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\title{Rp-Hplc Method for the Determination of Pramipexole Dihydrochloride in Tablet Dosage Form}
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             \author[1]{Dr.  P.Lavudu}

             \author[2]{A.Prameela  Rani}

             \author[3]{petikam  lavudu}

             \affil[1]{  }

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\date{\small \em Received: 18 February 2012 Accepted: 16 March 2012 Published: 28 March 2012}

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\begin{abstract}
        


A simple, sensitive, rapid, selective, precise and accurate high performance liquid chromatographic method was developed and validated for the determination of Pramipexole dihydrochloride in bulk and tablet dosage forms. HPLC separation was carried out by reversed phase chromatography on a Thermo Scientific C18 column (250 mm × 4.6 mm, 5 ?m), held at ambient temperature. The mobile phase consisted of methanol: acetonitrile (40:60 v/v), run at a flow rate of 1.0 ml/min and with UV detection at 263 nm. The method was found to be linear over an analytical range of 1-100 ?g/ml with LOD = 0.075 ?g/ml and LOQ = 0.227 ?g/ml, respectively. The proposed method was validated successfully and applied to the quantification of the drug in tablet dosage forms.

\end{abstract}


\keywords{Pramipexole dihydrochloride, RP-HPLC, development, Validation.}

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\let\tabcellsep& 	 	 		 \par
ramipexole dihydrochloride (PPD) \hyperref[b0]{[1]}\hyperref[b1]{[2]}\hyperref[b2]{[3]}\hyperref[b3]{[4]}\hyperref[b4]{[5]}\hyperref[b5]{[6]}, a nonergot dopamine agonist approved in the US (1997), is used as an antidyskinetic for treatment of Parkinson's disease. Its chemical name is (S)-N 6 propyl-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine dihydrochloride (Fig.  {\ref 1}). The ability of PPD to alleviate the signs and symptoms of Parkinson's disease is supposed to be linked to its ability to stimulate dopamine receptors in the striatum.\par
Various analytical methods have been reported in the literature for the assay of PPD in pure and in its pharmaceuticals preparations. Procedures using UVspectrophotometry \hyperref[b6]{[7]}, visible spectrophotometry \hyperref[b7]{[8,}\hyperref[b8]{9]}, HPTLC \hyperref[b9]{[10]} have been reported by several workers. High-performance liquid chromatography with mass spectrometer (HPLC-MS) \hyperref[b10]{[11]}\hyperref[b11]{[12]}\hyperref[b12]{[13]}, capillary electrophoresis with laser-induced fluorescence detection \hyperref[b13]{[14 ]}, gas chromatography with mass spectrometer (GC-MS) \hyperref[b14]{[15]} and Ultra-performance liquid chromatography with mass spectrometer (UPLC-MS) \hyperref[b15]{[16]} have been used for the analysis of PPD in biological samples.\par
Only few HPLC methods with UV detection have been described in the literature for determination of PPD. Pathare et al \hyperref[b16]{[17]} developed a chiral liquid chromatographic method for the enantiomeric resolution of Pramipexole dihydrochloride monohydrate on a Chiralpak AD (250 mm × 4.6 mm, 10 ?m) column using a mobile phase system containing n-hexane:ethanol: diethylamine (70:30:0.1 v/v/v). A method developed for determination of PPD and its impurities by Jan ?i? et al \hyperref[b17]{[18]} was carried out using a C18 column with mobile phases containing different ratios of acetonitrile and water phase (aqueous triethylamine/orthophosporic acid). Yau et al \hyperref[b18]{[19]} reported a HPLC method for the determination of pramipexole in human plasma and urine.\par
Separation is achieved by ion-pair chromatography on a Zorbax Rx C8 column (250 mm × 4.6 mm, 5 ?m) and a Brown lee RP-8 pre-column (15 mm x 3.2 mm, 7 ?m) with electrochemical detection at 0.6 V for plasma and ultraviolet detection at 286 nm for urine. A RP-HPLC \hyperref[b19]{[20]} method for PPD in pure and in its pharmaceutical dosage forms has been reported by RAO et al and was carried out on an hypersil ODS-C18 (250 mm × 4.6 mm, 5?m) column with acetonitrile and acetate buffer (90:10 v/v) as the mobile phase and a detection wavelength of 260 nm. Srinubabu et al \hyperref[b20]{[21]} have reported an RP-HPLC method for the assay of PPD in tablet formulations on an ODS-C18 column (250 mm × 4.6 mm, 5 ?m) with a mobile phase of acetonitrile and phosphate buffer (60:40 v/v) and detection at 260 nm.\par
The reported HPLC methods for the determination of PPD in pharmaceutical dosage forms suffer from one or more disadvantages like preparation of buffer, rigid pH control, narrow linear concentration range and less sensitivity.\par
In this paper, an attempt is made to develop and validate a simple, efficient and reliable method, without incorporating the use of an internal standard, for the determination of PPD in tablet dosage forms by HPLC using UV detection. All HPLC experiments were carried out on a isocratic High Pressure Liquid Chromatography system (Shimadzu HPLC class VP series, Shimadzu Corporation, Kyoto, Japan) with two LC-10 AT, VP pumps, variable wavelength programmable UV/Visible detector SPD-10A, VP, CTO-10AS VP column oven, SCL-10A, VP system controller. The HPLC system was equipped with the software "class VP series version 5.03" (Shimadzu). The analytical column used for the separation was 250 mm × 4.6 mm I.D., 5 ?m particle size, Thermo Scientific C18 (Phenomenex, Torrance, CA, USA). 
