Extractive Spectrophotometric Methods for Determination of Pioglitazone Hydrochloride Using Acidic Triphenylmethane Dyes

 

M. Chennaiah¹, T. Veeraiah², T. Charan Singh¹ and G. Venkateshwarlu¹*

¹Department of Chemistry, Nizam College (O.U), Hyderabad, 500001, India

²Department of Chemistry, SAP College Vikarabad, Ranga Reddy, A P, 501101, India

*Corresponding Author E-mail: venkateshwarlugoud@yahoo.com

ABSTRACT:

Three simple and sensitive extractive spectrophotometric methods have been described for the assay of Pioglitazone hydrochloride either in pure form or in pharmaceutical formulations. The developed methods involve formation of coloured chloroform extractable ion-pair complexes of the drug with bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol purple (BCP) in acidic medium. The extracted complexes showed absorbance maxima at 415 nm for all three methods. Beer’s law is obeyed in the concentration ranges 2.0-30, 3.0-30 and 2.5-25µg/ml with BTB, BPB and BCP respectively. The effect of concentration of dye, pH, and interference of excipients have been studied and optimized. The limits of detection and quantification have been determined for three methods. All the three methods have been validated as per the guidelines of ICH. The methods have been applied to the determination of drug in commercial tablets and results of analysis were validated statistically through recovery studies.

 

 

 

INTRODUCTION:

Pioglitazone hydrochloride [(±) - 5- [[4- [2- (5- ethyl- 2- pyridinyl)ethoxy] phenyl] methyl] - 2, 4-] thiazolidinedione monohydrochloride is an oral antidiabetic agent that has been shown to affect  abnormal glucose and lipid metabolism associated with insulin resistance by enhancing insulin action on peripheral tissues in animal models^1-5. This belongs to a different chemical class and has a different pharmacological action than the sulfonylureas, metformin, or α glucosidase inhibitors. Because of its physiological importance various physical methods including spectrophotometry have been applied for  quantification of this drug involve high performance liquid chromatography (HPLC)^6-7, RP-L⁸, RP-HPLC⁹, HPLC/MS¹⁰, HPLC/MS/MS¹¹, TLC¹², capillary electrophoresis(CE)¹³, Chemiluminescence¹⁴, membrane selective electrode¹⁵, potentiometry¹⁶, extyractive spectrophotometric method¹⁷ and  UV spectrophotometricmethod¹⁸.

 

Thorough survey of literature revealed that, although quantification of drugs based on ion-pair complexation is simple, sensitive and accurate, the studies involving bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol purple (BCP) as complexing agents have not been reported yet. In this communication quantitative determination of pioglitazone using BTB, BPB and BCP is described and the methods have been validated in the lines of ICH.

 

In this paper we report three simple and sensitive extractive spectrophotometric methods for the assay of Pioglitazone hydrochloride. The methods are based on ion-pair complexation of drug with dyestuffs such as bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol purple (BCP) and subsequent extraction into chloroform and measure the absorbance of colour complex.

 

MATERIALS AND METHODS:

Pioglitazone hydrochloride is procured from Hetero labs limited, Hyderabad as a gift sample. The dyestuffs viz., BTB, BPB and BCP (AR grade) supplied by SD Fine Chemicals Ltd. Mumbai, are used without any further purification.  The dyestuffs were used as 0.025% solutions in doubly distilled water. Sodium acetate-hydrochloric acid buffers¹⁹ of pH 2.5, 2.8 and 2.5 were prepared by mixing 50ml of 1.0M sodium acetate solution with 50.50, 49.50 or 50.50 ml, respectively, of 1.0 M HCl solution and diluted to 250 ml with doubly distilled water. The pH of each solution was adjusted to an appropriate value with the aid of a pH meter. Chlorofom (HPLC grade) supplied by SD Fine Chemicals Ltd. Mumbai is used throughout the work. Stock solutions were prepared for all the dyes and drugs (25mg/100ml).

 

The spectra (Fig. 1) of ion-pair complexes have been recorded on Shimadzu 140 double beam spectrophotometer, Thermo Nicolet 1000 and also on ELICO 159 UV-Visible single beam spectrophotometer using quartz cells of 10 mm path length. An Elico model Li-120 pH meter was used for pH measurement.

 

Fig. 1.  Absorption spectra of Pioglitazone hydrochloride-dye complex extracted into 10 ml chloroform: (a) drug = 25 mg  ml^-1 + 5 ml of 0.025% BTB + 5 ml of pH 2.8 buffer;  (b) drug = 20 mg ml^-1 + 5 ml of 0.025% BPB + 5 ml of pH 2.5 buffer; (c) drug = 22.5 mg ml^-1 + 5 ml of 0.025% BCP + 5 ml of pH 2.5 buffer

 

Fig. 2 Calibration graphs for Drug-BTB, BPB & BCP ion pair complexes

 

Concentration (µg ml^-1)

 

Calibration curve:

Different aliquots of drug solution were transferred into 125 ml separating funnel. To this 5 ml of buffer (pH 2.5 and 2.8), 5 ml of dye were added and total volume was made up to 20 ml with water. 10 ml of chloroform was added and the contents were shaken for 5 min. The two layers were allowed to separate for 5 min. The organic layer was separated and absorbance of yellow colored solution which is stable at least for 3 hrs is measured at 415 nm against blank similarly prepared. The same procedure of analysis is followed either for assay of pure drug or for dosage form. The calibration graphs (Fig. 2) are linear over the concentration ranges are within the permissible range. The optical characteristics and statistical data for the regression equation of the proposed methods are presented in (Table 1).

