APPLICATION OF STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD FOR QUANTITATION OF PRAMIPEXOLE IN PHARMACEUTICAL DOSAGE FORM

APPLICATION OF STABILITY INDICATING HIGH PERFORMANCETHIN LAYER CHROMATOGRAPHIC METHOD FOR QUANTITATIONOF PRAMIPEXOLE IN PHARMACEUTICAL DOSAGE FORM

Shubhangi M. Pawar and Sunil R. Dhaneshwar

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University,Poona College of Pharmacy, Pune, Maharashtra, India

& A sensitive, selective, precise, and stability-indicating high performance thin layer chromato-graphic method was developed and validated for the determination of pramipexole both as a bulkdrug and in formulation. The method uses aluminum plates precoated with silica gel 60F-254 as thestationary phase and solvent system ethyl acetate: toluene: methanol: ammonia 8:1.5:0.5:0.6,(v=v=v=v). This system gave compact spots for pramipexole (Rf: 0.22� 0.02). Pramipexole wassubjected to acid and alkali hydrolysis, oxidation, and photodegradation. The peaks of the degra-dation products were well resolved from that of the pure drug and had significantly different Rf

values. Densitometric analysis of pramipexole was performed in the absorbance mode at 263 nm.The linear regression analysis data for the calibration plots showed a good linear relationship overa concentration range of 200–2000 ng � spot�1. The mean values of the correlation coefficient,slope, and intercept were 0.9986� 1.42, 4.1411� 0.965, and 768.73� 1.24, respectively.The method was validated for precision, robustness, and recovery. The limit of detection and limitof quantitation were 30 and 200 ng � spot�1, respectively. Statistical analysis showed that themethod is repeatable, selective, and can separate the drug from its degradation products andcan be used to monitor stability.

Keywords high performance thin layer chromatography, pramipexole, quantitation,stability indicating degradation, validation

INTRODUCTION

Pramipexole is a new drug used in the therapy of Parkinson’s disease.Chemically, it is (S)-2-amino-4,5,6,7-tetrahydro-6-(propylamino) benzothia-zole (Figure 1). Pramipexole is a nonergot dopamine agonist with highrelative in vitro specificity and full intrinsic activity at the D2 subfamilyof dopamine receptors, binding with higher affinity to D3 than to D2

Address correspondence to Sunil R. Dhaneshwar, Department of Pharmaceutical Chemistry,Bharati Vidyapeeth University, Poona College of Pharmacy, Pune, Maharashtra, India 411038. E-mail:[email protected]

Journal of Liquid Chromatography & Related Technologies, 34:1664–1675, 2011Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.576301

or D4 receptor subtypes.[1] Several methods such as HPLC[2–6] and UVspectrophotometric[7] have been reported for the estimation ofpramipexole. Gradient Ultra-Fast Liquid Chromatographic analysis ofpramipexole,[8] chromatographic method for determination of dis-sociation constants of pramipexole and its impurities,[9,10] stability studiesand structural characterization of pramipexole,[11] and HPLCMS=MS[12]

method have been reported in literature.To our knowledge, no article related to the stability indicating HPTLC

determination of pramipexole in pharmaceutical dosage forms hasappeared in the literature. The International Conference onHarmonization(ICH) guideline entitled Stability Testing of New Drug Substances and Productsrequires testing to be performed to elucidate the inherent stability character-istics of the active substance.[13]

Now-a-days HPTLC is becoming a routine analysis technique due toadvantages of lowoperating cost, high sample throughput, and need forminimum sample clean up. The major advantage of HPTLC is that severalsamples can be run simultaneously using a small quantity of mobile phase,unlike HPLC, thus lowering analysis time and cost per analysis.[14]

The aim of the present work was to study the degradation behaviorof pramipexole under stress conditions and to develop an accurate,specific, repeatable, and stability-indicating HPTLC method for the deter-mination of pramipexole in the presence of its degradation products andrelated impurities. The proposed method was validated according to ICHguidelines[15] and its updated international convention.