\section[{b) Chemicals and reagents}]{b) Chemicals and reagents}\par
All chemicals and reagents were of HPLC grade quality. Milli-Q-water was used throughout the process and it was obtained from Merck Specialties Private Ltd, Hyderabad, and Andhra Pradesh, India. Methanol and acetonitrile of HPLC grade were from Rankem laboratories, Mumbai, India. 
\section[{c) Preparation of Mobile phase}]{c) Preparation of Mobile phase}\par
Mobile phase 'A' consisted of methanol. Mobile phase 'B' was acetonitrile. The mobile phase used for analysis was prepared by mixing mobile phase 'A' and mobile phase 'B' in the ratio, 40:60 v/v. The same mobile phase was also used as a diluent for the sample preparations. 
\section[{d) Standard solutions and tablet dosage forms}]{d) Standard solutions and tablet dosage forms}\par
Pharmaceutical grade PPD was kindly gifted by Matrix laboratories, Hyderabad, India, and was used as received. The following available pharmaceutical dosage forms containing 0.5 mg and 1 mg of active ingredient were purchased from the local pharmacy and used in the present investigation:\par
? Parpex (1 mg, Zydus cadila, Ahmedabad, India) ? Pramipex (0.5 mg and 1 mg, Sun pharma, Mumbai, India) Stock solution of PPD (1 mg/ml) was prepared by dissolving 100 mg of PPD in 50 ml of diluent in a 100 ml volumetric flask and then made up to the mark with diluent. 
\section[{e) Chromatographic conditions}]{e) Chromatographic conditions}\par
The mobile phase was a mixture of methanol and acetonitrile (40:60 v/v). The contents of the mobile phase were filtered before use through 0.45 ?m membrane filter, degassed with a helium sparge for 15 min and pumped from the respective solvent reservoirs to the column at a flow rate of 1 ml/min. The column temperature was maintained at 25±10C. The injection volume of samples was 20 ?l. The analyte was monitored at a wavelength of 263 nm. f) Recommended procedure Working standard solutions equivalent to 1 to 100 ?g/ml PPD were prepared by appropriate dilution of the stock standard solution (1 mg/ml) with the diluent. Prior to injection of the drug, the mobile phase was pumped for about 30 minutes to saturate the column thereby to get the base line corrected. 20 ?l of each solution was injected automatically onto the column in triplicate and the peaks were determined at 263 nm. The peak areas of PPD were plotted against the corresponding nominal concentration to obtain calibration graph. The concentration of the drug was obtained from the calibration graph or the regression equation.\par
g) Procedure for tablet dosage forms Fifty tablets containing PPD were exactly weighed and ground into a fine powder. From this powder, an amount of the tablet powder equivalent to 25 mg PPD was transferred to a 25 ml standard flask containing 10 ml of diluent and shaken for 10 minutes. The volume was made up to the mark with diluent and mixed well. The solution was filtered through a 0.45 ?m membrane filter. The filtered solution was appropriately diluted with diluent to obtain a concentration of 100 ?g/ml. From this solution, 20 ?L was injected into the HPLC system. The area under the peak was noted and the drug content in the tablets was quantified using the calibration graph or regression equation. 
\section[{a) Method development}]{a) Method development}\par
In order to develop an efficient and simple RP-HPLC method for the analysis of the drug in bulk and in its tablet dosage forms, preliminary tests were conducted to select satisfactory and optimum conditions. HPLC parameters, such as detection wavelength, ideal mobile phase \& their proportions and flow rate were carefully studied.\par
Preliminary experiments indicated that the Thermo Scientific C18 (250 mm × 4.6 mm, 5 ?m) column provides efficient and reproducible separation of PPD at ambient temperature. Hence Thermo Scientific C18 column was selected for method development and validation. PPD was determined by injecting the drug solution on to Thermo Scientific C18 column with UV detector set at 263 nm. After trying different ratios of mixtures of methanol and acetonitrile, the best results were achieved by using a mixture of methanolacetonitrile (40:60 v/v) as mobile phase. At a flow rate of 1.0 ml/min, the retention time for PPD was 4.458 min. The analyte peak area was well defined and free from tailing under the described experimental conditions. b) System suitability System suitability test was carried out on freshly prepared solution of PPD (50 ?g/ml) to ensure the II. 
\section[{Materials and Methods}]{Materials and Methods} 
\section[{III.}]{III.} 
\section[{Results and Discussion}]{Results and Discussion}\par
validity of the analytical procedure. Data from five injections were used to confirm system suitability parameters like retention time, peak area, peak asymmetry, theoretical plates, plates per meter and height equivalent to theoretical plate. The results are presented in Table \hyperref[tab_0]{1}. The values obtained demonstrated the suitability of the system for the analysis of the PPD. 
\section[{c) Selectivity}]{c) Selectivity}\par
