diluent.

\textbf{Preparation of Standard solution (further dilution)}

The typical answer is organized by adding 4ml of standard stock solution is pipetted out into 100 ml volumetric flask and the volume is made upto the mark by diluent.

\textbf{Preparation of Sample solution}

Weighed accurately about five tablets and relocated into a 250 ml volumetric flask. To that 200 ml of dilutant is added and it is sonicated for 30 min with vigorous trembling. Then it was cooled to room temperature and the volume was made-up with diluents upto 100 ml. From that 4 ml of the solution is taken and dilute to 100 ml by diluents and mixed. The obtained solution is filtered by 0.45 \ensuremath{\mu} nylon filter.

\textbf{Validation procedure}

The standard preparation of 20\ensuremath{\mu}l for 5 times and also the sample research was inserted into the chromatographic system. Chromatograms are read and peak retorts for HCTZ + Losartan potassium were measured. The system fitness strictures are met from the peak replies the satisfied of HCTZ + Losartan potassium in the sample was calculated.

\textbf{Classification Appropriateness}

The structure correctness restrictions are appraised by typical chromatograms attained. The parameters include preservation times, tracking factor, theoretical plates and \% RSD are calculated and found to be in limits.

\textbf{Linearity and Range}

Linearity means an analytical method in which the attain consequences would be specifically proportional of the focus of the analyte in the test inside a range. The 6 arrangement for standard results are chose for those estimation of linearity extent. Those alignment bend might have been plotted by utilizing An top territory (vs) focus of the standard result and the relapse comparison will be ascertained. 

\textbf{Precision.}

An explanatory system is those degree from claiming reproducible "around distinctive test effects is known as Precision. It might have been measured by the Investigation for different inspecting of homogeneous example. Those precision of the available technique might have been ascertained As far as repeatability, intraday What's more interday variations. It was estimated by analysing the samples at various time intervals of the same day as well as on other days.

\textbf{Accuracy}

The nearness of the test consequences found by the technique to the true value is known as Accuracy. The accuracy is measured by the recovery studies at three different concentration levels. In each level, the lowest of three vaccinations are assumed and the quantity of the drug current, \% of retrieval and connected typical deviation (Sd) are considered.

\textbf{Specificity}

Specificity is clear as the aptitude to assess unmistakable the analysis in the occurrence of other workings like impurities, degraded products (or) excepients. These components might be predictable to current in the test example and standard solution. It was observed by checking the chromatograms of blank sample.

\textbf{Robustness}

Robustness is defined as an investigative process is amount of ability to continue genuine by thoughtful changes in technique limits and delivers an suggestion of its dependability through usual conditions. This study is performed by injecting standard solution into the HPLC system and altered chromatographic conditions that is flow rate   wavelength (\ensuremath{\pm}5 nm), and change of pH (\ensuremath{\pm}0.5), mobile period (\ensuremath{\pm}2\%). The retention time and peak asymmetry are calculated by determining the effect of the obtained parameters.

\textbf{LOD and LOQ}

In a sample solution the lowest amount of analyte can be detected as LOD and as LOQ is the last quantity of analyze in a sample solution that could be quantified can be resolute. LOD and LOQwere separately estimated based on the calibration curves. The LOD and LOQ are intended as 3.3 Sd/s and 10 Sd/s correspondingly as per ICH guidelines, are Sd/s indicates standard deviation and slope method.

\textbf{Statistical Analysis}

The data obtained were analysed by chromatograms. The data is subjected to regression analysis to obtain the line of equation in linearity studies.
    
\section{Results and Discussion}
\textbf{Method validation}

In this method system suitability, linearity, precision, accuracy, lod, loq, robustness are validated. The proposed method is having blank solution, standard solution and sample solution chromatograms are shown in the figures.


\bgroup
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\caption{\boldmath {Linearity Graph}}
\label{f-008371bb5c9f}
\end{figure*}
\egroup

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\makeatother 
\caption{\boldmath {(a) Chromatographs of Optimized conditions of HCTZ \& (b) LSR}}
\label{f-d50fae4c441f}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {For Linearity} }
\label{tw-9875583391eb}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.17140000000000004\linewidth-2\tabcolsep}p{\dimexpr.3286\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Analyte & Linearity Range & Equation of Calibration \mbox{}\protect\newline Curve & Correlation Coefficient\\
\tblmidrule 
HCTZ &
  10-300 \mbox{}\protect\newline \ensuremath{\mu}g/ml &
  Y=4920x &
  NLT 0.990\\
LSR &
  10-300 \mbox{}\protect\newline \ensuremath{\mu}g/ml &
  Y=9033x &
  NLT 0.990\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {For Precision (System, Method, Intermediate)} }
\label{tw-ee77bef09fc2}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.44230000000000004\linewidth-2\tabcolsep}p{\dimexpr.30309999999999995\linewidth-2\tabcolsep}p{\dimexpr.2546\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Analyte & Amount Present & \% RSD\\
\tblmidrule 
Hydrochlorothiazide &
  12.50 Mg &
  0.29\\
HCTZ &
  12.50 Mg &
  0.657\\
HCTZ &
  12.50 Mg &
  0.31\\
Losartan Potassium &
  50 Mg &
  0.37\\
LSR &
  50 Mg &
  0.515\\
LSR &
  50 Mg &
  0.23\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {For Accuracy} }
\label{tw-f5cc9e7d6fce}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.30750000000000004\linewidth-2\tabcolsep}p{\dimexpr.3525\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\% of Target Conc. & Hydrochlorothiazide (\% Recovery) & Losartan Potassium (\% Recovery)\\
\tblmidrule 
50 &
  100.1 &
  100.0\\
100 &
  100.7 &
  100.2\\
150 &
  100.3 &
  100.8\\
Mean &
  100.5 &
  100.3\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {For Robustness} }
\label{tw-57b2a413997b}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1946\linewidth-2\tabcolsep}p{\dimexpr.2054\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Drug \mbox{}\protect\newline Name & Flow \mbox{}\protect\newline Plus & Flow \mbox{}\protect\newline Minus & Org \mbox{}\protect\newline Plus & Org \mbox{}\protect\newline Minus\\
\tblmidrule 
HCTZ &
  1.7 &
  1.3 &
  65:35 &
  75:25\\
LSR &
  1.7 &
  1.3 &
  65:35 &
  75:25\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{System suitable}