EXPERIMENTAL

Materials

Dr. Reddy’s Laboratories Ltd. (Hyderabad, Andhra Pradesh, India),kindly supplied pure drug samples of pramipexole as a gift sample ofBatch No.: PH0051109. Twenty tablets of pramipexole (Brand name:PRAMIPEX, label claim: 1mg pramipexole per tablet) was procured froma local Pharmacy in Pune. All chemicals and reagents used were ofanalytical grade and purchased from Merck Chemicals, Mumbai,Maharashtra, India.

FIGURE 1 Structure of pramipexole.

HPTLC Quantitation of Pramipexole in Pharmaceuticals 1665

HPTLC

The samples were spotted in bands widths of 6mm with a Camag 100microliter sample (Hamilton, Bonaduz, Switzerland) syringe on silica gelprecoated aluminum plate 60 F254, [20 cm� 10 cm with 250 mm thickness;E. Merck, Darmstadt, Germany, using a Camag Linomat V (Switzerland)sample applicator]. The plates were prewashed by methanol and activatedat 110�C for 5min prior to chromatography.

Themobile phase consisted of ethyl acetate:toluene:methanol:ammoniain the ratio of 8:1.5:0.5:0.6 (v=v=v=v). Linear ascending development was car-ried out in 20 cm� 10 cm a twin-trough glass chamber (Camag, Muttenz,Switzerland) saturated with the mobile phase. The optimized chamber satu-ration time for themobile phase was 30min at room temperature (25� 2�C)at relative humidity of 60%. The length of chromatogram run was 8 cm. Sub-sequent to the development, TLC plates were dried in current of air with thehelp of dryer in wooden chamber with adequate ventilation. The flow rate inlaboratory was maintained unidirectional (laminar flow, toward exhaust).

Densitometric scanning at 263 nm was performed on Camag TLCscanner III in the reflectance absorbance mode and operated by CATSsoftware (V 3.15, Camag). The source of radiation utilized was deuteriumlamp emitting continuous UV spectrum between 200 and 400nm. A con-stant application rate of 0.1mL � s�1 was employed. The slit dimension waskept at 5mm� 0.45mm and 10mmsec�1 scanning speed was employed.The monochromator bandwidth was set at 20 nm, each track was scannedthrice and baseline correction was used.

For calibration, standard stock solution of 0.2mg �mL�1 concentrationof pramipexole was prepared using methanol. Different volumes of thestock solution (1, 2, 3, 4, 5, 7, and 10mL) were applied to TLC plates tofurnish 200, 400, 600, 800, 1000, 1400, and 2000 ng pramipexole per band.This was repeated six-fold. Peak area and drug concentration data weretreated by linear regression analysis.

Forced Degradation Studies

A stock solution containing 25mg pramipexole in 25mL methanol wasprepared. This solution was used for forced degradation to provide anindication of the stability indicating property and specificity of theproposed method. In all the degradation studies, the average peak areaof pramipexole after application (2000ng � spot�1) of seven replicates wasobtained. In order to study the degradation products of pramipexole usingthe HPTLC method, most of the study was carried out by single develop-ment of a TLC plate in order to prevent the movement of the nonpolardegradates to the extreme end of the plate.

1666 S. M. Pawar and S. R. Dhaneshwar

Preparation of Acid and Base Induced Degradation ProductAcid decomposition studies were performed by refluxing the solution of

drug in 3N hydrochloric acid at 80�C for 48hr. The studies in alkaline con-ditions were carried out in 2N sodium hydroxide and the solution wasrefluxed for 24hr at 80�C. The resultant solutions were applied on TLCplate in such a way that final concentration achieved was 2000ng � spot�1

and the chromatograms were run as described in the HPTLC section.

Preparation of Hydrogen Peroxide-Induced Degradation ProductTo study hydrogen peroxide-induced degradation, the initial studies

were performed in 3% hydrogen peroxide at room temperature for 24hr.Then, the drug was exposed to 6% hydrogen peroxide at room tempera-ture (25� 2�C) for a period of 8 days. For the HPTLC study, resultant solu-tions were applied on the TLC plate in such a way that final concentrationachieved was 2000ng � spot�1 and the chromatograms were run asdescribed in the HPTLC section.

Photochemical Degradation ProductThe photochemical stability of the drug was studied by exposing the stock

solution (1mg.mL�1) to direct sunlight for 8 days on a wooden plank kept ona terrace. Two microliters of the solution (2000ng � spot�1) were applied onTLC plate and chromatograms were run as described in the HPTLC section.