Selectivity is the ability of an analytical method to distinguish between the analyte of interest and other components present in the sample. To identify the interference by the excipients in the tablet dosage form, the tablet extract was prepared according to procedure described under "Procedure for tablet dosage forms" and injected. The resulting chromatogram (Fig.  {\ref 2}) did not show any peak other than that of PPD, which confirmed the selectivity of the method. The selectivity of the method was also demonstrated by interference check by injecting the diluent blank to determine whether any peaks in the diluent are co-eluting with PPD peak. No interference of peaks eluted in the diluent blank with PPD peak was observed (Fig. \hyperref[fig_3]{3}). 
\section[{d) Linearity}]{d) Linearity}\par
The linearity was determined by constructing calibration curve. A calibration curve was constructed using least squares method by plotting the peak area vs concentration of PPD. The calibration curves (Fig.  {\ref 5}) for PPD show good linearity with excellent regression coefficient (0.9993) in the concentration range of 1-100 ?g/ml. The linear regression equation and regression coefficient of the calibration curve is presented in Table \hyperref[tab_1]{2}.\par
e) LOD and LOQ The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of y-intercepts of regression lines or standard deviation of blank readings and the slope of the calibration curve by using three calibration curves. Results of LOD and LOQ for PPD are shown in Table \hyperref[tab_1]{2}. 
\section[{f) Accuracy and precision}]{f) Accuracy and precision}\par
The precision and accuracy of the method was determined by performing five repeated analysis of three different standard solutions containing 5, 50, 90 ?g/ml PPD, on the same day, under the optimized experimental conditions. The precision and accuracy are expressed as RSD and relative error, respectively. The results of this study are presented in Table \hyperref[tab_2]{3}. The values of the relative standard deviation and relative error were found satisfactory. Hence the proposed method is precise and accurate. 
\section[{g) Recovery studies}]{g) Recovery studies}\par
The accuracy of the proposed method was also further assessed by performing recovery experiments using the standard addition method. Known amount of the pure PPD was added to pre-analyzed formulation and the total concentration was once again determined by the proposed method. The obtained mean recoveries and relative standard deviations were in the range 99.66-100.33 and 0.378-0.614 \%, respectively (Table \hyperref[tab_3]{4}). The results revealed that any small change in the drug concentration in the solutions could be accurately determined by the proposed method. The closeness of the recoveries suggests lack of interference from tablet excipients and thereby establishes some degree of selectivity. 
\section[{h) Application to tablet dosage forms}]{h) Application to tablet dosage forms}\par
To find out the suitability of the proposed method for the assay of tablet dosage forms containing PPD was analyzed by the proposed method. The results obtained from the proposed method were compared statistically with reference method7 by applying Student's t-test for accuracy and F-test for precision. From the results (Table \hyperref[tab_4]{5}) it was found that the proposed method does not differ significantly in precision and accuracy from the reference method.\par
A simple, sensitive, selective, accurate and precise RP-HPLC method was developed for the determination of PPD in bulk and in tablet dosage forms. It should be emphasized it is isocratic and the mobile phase do not contain any buffer. The short chromatographic time makes this method appropriate for the processing of numerous samples in a limited time. The method has wider linear range with good accuracy and precision. The method shows no interference from tablet excipients. Hence, the proposed method could be useful and fit for the quantification of PPD in bulk and tablet dosage forms.     \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-2.png}
\caption{\label{fig_1}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{12}\includegraphics[]{image-3.png}
\caption{\label{fig_2}Figure 1 :Figure 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3}\includegraphics[]{image-4.png}
\caption{\label{fig_3}Figure 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4}\includegraphics[]{image-5.png}
\caption{\label{fig_4}Figure 4 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1} \par 
\begin{longtable}{}
\end{longtable} \par
 
\caption{\label{tab_0}Table 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{2} \par 
\begin{longtable}{}
\end{longtable} \par
 
\caption{\label{tab_1}Table 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3} \par 
\begin{longtable}{}
\end{longtable} \par
 
\caption{\label{tab_2}Table 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4} \par 
\begin{longtable}{}
\end{longtable} \par
 
\caption{\label{tab_3}Table 4 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{5} \par 
\begin{longtable}{}
\end{longtable} \par
 
\caption{\label{tab_4}Table 5 :}\end{figure}
 			\footnote{Rp-Hplc Method for the Determination of Pramipexole Dihydrochloride in Tablet Dosage Form © 2012 Global Journals Inc. (US) © 2012 Global Journals Inc. (US)} 			\footnote{© 2012 Global Journals Inc. (US)} 			\footnote{© 2012 Global Journals Inc. (US) © 2012 Global Journals Inc. (US)} 		 		\backmatter  			 			 			  				\begin{bibitemlist}{1}
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\end{document}