 

Procedure for the assay of pure drug:

Five different solutions of pure drug in the range of calibration curve were selected and the recovery experiments were performed. The recoveries and their relative standard deviations are tabulated in (Table 2).

 

TABLE -1: OPTICAL CHARACTERISTICS AND STATISTICAL FOR THE REGRESSION EQUATION OF THE PROPOSED METHODS

Parameters	Extraction methods with
	BTB	BPB	BCP
λmax (nm) Beer’s law limit (μg ml-1) Molar absorptivity (L mol-1 cm-1) Formation constant, K, M-1 Sandell sensitivity (μg cm-2) Slope (specific absorptivity),  b Intercept (a) Correlation coefficient (r) Standard deviation of intercepts (% n=6) Limit of detection, μgml-1 Limit of quantification, μgml-1 Regression equation	415 2.0 - 30 17234 1.44 x 106 0.023 0.0435 0.0189 0.9978 0.0058 0.44 1.32 Y= 0.0435C+ 0.0189	415 3.0 - 30 16943 1.25x 106 0.0229 0.0436 -0.094 0.9977 0.0071 0.5374 1.612 Y= 0.0436C -0.094	415 2.5-25 20576 1.51x 106 0.0171 0.0584 0.0088 0.999 0.00103 0.578 1.734 Y= 0.0584C +0.0088

^aWith respect to Y=bc+a, where C is the concentration (μg ml^-1) and Y is absorbance ^bSix replicate samples.

 

 

TABLE – 2: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF PIOGLITAZONE HYDROCHLORIDE IN PURE FORM

Taken (μg ml-1)	Proposed methods						Reference method[18]
	Found (μg ml-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCP	BTB	BPB	BCP
4 8 12 16 20 RSD (%)	4.002 8.089 12.024 15.913 20.002	4.029 7.94 12.05 15.93 19.948	3.98 8.013 12.02 15.96 20.07	100.05 101.12 100.2 99.45 100.01 0.6	100.74 99.29 100.42 99.57 99.74 0.61	99.42 100.17 100.197 99.76 100.36 0.38	99.11         1.1865
Mean±SD t-test F-test				100.2±0.6 1.92 0.26	99.95±0.61 1.53 0.266	99.9±0.38 2.04 0.105	99.11±1.18

 

 

TABLE – 3: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF PIOGLITAZONE HYDROCHLORIDE IN PHARMACEUTICALS FORM

Taken (μg ml-1)	Proposed methods						Reference method[18]
	Found (μg ml-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCP	BTB	BPB	BCP
Pioglit-15 15mg/tab 4.5 9 13.5 18 22.5 RSD (%)	4.5 9.01 13.49 18.002 22.49	4.49 9.08 13.48 18.005 22.47	4.49 9.01 13.74 17.85 22.5	100.19 100.15 99.97 100.01 99.94 0.1083	99.8 100.87 99.88 100.03 99.89 0.4416	99.78 100.11 101.75 99.15 100.01 0.9633	99.11         1.186
Mean±SD t-test F-test				100.05±0.11 1.95 0.0085	100.1±0.44 1.89 0.14	100.2±0.9 1.8 0.67	99.11±1.176

 

 

Procedure for the assay of dosage forms:

Ten tablets of pioglit-15 mg are powdered and dissolved in doubly distilled water and stirred thoroughly, filtered through a Whatman No. 42 filter paper. This solution was transferred into 100 ml standard volumetric flask and diluted with doubly distilled water as required. Different solutions of drug in the range of calibration curve were chosen and the assay was estimated using the calibration curve. The results of the recovery experiments are tabulated in (Table 3).

 

RESULTS AND DISCUSSION:

Pioglitazone hydrochloride forms ion-pair complexes in acidic buffer with dyestuffs such as bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol purple (BCP) and these complexes are quantitatively extracted into chloroform. Ion-pair complexes of drug with BTB, BPB and BCP absorbed maximally at 415 nm. The reagent blank under similar conditions showed no absorption.