Optimization of a Stability Indicating HPTLC Method

The HPTLC procedure was optimized with a view to develop stabilityindicating assay method. Both the pure drug and degraded drug solutionwere spotted on TLC plates and run in different solvent systems. Initially,ethyl acetate, toluene, and methanol were tried, but pramipexole spotremained at the original place. When the volume of ethyl acetate wasincreased by 2mL and the volume of toluene was decreased by 1mL, theresulting Rf was 0.32 but compactness of spot was lacking; as a result, a con-siderable amount of peak tailing was observed. Therefore, to reduce thetailing and improve compactness of the spot 0.6mL, ammonia solutionwas added. Ultimately, the mobile phase consisting of ethyl acetate:toluene:methanol:ammonia in the ratio 8:1.5:0.5:0.6 (v=v=v=v) was optimized(Figure 2). The chamber was saturated with mobile phase vapor for30min and run up to a distance of 8 cm.

Validation of the Method

Validation of optimized HPTLC method was done with respect tofollowing parameters.


Linearity and RangeThe standard solutions of pramipexole were prepared to reach a con-

centration range of 200–2000ng �mL�1. One micro liter from each stan-dard solution was spotted on the TLC plate to obtain final concentrationof 200–2000ng � spot�1. Each concentration was spotted six times on theTLC plate. The plate was developed in the previously described mobilephase. The peak areas were plotted against the corresponding concentra-tions to obtain the calibration graphs.

PrecisionPrecision of the method was verified by repeatability and intermediate

precision studies. Repeatability studies were performed by analysis of threedifferent concentrations (200, 1000, and 2000ng � spot�1) of the drug inhexaplicate on the same day. Intermediate precision of the method waschecked by repeating the studies on three different days.

Limit of Detection and Limit of QuantitationLOD and LOQ values represent the concentrations of analyte that

would yield signal-to-noise (S=N) ratios of 3 for the LOD and 10 for theLOQ. LOD and LOQ were determined by measuring the magnitude ofanalytical background; a blank was spotted and the S=N for pramipexolewas calculated by spotting a series of solutions until the S=N values were3 for LOD and 10 for LOQ. To determine the LOD and LOQ, serialdilutions of pramipexole was made from the standard stock solution inthe range of 10–200 ng � spot�1. The samples were applied to the TLC plate,the chromatograms were run as described in the instrumentation section,and the measured signals from the samples were compared with those fromblank samples.

FIGURE 2 Densitogram of standard pramipexole; Peak 1 (Rf: 0.22� 0.02), Mobile phase: ethyl acetate-toluene-methanol-ammonia (8:1.5:0.5:0.6, v=v=v=v).


Robustness of the MethodBy introducing small changes in themobile phase composition (�0.1mL

for each component) the effects on the results were examined. Mobilephases having different composition like ethyl acetate: toluene: methanol:ammonia (8.1:1.5:0.5:0.6, v=v=v=v), ethyl acetate:toluene:methanol:ammonia (7.9:1.5:0.5:0.6, v=v=v=v), ethyl acetate:toluene:methanol:ammo-nia (8:1.6:0.5:0.6, v=v=v=v), ethyl acetate:toluene:methanol:ammonia (8:1.4:0.5:0.6, v=v=v=v) and so on were tried and chromatograms were run. Theamount of mobile phase was varied in the range of�5%. The plates were pre-washed by methanol and activated at 60�C for 2, 5, and 7min respectivelyprior to chromatography. Time from spotting to chromatography and fromchromatography to scanning was varied by �10min. Robustness of themethod was done at three different concentration levels 200, 1000, and2000ng � spot�1 for pramipexole.

SpecificityThe specificity of the method was ascertained by analyzing standard

drug and sample. The spot for pramipexole in sample was confirmed bycomparing the Rf and UV spectrum of the spot with that of standard.The peak purity of pramipexole was assessed by comparing the spectrumat three different levels, that is, peak start (S), peak apex (M), and peakend (E) position of the spot.

AccuracyAccuracy of the developed method was determined by applying the

method to a drug sample (pramipexole tablets) to which a known amountof pramipexole standard powder corresponding to 80, 100, and 120% oflabel claim were added (standard addition method); after mixing andextraction, the extract was analyzed as described in the HPTLC section.

