INTRODUCTION:

Stability studies of drug substances via acid hydrolysis, base hydrolysis, oxidation, and thermal and photolytic stress testing are a part of development strategy under the International Conference on Harmonization (ICH) requirements. These studies provide information on a drug's inherent stability and help to validate analytical methods to be used for evaluation of stability. Stability-indicating assays are currently being developed by using the stress-testing approach of the ICH guidelines, Q1A [R2]. The method distinctly separated the drug and degradation products, even in actual sample^1,2. The approach has been further extended to stress tests of drug combinations also. These tests allow accurate and precise quantification of drug, its degradation products, and its interaction products^3,4,5.

Pramipexole (INN, trade names Mirapex® and Sifrol®) is a medication indicated for treating Parkinson’s disease and restless legs syndrome (RLS). It is also sometimes used off-label as a treatment for cluster headache or to counteract the problems with low libido experienced by some users of SSRI antidepressant drugs. Pramipexole has shown robust effects on pilot studies in bipolar disorder. Pramipexole is classified as a non-ergoline dopamine agonist.

Pramipexole

High-performance liquid chromatographic (HPLC) method has been reported for the determination of Pramipexole. To our knowledge, no stability-indicating HPLC assay method has been reported for the determination of Pramipexole in tablets in the presence of its degradants by using the ICH approach of stress testing. The focus of the present study is to develop a simple, rapid, precise, and accurate isocratic reversed-phase stability-indicating HPLC method for the determination of Pramipexole in tablet dosage form^{.6 .}

MATERIALS AND METHODS:

Apparatus:

Chromatography was performed with The HPLC system (Shimadzu Corporation, Japan), model Shimadzu VP, consisted of a system controller (CLASS-VP), on-line degasser (LC 2010C, Shimadzu), low pressure gradient valve (LC 2010C, Shimadzu), solvent delivery module (LC 2010C, Shimadzu), auto injector (LC 2010C, Shimadzu), column oven (LC 2010C, Shimadzu), and CLASS – VP software version = SPI, binary pump, auto injector (SIL-10AD VP, Shimadzu), column oven (CTO-10AS VP, Shimadzu) and PDA detector (PDA-SPD-M10A VP, Shimadzu Diode Array Detector) and Chem station (software). The seperation wera performed at room temperature on a Zorbex SB CN(250 ×4.6 mm , 5 µm ) column. The mobile phase was degassed by Oscar Ultra Sonics, OU- 72 (SPL) sonicater. The standard substances were weighed on analytical balance(AX 205, METTLER TOLEDO). Stability studies were carried out in Proto – Tech oven.

Reagent and Material:

Pure Pramipexole sample from Zydus Cadila (Ahmedabad, Gujarat) with 97.4 % purity. Marketed products, label claim: 0.5 mg Pramipexole per tablet, Zydus Cadila LTD was taken.

Acetonitrile, Methanol, and water (HPLC grade) were purchased from spectrochem Pvt. Ltd. (Mumbai, India). Hydrochloric acid, sodium hydroxide and hydrogen peroxide were purchased from Rankem (Mumbai); Tri Ethyl Amine and phosphoric acid was purchased from Spectrochem PVT Ltd., Mumbai.

Preparation of Standard Solutions:

Standard stock solution of Pramipexole (500µg/ml) was prepared in mobile phase. For the calibration plot of Pramipexole, varies dilution was prepared from the stock solution in range of 2-24 µg/ml. All the dilution was made with mobile phase.

Forced Degradation Studies of Standard Drug Solution and Sample:^7,8

A series of standard solution of Pramipexole was prepared by dissolving 50 mg standard Pramipexole in required mobile phase to dissolve it in a 100 ml volumetric flasks. 5 ml 5 N HCL, 5 ml 1 N NAOH and 5 ml 3% H2O2 were added to each flask. Flask containing 5 N HCL was heated for 2 hour at 80 ·c and flask containing 1 N NaOH was refluxed for 1 hour at 80·c. Flask containing H202 was allowed to react for 30 min at room temperature. Then all volumes were make up to the mark with mobile phase. 2 ml from each flask was further diluted to 100 ml by mobile phase. Same treatment was done with the samples taking 6 gms of sample, and following the same dilution procedure. Degradation was also carried out in the solid state by exposing pure drug to dry heat at 100 · C for 120 hours. For photolytic condition pure drug was exposed to UV light for 36 hours.