 

In order to establish molar ratio between Pioglitazone hydrochloride and dyestuffs used, the Job’s method of continuous variation²⁰ has been applied. In this method, solutions of drug and dyestuff with identical molar concentrations [8 x 10^-5M] were mixed in varying volume ratios in such a way that the total volume of each mixture was the same. The absorbance of each solution was measured and plotted against the mole fraction of the drug, [drug]/ [drug] + [dyestuff] (Fig. 3). This measurement showed that 1:1 complex was formed with each dyestuff. The formation constants^21,22 were also estimated and found to be 1.25x 10⁶, 1.44 x 10⁶ and 1.31x 10⁶ K M^-1 for complexes with BTB, BPB and BCP  respectively.

 

Fig. 3 Continuous-variations study of drug-dye systems: [Drug] = [Dye] = 8x10^-5M

 

Pioglitazone hydrochloride contains pyridine nitrogen which is protonated in acid medium, while sulphonic acid group is present in BTB, BPB and BCP, that is the only group undergoing dissociation in the pH range 1-5. The colour of such dyes is due to the opening of lactoid ring and subsequent formation of quinoid group. It is supposed that the two tautomers are present in equilibrium but due to strong acidic nature of the sulphonic acid group, the quinoid body must predominate. Finally the protonated Pioglitazone hydrochloride forms ion-pairs with the dyestuffs which are quantitatively extracted into chloroform. The possible reaction mechanisms are proposed and given in (Scheme 1).

 

The influence of pH on the ion-pair formation of Pioglitazone hydrochloride with various dyestuffs has been studied using sodium acetate-hydrochloric acid buffer. The results are shown in (Fig.  4). It is evident that absorbance of complexes with BTB, BPB and BCP was found to be constant within the pH ranges 2.2-3.3, 2.0-3.0 and 2.0-3.0 respectively. Thus, all the absorbance measurements were made at pH 2.8, 2.5 and 2.5 with BTB, BPB and BCP respectively.

 

Scheme 1 Drug-dye complex

 

Fig. 4 Effect of pH

[Drug] = [12.5µg ml^-1, [Dye] = 5ml of 0.025%

 

The effect of dyestuff concentrations was also studied by adding different volumes of dyestuff to a constant amount of Pioglitazone hydrochloride (12.5 µg ml^-1). It is apparent from (Fig. 5). That the maximum absorbance, in each case, was found with 3.0 ml of dyestuff, beyond which absorbance was constant. Thus, 5 ml of each dyestuff was used for ion-pair formation throughout the experiment.

 

Fig. 5 Influence of the volume of 0.025% Dye

[Drug] = [12.5µg ml^-1]

 

A systematic study of the effect of foreign species present along with Pioglitazone hydrochloride on the determination of Pioglitazone hydrochloride at 12.5 µg ml^-1 levels was undertaken. This study was carried out by following the proposed procedures for a 10 ml sample system, by adding a known amount of foreign species to a Pioglitazone hydrochloride solution of 12.5 µg ml^-1. (Table 4) summarizes the results obtained. However, the drug content from the powdered capsules was extracted into chloroform, which completely removes any interference by the common excipients found in formulations.

 

TABLE – 4: INTERFERENCE STUDY

S. No.	Excipients	Tolerance limit (μg ml-1)
1 2 3 4 5 6	Microcrystalline cellulose Starch Lactose Magnesium stearate   Colloidal silicon dioxide Titanium dioxide	85 165 130 85 65 20

Validation of the proposed method:

All the three proposed methods have been validated in terms of guideline proposed by ICH²³ viz. selectivity, specificity, accuracy, precision, limits of calibration curve, LOD, LOQ, robustness, ruggedness and regression equation. The student t-test and variance F-test have been performed in comparison with a reference method. (Table 1) summarizes the values for Beer’s law limits, molar absorptivity, regression equation, correlation coefficients, relative standard deviation and recoveries. To test the reproducibility of the proposed methods, six replicate determinations of 12.5µg ml^-1 of Pioglitazone hydrochloride were made. The coefficient of variation was found to be less than 1.2% for all the procedures.

 

The proposed methods have been successfully applied to the determination of Pioglitazone hydrochloride in pharmaceutical preparations. The performance order of the proposed methods is BCP>BTB>BPB. The results obtained and shown in (Table 2 & Table 3) were compared to those obtained by a reference method¹⁸ by means of t-test at 95% confidence level. In all cases, the average results obtained by proposed methods and reference method were statistically identical, as the difference between the average values had no significance at 95% confidence level.

The proposed methods are simple, sensitive and reproducible and can be used for routine analysis of Pioglitazone hydrochloride in pure form and in formulation.

 

CONCLUSION:

Pioglitazone hydrochloride formed ion pair complexes with acidic dyes with 1:1 composition and extractable in to chloroform for assay of the drug. The method is validated and applied to pharmaceuticals.

 

ACKNOWLEDGEMENTS:

The authors are grateful to Head, Department of Chemistry and Principal, Nizam College for providing facilities. MC is thankful to UGC for FDP fellowship. TV is thankful to the Management of SAP College, Vikarabad for providing facilities and to the UGC for financial assistance under Major Research Project.

 

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Received on 28.03.2011        Modified on 08.04.2011

Accepted on 11.04.2011        © AJRC All right reserved