Analysis of Marketed Formulation

To determine the content of pramipexole in conventional tablets[Brand name: PRAMIPEX (Sun Pharmaceuticals Industries Ltd.) (BatchNo. PXPF0934), label claim: 1mg pramipexole per tablet], the contentsof twenty tablets were weighed, their mean weight determined and finelypowdered. An equivalent weight of the powder=triturate was transferredto a 10mL volumetric flask containing 7mL methanol, sonicated for30min, and diluted to 10mL with methanol. The resulting solution wascentrifuged at 3000 rpm for 5min and supernatant was analyzed for drugcontent (100mg �mL�1). Nine microliters of the aforementioned solution(900 ng � spot�1) was applied to the TLC plate followed by development


and scanning as described in the HPTLC section. The analysis was repeatedin triplicate. The possibility of excipient interference in the analysis wasstudied.

RESULTS AND DISCUSSION

Stability Indicating Property

HPTLC studies of samples obtained on stress testing of pramipexoleunder different conditions using ethyl acetate:toluene:methanol:ammonia(8:1.5:0.5:0.6, v=v=v=v) as the mobile solvent system indicated the followingdegradation behavior.

Acid Induced Degradation ProductThe rate of degradation in acid was slower as compared to that of alkali.

Initially, 0.1N and 1N hydrochloric acid was used at 80�C for 24hr but nodegradation was observed; hence, the strength of acid was increased,10–20% degradation was observed by heating drug solution with 3N hydro-chloric acid at 80�C for 48hr. The two acid degradation products wereobserved at Rf 0.12 and 0.65 (Figure 3).

Base Induced Degradation ProductThe drug was found to be highly labile to alkaline degradation. Initially,

0.1N and 1N sodium hydroxide was used at 80�C for 24 hr but no degra-dation was observed; hence, the strength of alkali was increased, 10–20%degradation was observed by heating the drug solution with 2N sodiumhydroxide at 80�C for 24hr. The densitogram of two base degradationproducts were observed at Rf 0.13 and 0.67 (Figure 4).

FIGURE 3 Densitogram of acid degradation product; Condition: 3N HCl at 80�C for 48 hr; Peak 1(degraded, Rf: 0.12), Peak 2 (pramipexole, Rf: 0.20), and Peak 3 (degraded, Rf: 0.65).


Hydrogen Peroxide Induced Degradation ProductIn 6% hydrogen peroxide at room temperature the drug was found to

be stable to degradation as negligible degradation was seen after exposingthe drug to 6% hydrogen peroxide for 8 days.

Photochemical Degradation ProductWhen drug was exposed to photodegradation for 1 day, no significant

degradation was observed, then time of exposure was increased. Tenpercent degradation was observed after exposure for 8 days with thegeneration of one peak at Rf 0.23 (Figure 5).

Validation of the Stability Indicating Method

The results of validation studies on the stability indicating HPTLCmethod developed for pramipexole in the current study involving ethyl

FIGURE 4 Densitogram of base degradation product; Condition: 2N NaOH 80�C for 24 hr; Peak 1(degraded, Rf: 0.13), Peak 2 (pramipexole, Rf: 0.22), and Peak 3 (degraded, Rf: 0.67).

FIGURE 5 Densitogram of pramipexole exposed to photodegradation for 8 days; Peak 1 (degraded, Rf:0.23) and Peak 2 (pramipexole, Rf: 0.29).


acetate:toluene:methanol:ammonia (8:1.5:0.5:0.6, v=v=v=v) as the mobilephase are given in the following sections.

LinearityThe response for the drug was linear (r2¼ 0.9986) in the concentration

range between 200–2000ng � spot�1. The mean (�RSD) values of slope,intercept, and correlation coefficient were 4.1411 (�0.965), 768.73(�1.24) and 0.9986 (�1.42), respectively.

PrecisionThe results of the repeatability and intermediate precision experiments

are shown in Table 1. The developed method was found to be precise as theRSD values for repeatability and intermediate precision studies were <2%,respectively, as recommended by ICH guideline.

LOD and LOQThe signal:noise ratios of 3:1 and 10:1 were considered as the LOD and

LOQ, respectively. The LOD and LOQ were found to be 30ng � spot�1 and200ng � spot�1, respectively.