Chromatographic Separation:

HPLC studies were carried out with all the reaction solution individually, and with a mixture of the solution in which decomposition was observed. The stressed samples were initially analyzed by HPLC using a reversed- phase Zorbex SB CN column by using Tri Ethyl Amine buffer (PH 7): Methanol (65:35%V/V) as the mobile phase. During these studies, the injection volume was 50 µl. and the mobile phase flow rate was constant at 1 mi/min; the analytical wavelength was 263 nm. (Fig.1)

Method Validation:

Linearity was established by triplicate injection of solution containing standard Pramipexole in the concentration range of 2-24 µg/ml. The system precision was established by making 6 replicate injection of standard solution containing 10µg/ml Pramipexole. The method precision was established by making 6 replicate injection of test solution containing 10µg/ml Pramipexole. Accuracy was determined by fortifying e mixture of test solution with 3 standard solution containing known concentration of Pramipexole (5, 10, 15 µg/ml) and percent recoveries of added drug were determined. The specificity of the method was established through study of resolution factor of drugs peaks from the nearest peaks, and also from all other peaks. The specificity of the method toward the drug also established through determination of purity of Pramipexole peaks from HPLC analysis of mixture of stressed samples through study of purity plots by using a PDA detector.

Robustness of the method was determined by deliberately varying certain parameter like flow rate (ml/min), concentration of methanol (ml) in mobile phase. Each parameter was evaluated at 3 levels (-1, 0, +1).

RESULT AND DISCUSSION:

Chromatogram of optimized condition in HPLC.

Figure 1 Chromatogram showing the separation of Pramipexole in sample

Forced Degradation Studies:

Condition used for forced degradation were attenuated to achieve degradation in the range of 5-30 % . The following behavior of drugs was observed during the HPLC studies

Acidic condition:- The drug and was heated in HCL for varies conditions . About 10-25% degradation of Pramipexole was observed. The major degradation product formed from pramipexole in the product was at retention times of 3.3 min.(Fig.2)

Figure 2 Chromatogram showing the separation of Pramipexole in stressed sample of the synthetic mixture subjected to acid Hydrolysis

Degradation in Alkali:

Pramipexole underwent alkali hydrolysis but the rate of hydrolysis was faster than that under acidic condition.The degradation patterns was similar to that seen under acidic condition(Fig. 3)

Figure 3 Chromatogram showing the separation of Pramipexole in stressed sample of the synthetic mixture subjected to alkali Hydrolysis.

Oxidative Condition:

The drugs was highly liable to oxidative hydrolysis in 3% Peroxide , 5-30 % degradation was achieved in peroxide(Fig 4)

Figure 4 Chromatogram showing the separation of Pramipexole in stressed sample of the synthetic mixture subjected to Oxidative Hydrolysis.

Thermal Degradation:

The drug was relatively stable when exposed to dry heat at 80 ·C for 120 hours. The percentage of drug remaining after 120 hours of exposure to dry heat were in the range of 99-100%.(Fig 5)

Figure 5 Chromatogram showing the separation of Pramipexole in stressed sample of the synthetic mixture subjected to Thermal condition.

Photolytic Conditions

The drug was relatively stable when exposed to UV for 36 hours. The percentage of both drugs remaining after 36 hours of exposure to UV were in the range of 99-100%.(Fig 6)

Figure 6 Chromatogram showing the separation of Pramipexole in stressed sample of the synthetic mixture subjected to Photolytic condition.

Table 1. System suitability parameters for the determination of Pramipexole by proposed HPLC method

Parameter	Pramipexole
Linearity Range	2-24 µg/ml
Regression Coefficient	0.999
Slop	64.82
LOD	0.085
LOQ	0.259
Retention Time	7.3
Tailing Factor	1.33
Resolution Factor	-
Theoretical Plates	3136

*Average of Six Replicate

Table 2.Assay result obtained for solid dosage form by proposed HPLC method.

Tablet	Drug,mg/Tablet	Drug found ± SD
MIRAPREX	Pramipexole 0.5 mg	99.04±.0.79

*n = 3

Table 3. Recovery studies for determination of Pramipexole by proposed HPLC method.