The HPLC framework might have been settled to 30 min by Emulating the chromatographic states on get a stable build accordance. A spotless result emulated eventually Tom's perusing 6 replicates of a standard result is injected to perform those framework suitable. The framework suitable parameters were controlled from standard chromatograms obtained, Toward figuring those maintenance times, tailing factors, hypothetical plates also \% RSD from claiming crest zones. 

\textbf{Linearity}

Arrangement for results would readied utilizing Hydrochlorthiazide Also Losartan working standard at An centralization levels from 10 {\textendash} 300 \ensuremath{\mu}g/ml Furthermore 10 {\textendash} 300 \ensuremath{\mu}g/ml separately. The results need aid injected under the HPLC framework as for every technique. Measured those top zone of the injected result. An alignment bend might have been plotted for crest range in the y hub Also focus of standard result in the x hub. Those linearity from claiming both the medication results are evaluated Toward ascertaining those correspondence co-efficient. Those information of the alignment bend might have been provided for in Table~\ref{tw-9875583391eb}. The mathematical statement for alignment bend What's more relationship coefficient is indicated for figure x linearity plot to Hydrochlorothiazide, figure x linearity plot to Losartan potassium. Those relationship coefficient values for these analytes were not under 0. 990. 

\textbf{Limit of identification and Quantification}

Farthest point from claiming identification Also quantification were decided starting with standard deviation Furthermore slant technique Concerning illustration for every ICH rules. LOD What's more LOQ were computed from those Normal incline Furthermore standard deviation from the alignment bend. LOD for HCTZ Furthermore LSR were discovered will be 1. 14 \ensuremath{\mu}g/ml Also 1. 34 \ensuremath{\mu}g/ml. LOQ of HCTZ Furthermore LSR were discovered to a chance to be 3. 414 \ensuremath{\mu}g/ml Also 4. 066 \ensuremath{\mu}g/ml.

\textbf{Precision}

Framework precision might have been conveyed crazy utilizing 6 replicates of the same standard concentration (8 \ensuremath{\mu}g/ml of HCTZ and 32 \ensuremath{\mu}g/ml of LSR). The data of the system precision. Method precision was carried out using 6 different sample preparations from same homogenous blend of marketed sample (8 \ensuremath{\mu}g/ml of HCTZ and 32 \ensuremath{\mu}g/ml of LSR). 

The data of the method precision. The data of the intermediate precision was also given in the Table~\ref{tw-ee77bef09fc2}.

\textbf{Accuracy}

Accuracy of established technique was calculated by examine and retrieval procedures. The retrieval studies are approved out at 3dissimilar levels. The pre analysed sample solution was spiked with 50\%, 100\%, 150 \% of mixed standard solution. The prepared mixture of solutions are analyzed by the projected method. The study was carried out in triplicate. Then the obtained retrieval results are given in the Table~\ref{tw-f5cc9e7d6fce}.

\textbf{Specificity}

The impact of extensive variety from claiming excipients What's more different additives Typically introduce in the consolidation from claiming HCTZ What's more LSR in the determinations under ideal states were investigated. 

Placebo solutions, example Furthermore standard results were analysed separately. Likewise for every the technique should analyze obstruction. Chromatograms of placebo, standard and test Concerning illustration in the Figure~\ref{f-008371bb5c9f}. 

\textbf{Robustness}

Should exhibit the heartiness of the method, get ready standard result Similarly as for every test technique What's more injected during separate variable states in utilizing separate stream rates What's more progress in the versatile period creation in the versatile stage. Those got system might have been hearty and it might have been enough with withstand the slight progressions Also permitted those schedule Investigation of the tests. The observed data was given in Table~\ref{tw-57b2a413997b}.

\textbf{LOD and LOQ Formula}

LOD = 3.3 (S\ensuremath{_{d}}/s) LOQ = 10 (S\ensuremath{_{d}}/s)

The Limit of Detection and Limit of Quantification are estimated from standard deviation and slope method as per ICH guidelines. LOD were found to be 1.14 \ensuremath{\mu}g/ml and 1.34 \ensuremath{\mu}g/ml for HCTZ and LSR. LOQ were found to be 3.414 \ensuremath{\mu}g/ml and 4.066 \ensuremath{\mu}g/ml for HCTZ and LSR. In Figure~\ref{f-d50fae4c441f}.
    
\section{Conclusion}
This chapter deals with development of simultaneous RP-HPLC technique to the estimation for hydrochlorothiazide Also Losartan potassium to heft Also pharmaceutical measurement type. Those effects gotten for recommended systems affirm those suitable for these strategies for pharmaceutical measurement structures. Alternate animated parts \& excipients as a rule available in the pharmaceutical measurement manifestations didn't obstruction in the estimation. That exactness of the systems might have been affirmed Toward the recuperation investigations. These strategies could make utilized for the synchronous estimation for hydrochlorothiazide furthermore Losartan potassium Toward RP-HPLC technique done both greater part medication regardless \& pharmaceutical formulations.

\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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