Robustness of the MethodThe standard deviation of peak areas was calculated for each parameter

and RSD (%) was found to be less than 2%. The low values of RSD (%) asshown in Table 2 indicated robustness of the method.

SpecificityThe peak purity of pramipexole was assessed by comparing its respect-

ive spectrum at peak start, apex, and peak end positions of the spot, that is,r (S, M)¼ 0.9994 and r (M, E)¼ 0.999. Good correlation (r¼ 0.9996) wasalso obtained between standard and sample spectra of pramipexole.

Recovery StudiesAs shown from the data in Table 3. Good recoveries of the drug in the

range from 98.5 to 99.1% were obtained at various added concentrations.

TABLE 1 Precision Study of Proposed Stability Indicating HPTLC Method

Repeatability (n¼ 6) Intermediate Precision (n¼ 6)

Conc. ng � spot�1 Found Conc.� SD RSD (%) Found Conc.� SD RSD (%)

200 197.21� 1.45 0.736 198.39� 0.76 0.3801000 997.4� 0.81 0.217 998.315� 1.8 0.1802000 1997.4� 0.81 0.040 1998.35� 1.8 0.090


TABLE 2 Robustness Testing of Proposed Stability Indicating HPTLC Methoda

Parameter SDb of Peak Area RSD (%)b

Mobile phase composition 2.64 0.192Amount of mobile phase 3.50 0.250Time from spotting to chromatography 1.52 0.102Time from chromatography to scanning 1.36 0.098

an¼ 6.baverage of three concentrations 200, 1000, and 2000ng � spot�1.

TABLE 3 Recovery Studies of Proposed Stability IndicatingHPTLC Methoda

Actual Conc.ng � spot�1

CalculatedConc.� SD RSD (%) Recovery (%)

900 885.86� 1.025 0.114 98.421000 985.86� 0.16 0.162 98.581200 1189.2� 1.211 0.102 99.10

an¼ 6.

TABLE 4 Applicability of the Stability Indicating HPTLC Methodfor the Analysis of the Pharmaceutical Formulation

Pramipexole Found (mg per tablet)Commercial FormulationPRAMIPEX (1mg) Mean� SD n¼ 6 Recovery (%)

1st Lot 0.983� 0.569 98.32nd Lot 0.978� 0.247 97.8

TABLE 5 Summary of Validation Parameters

Parameter Data

Linearity range 200–2000ng � spot�1

Correlation coefficient 0.9986Limit of detection 30ng � spot�1

Limit of quantitation 200ng � spot�1

% Recovery (n¼ 6) 98.7%Precision [RSD (%)]Repeatability (n¼ 6) 0.331Inter day (n¼ 6) 0.22Robustness RobustSpecificity Specific


Analysis of Marketed Formulation

Experimental results of the amount of pramipexole in tablets,expressed as a percentage of the label claim were in good agreement withthe label claims, thereby suggesting that there is no interference from anyexcipients, which are normally present in tablets. The drug content wasfound to be 98.05%. Two different lots of commercially available pramipex-ole tablet were analyzed using the proposed procedures and the results aresummarized in Tables 4 and 5.

CONCLUSION

Introducing HPTLC in pharmaceutical analysis represents a major stepin terms of quality assurance. The developed HPTLC technique is precise,specific, accurate, and stability indicating. Statistical analysis proves that themethod is suitable for the analysis of pramipexole as a bulk drug and inpharmaceutical formulation without any interference from the excipient.This study is a typical example of stability indicating assay, establishedfollowing the recommendations of ICH guidelines. The method can beused to determine the purity of drug available from various sources bydetecting the related impurities. It is proposed for the analysis of drugand degradation products in stability samples in industry.

ACKNOWLEDGMENTS

Authors thank Dr. Reddy’s Laboratories Ltd. (Hyderabad, AndhraPradesh, India) for providing gift sample of standard pramipexole andDr. K. R. Mahadik, Principal, Poona College of Pharmacy, Pune, India forproviding necessary facilities to carry out the work.

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APPLICATION OF STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD FOR QUANTITATION OF PRAMIPEXOLE IN PHARMACEUTICAL DOSAGE FORM