Development and validation of extractive visible spectrophotometric methods for determination of ropinirole hydrochloride in pharmaceutical dosage forms based on ion-association complex formation

K. Raghubabu¹*, V. Jagannadharao², B. Kalyana Ramu³

¹Department of Engineering Chemistry, AU College of Engineering (A), Andhra University, Visakhapatnam -530003 Andhra Pradesh (India)

²Department of Chemistry, Anil Neerukonda Institute of Technology and Sciences, Sangivalasa (AP) India.

³Department of Chemistry, Maharajah’s College (Aided and Autonomous), Vizianagaram-535002 (AP) India.

ABSTRACT:

Two simple and sensitive extractive visible spectrophotometric methods (A&B) for the assay of ropinirole hydrochloride in pure and dosage forms based on the formation of colored chloroform soluble ion-association associates under specified experimental conditions are described. Two dyes namely acidic dye Tropaeoline ooo (TPooo, method A), Azo carmine- G (ACG, method B) are utilized. The extracts of the ion-associates exhibit absorption maxima at 485nm and 550nm for methods A and B respectively. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (5.0-25) µg/ml for method A, (10-50) µg/ml for method B respectively. The proposed methods are applied to commercial available tablets and the results are statistically compared with those obtained by the UV reference method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the ropinirole hydrochloride in the presence of other ingredients that are usually present in dosage forms. These methods offer the advantages of rapidity, simplicity and sensitivity and low cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

KEYWORDS: Assay, ACG, Anti Parkinson’s agent, Ion-Association methods, Statistical analysis, TPOOO, Tablets.

INTRODUCTION:

Ropinirole hydrochloride (RPR) is an orally administered specific D₂and D₃ receptor non-ergoline dopamine antagonist. Chemically it is hydrochloride salt of 4-[2-(dipropyl amino) ethyl]-1,3-dihydro-2H-indol-2-one (Fig.1). It is used in the treatment of early and advanced Parkinson’s disease caused by deficit of dopamine. It has high relative in vitro specificity and acts by binding with higher affinity to D₃ than to D₂ or D₄ receptor subtypes. The mechanism of ropinirole induced postural hypertension is presumed to be due to a D₂-mediated blunting of the noradrenergic response to standing and subsequent decrease in peripheral vascular resistance. The drug is listed in Merck Index [1], Martindale the Complete drug reference [2].

Fig.1: Chemical structure of ropinirole hydrochloride

Several analytical techniques like HPLC [3-6], UPLC [7], TLC [8], Chemo metric [9], LC-MS [10], UV [11-13], capillary LC [14] visible spectrophotometric [15] and spectrophotometric and spectro fluorometric method [16] have been reported for its determination in plasma and tablet dosage forms. For routine analysis, simple, rapid and cost effective visible spectrophotometric methods are required and preferred. As the extraction spectrophotometric procedures are popular for their sensitivity and selectivity in the assay of drugs, the extractive spectrophotometric acid- dye technique [17] was therefore, utilized in the present work for the estimation of RPR. The present paper describes two simple and sensitive extraction visible spectrophotometric methods for the determination of RPR, based on its tendency to form chloroform extractable ion-associates with acidic dye TP ooo (C.I No.14600) belonging to azo category dye (method A) or Azocarmine G (C.I No.50085) belonging to Phenazine category dye (method B) under experimental conditions by exploiting the basic nature (tertiary amine) of the drug molecule.

The proposed methods for RPR determination have many advantages over other analytical methods due to its rapidity, lower cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are inexpensive and available in any analytical laboratory. These methods can be extended for the routine assay of RPR formulations.

MATERIALS AND METHODS:

A Shimadzu UV-Visible spectrophotometer 1601 with1cm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-361 was used for pH measurements. All the chemicals used were of analytical grade. Tablets were purchased from local market. Tropaeolin 000 (Fluka, 0.2%, 5.7x10^-3M prepared by dissolving 200mg of Tropaeolin 000 in 100ml distilled water and subsequently washed with chloroform to remove chloroform soluble impurities), 0.1M HCl (prepared by diluting 8.7ml of Con. Hydrochloric acid to 1000ml with distilled water and standardized) ACG solution ( Gurr, 0.05%, 8.75x10^-4M prepared by dissolving 50mg of azocarmine G in 100ml of distilled water containing traces of sodium hydroxide and subsequently washed with chloroform to remove chloroform soluble impurities), pH 1.5 Buffer solution (prepared by mixing 289ml of 0.1M glycine solution(7.507g of glycine and 5.85 g NaCl was dissolved in 100ml of distilled water) with 711 ml of 0.1M HCl and the pH of the solution was adjusted to 1.5) were prepared.

Preparation of Standard stock solution: The standard stock solution (1mg/ml) of RPR was prepared by dissolving initially in 10ml of 0.1M NaOH and followed by dilution to 100 ml with distilled water. The working standard solutions of RPR were obtained by appropriately diluting the standard stock solution with the same solvent.

Preparation of Sample solution: About 10 tablets were pulverized and the powder equivalent to 100mg of RPR was weighed, dispersed in 25ml of IPA, sonicated for 30 minutes and filtered through Whatman filter paper No 41.The filtrate was evaporated to dryness and the residue was dissolved as under standard solution preparation.