Value	Set	mg added	Response	Percentage	% Recovered	Average
50	Set-1	25	324.851	51.33135604	102.6627121	101.84
	Set-2	25	320.544	50.6507851	101.3015702
	Set-3	25	321.348	50.77782922	101.5556584
100	Set-1	50	632.005	99.86631924	99.86631924	99.76003
	Set-2	50	630.96	99.70119349	99.70119349
	Set-3	50	631.032	99.71257057	99.71257057
150	Set-1	75	945.034	149.329621	99.55308069	99.60308
	Set-2	75	945.957	149.4754689	99.65031263
	Set-3	75	945.535	149.4087866	99.60585772
Standard for Calculation		632.851

*n = 3

Validation of Proposed Method:

The method was validated with respect to the following parameters given below;

Linearity:- Linearity calibration plot for above method was obtained in the calibration ranges of 2-24 µg/ml, and the correlation coefficient obtained were greater than 0.999(Table 1).

LOD and LOQ – The LOD value for Pramipexole was 0.070µg/ml and the LOQ value for Pramipexole was 0.213 µg/ml.

Precision :- Data obtained from analysis of the sample on the same day (n=6) and on consecutive days(n=6) are given in the Table 4. The RSD values obtained were well below 2%. The RSD value indicate that the method is sufficiently precise. The intermediate precision established for the method showed that similar resolution was obtained when the experiment was conducted with 2 different days.

Table 4 . Statistical evaluation of Precision for determination of Pramipexole by proposed HPLC method.

Parameter Value	Precision
Parameter Value	System Precision	Method Precision	Method Precision
1	636.292	633.937	630.182
2	636.815	632.665	631.254
3	634.526	632.542	633.254
4	633.12	631.937	633.657
5	633.12	634.199	633.128
6	637.21	632.78	633.348
Average	635.1805	633.01	632.4705
Std. Dev.	1.680554646	0.797425859	1.28718501
%RSD	0.264579068	0.125973659	0.203517

Accuracy:- Percent recoveries were calculated from the differences between the peak area obtained for the fortified and unfortified solutions. Good recoveries were obtained for each fortification level (Table 3) ,indicating that method is accurate.

Specificity: - The specificity of the HPLC method was shown by the complete separation of Pramipexole from its degradation products. Studies using PDA detection to determine the purity of Pramipexole peak showed peak purity index was always greater than 0.9999, thereby indicating that the Pramipexole peak was free from any coeluting peaks.

Robustness:- The method remained robust even with small variation in flow rate(±0.1 ml/min) and the concentration of Methanol; (±2 ml) in the mobile phase. There was no significant influence on the determination. Insignificant differences in peak area and less variability in retention times were observed(Table 5)

Table 5. Robustness studies for determination of Pramipexole by proposed HPLC method.

Compound	% RSD (n= 6)
Compound	Normal Condition	Changed Condition
Temperature	Normal	(-5°C)	(+5°C)
Pramipexole	0.26	0.14	0.24
pH	Normal	(-0.2 unit)	(+0.2 unit)
Pramipexole	0.26	0.33	0.23
Flow Rate	Normal	(-10%)	(+10%)
Pramipexole	0.26	0.031	0.27
Mobile phase ratio	Normal	(-2%)	(+2%)
Pramipexole	0.26	0.24	0.35

Applicability of Developed Method to Marketed Formulations:

A clear separation of the drugs and degradation products was obtained for tablets with no interference from excipients. This result indicate that the method could be extended to the study of available drug content in commercial products (Table 2)

CONCLUSION:

The stability indicating RP-HPLC methods for estimation of Pramipexole in its solid dosage forms was established as per the ICH guidelines. The forced degradation study and peak purity data confirm that there is no merging between peaks of active ingredients and any other degradation products as well as other additives. So the specificity of the proposed methods can be proved. The linearity of developed method for estimation was achieved in the range of 2-24 µg/ml (r²=0.999) for Pramipexole. From the validation results, we can conclude that our methods are simple, sensitive, rapid, linear, precise, rugged, accurate, and robust and hence they can be used for the routine analysis of Pramipexole in quality control department.

The work described in this paper has shown that the developed method is precise, accurate, linear and stability indicating. The method was found to be specific to the drugs, because the peaks of degradation products did not interfere with the drugs peaks. Application of this method to the determination of Pramipexole in tablet dosage form shows that neither the degradation nor the excipients interfere with the analysis. The finding indicates that the proposed method could be used as a stability-indicating method for simultaneous determination of Pramipexole either in the bulk drug or in pharmaceutical formulations.

REFERENCES:

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Received on 06.06.2011 Modified on 23.06.2011

Asian J. Research Chem. 4(9): Sept, 2011; Page 1393-1397