Assay: Aliquots of the standard RPR solution [1.0-5.0 ml, 50µg/ml (method A) and 1.0-5.0ml, 100µg/ml (method B)] were placed in a series 125ml separating funnels. Then 6.0ml of 0.1M HCl and 2.0ml of TPooo solution 5.70x10^-3M(for method A) or 6.0 ml of pH 1.5 buffer solution and 2.0 ml of ACG solution (8.75x10^-4M)(for method B) were added. The total volume of aqueous phase in each separating funnel was adjusted to 15.0ml with distilled water. Then 10.0ml of chloroform was added to each separating funnel and the contents were shaken for 2 minutes. The two phases were allowed to separate. The absorbance of the separated chloroform layer were measured at 485nm (method A) or 550 nm (method B) (Fig. 2and3 showing absorption spectra) against a reagent blank within the stability period (5 minutes to 1hour). The amount of drug was computed from its calibration graph (Fig.4 and 5).

Fig.2: Absorption spectra of RPR-TPOOO

Fig.3: Absorption spectra of RPR-ACG

Fig.4: Beer’s Law Plot of RPR-TPOOO

Fig.5: Beer’s Law Plot of RPR-ACG

RESULTS AND DISCUSSIONS:

Optimum operating conditions used in the procedure were established by adopting variation of one variable at a time (OVAT) method. The effect of various parameters such as time, volume and strength of TPooo, ACG reagents, 0.1M HCl, pH buffer solutions and solvent for final dilution of the colored species were studied. TPooo and ACG were preferred for this investigation as they yield high molar absorptivity values among six dyes belonging to different chemical classes. The water immiscible solvents tested for the extraction of colored complex into organic phase include chloro benzene, dichloromethane, carbon tetrachloride, benzene, nitro benzene, n-butanol or chloroform. Chloroform was preferred for its selective extraction of colored drug -dye complex into organic layer from the aqueous phase. The stoichiometric ratio of the dye-drug was determined by the slope ratio method and was found to be 1:1 for methods A and B respectively. The optical characteristics such as Beer’s law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation, (calculated from the six measurements containing 3/4^th of the amount of the upper Beer’s law limits), Regression characteristics like standard deviation of slope (S_b), standard deviation of intercept (S_a), standard error of estimation (S_e) and % range of error (0.05 and 0.01 confidence limits) were calculated and the results are summarized in Table-1. Commercial formulations containing RPR were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table-2.

Table 1: Optical characteristics, precision and accuracy of proposed methods

Parameters	Method A	Method B
λ max(nm)	485	550
Beer’s law limit (µg/ml)	5- 25	10-50
Sandell’s sensitivity (µg/cm^2/0.001 abs. unit)	0.0041	0.0038
Molar absorptivity (Litre/mole/cm)	6.36x10^4	2.09x10^4
Regression equation (Y) ^*= a +b c
Intercept (a)	-0.001	0.012
Slope(b)	0.024	0.007
%RSD	0.95	1.902
% Range of errors (95% Confidence limits) 0.05 significance level 0.01 significance level	0.997 1.641	1.997 3.13

*Y= a + b c; Where Y= absorbance, c= concentration of RPR in µg/ml.

Table 2: Analysis of RPR in pharmaceutical formulations by proposed and reference methods.

Method	*Formulations	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
			**Amount found ± SD	t	F
A	Batch-1	2	1.992±0.003	0.43	1.53	1.993 ± 0.002	99.61 ± 0.15
	Batch-2	2	1.987±0.004	0.87	1.54	1.99± 0.003	99.37 ± 0.20
B	Batch-1	2	1.991±0.0026	1.53	1.65	1.993 ± 0.002	99.563 ± 0.13
	Batch-2	2	1.987±0.0058	0.55	3.53	1.99± 0.003	99.406 ± 0 .29

* Different batches from two different companies (Batch-1 Ropin tablets of East west, Batch 2: Ropitor tablets of Torrent)

**Average ± Standard deviation of six determinations, the t- and F-values refer to comparison of the proposed method with reference method (UV). Theoretical values at 95% confidence limits t =2.57 and F = 5.05.

# Recovery of 10mg added to the pre-analyzed sample (average of three determinations). Reference method (reported UV method) using double distilled water (λ _max=249nm).

Fig.6: Scheme of the reaction

Chemistry of colored species:

The protenated tertiary nitrogen (positive charge) of the drug molecule in acid medium is expected to attract the oppositely charged part (negative charge) of the dye and behave as a single unit being held together by electrostatic attraction as given in scheme (Fig.6).

CONCLUSION:

A significant advantage of an extraction spectrophotometric determination is that it can be applied to the determination of individual compounds in a multi component mixture. This aspect of spectrophotometric analysis is of major interest in analytical chemistry, since, it offers distinct possibilities in assay of a particular component in a complex dosage formulation. In the present study, RPR was determined successfully as pure compound as well as a single component in representative dosage formulations. The proposed methods applicable for the assay of drug and the advantage of wider range under Beer’s law limits. The proposed extractive visible spectrophotometric methods are validated as per ICH guide lines and possess reasonable precision, accuracy and simple, sensitive. These methods can be extended for the routine assay of RPR in formulations.

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Received on 02.04.2012

Accepted on 10.05.2012

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Research Journal of Pharmaceutical Dosage Forms and Technology. 4(3): May-June 2012,172-176