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SPECTROPHOTOMETRIC DETERMINATION OF ETHAMBUTOL IN PURE AND PHARMACEUTICAL FORMS USING TRIPHENYL METHANE DYES

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SPECTROPHOTOMETRIC DETERMINATION OF ETHAMBUTOL IN PURE AND PHARMACEUTICAL FORMS USING TRIPHENYL METHANE DYES

Sayanna 1, M. Jyothi 2, T. Veeraiah *1 and CH. Venkata Ramana Reddy 3

Department of Chemistry 1, SAP College, Vikarabad -501101, Ranga Reddy Dist., Telangana, India.

Department of Chemistry 2, RBVRR Women’s College, Narayanguda, Hyderabad - 500029, Telangana, India.

Department of Chemistry 3, JNTUH College of Engineering, Kukatpally, Hyderabad-500085, Telangana, India.

ABSTRACT: Simple and sensitive extractive spectrophotometric methods for the determination of Ethambutol in pure form and in pharmaceutical formulations using triphenyl methane dyes have been developed. The developed methods involve formation of coloured chloroform extractable ion-pair complexes of the drug with triphenyl methane dyes viz., bromocresol green (BCG), bromocresol purple (BCP) and bromophenol blue (BPB) in acidic medium. The extracted complexes formed with BCG, BCP and BPB showed absorbance maxima at 420, 419 and 415 nm respectively.  The stoichiometry of the ion- pair complex is found to be 1:1 in each case.  Beer’s law is obeyed in the concentration ranges 2.0-25, 3.0-30 and 4.0-40 μg/ml with BCG, BCP and BPB respectively. The effect of concentration of dye, pH, and interference of excipients have been studied for optimization. The limits of detection and quantification have been determined for all the three methods. These methods have been validated as per the guidelines of ICH. The results of analysis were validated statistically through recovery studies.

Keywords:

Spectrophotometry,

Ethambutol, Bromocresol green, Bromocresol purple, Bromophenol blue, Ion-pair complex, Validation

 

INTRODUCTION:  Ethambutol is a white crystalline powder, soluble in chloroform, sparingly soluble in water, it has pKa 6.35 and 9.35, chemically called 2, 2’-(1, 2-Ethylenediimino) bis- 1-butanol [Fig.1]. It is an oral chemotherapeutic agent which is specifically effective against mycobacterium, which was the recent discovered (1961) first line treatment as anti-TB drug 1. It has excellent activity in vitro and in vivo against Mycobacterium tuberculosis.

fig-1

FIG. 1: CHEMICAL STRUCTURE OF ETHAMBUTOL

Ethambutol is bacteriostatic to both intracellular and extracellular organisms 2. Many reports have documented the efficacy of Ethambutol for the treatment of bronchopulmonary, urogenital and osteoarticular tuberculosis and Mycobacterium avium complex 3. Microbiological and gas liquid chromatographic methods are available for assaying Ethambutol concentrations in serum and cerebrospinal fluid. In microbiological assay, when Ethambutol (EMB) is present along with other drugs in plasma, it cannot be assayed without the interference of other drugs 4. Gas liquid chromatography methods using capillary columns are available but they involve a series of steps for preparation of the sample before analysis and needs internal standard 5. Ethambutol has been routinely assayed using a non-aqueous titration method 6, 7. It was observed that with certain pharmaceutical preparations, the values were higher than the stated contents. Another method for estimation of Ethambutol content in pharmaceutical preparations involves reaction with cupric chloride and measurement of the absorption in the visible range, which is again a time consuming procedure 8, 9, 10. In drug formulations containing EMB in addition to other anti-TB drugs, EMB has to be exclusively estimated without the interference of other drugs.

According to the literature available, methods found for the determination of Ethambutol in plasma were in High Performance Liquid Chromatography (HPLC) 11, 12, 13 where, extraction process involves more time consuming and tedious steps like derivatization. Though methods are available for Liquid Chromatography Mass Spectrometer tandem Mass Spectrometry (LC-MS/MS)14-16, it is found that the chromatographic conditions and extraction processes were highly expensive as they use cartridges, and high solvent usages 17. The assay method of Ethambutol hydrochloride in the tablets given in the Chinese Pharmacopoeia (CHP) involves repeated chloroform extraction from a basic aqueous solution and subsequent nonaqueous titration18.

A gas chromatography (GC)–mass spectrometric (MS) method has been used for the determination of Ethambutol in tablets 19. The determination of Ethambutol by reverse-phase (RP) high-performance liquid chromatography (HPLC) is difficult because of its high pose, small molecule, and its lack of strong absorption by UV. Ethambutol derivatives have a strong UV absorbance and can be measured by RP-HPLC. A number of methods for the derivatization of Ethambutol have been reported. Ming Yan et al 20 reported the determination of Ethambutol hydrochloride in the combination tablets by precolumn derivatization. The determination of Ethambutol in presence of fixed dose combination molecules from human plasma by direct injection to Liquid Chromatography Tandem Mass Spectrometry has also been reported 21.  A precise colorimetric procedure was proposed for the estimation of Ethambutol hydrochloride, based on reaction of the drug with 2,4-dinitro-1-fluorobenzene under stipulated conditions 22. A method for estimation of Ethambutol in pharmaceutical preparations and biological fluid was standardized 23.

An alternative method for Ethambutol determination by UV spectrophotometry in pharmaceutical formulations using complexation with Cu(II) was optimized 24. Another method of determination depends on the interaction of Ethambutol with Co(II) ions in the presence of ammonia solution 25. Spectrofluorimetric quantification of Ethambutol hydrochloride in bulk and oral dosage forms was reported 26. Micro determination of Ethambutol with AHC (Ammonium Hexanitrate Cerate) was also carried out and reported 27.

The chemical features of the Ethambutol drug molecule offers a lot of scope for the development of new methods for its determination with better sensitivity, specificity, precision and accuracy. The reported chromatographic techniques require expensive experimental set-up and are not affordable in every laboratories for routine analysis. Although several analytical methods for the estimation of Ethambutol are available in literature, a little attention was paid to the development of spectrophotometric methods for its determination using dyes. Spectrophotometry is considered as the most convenient analytical technique because of its inherent simplicity, low cost, and wide availability in most quality control laboratories.

So the present study reports on newly developed and validated spectrophotometric methods for the  estimation of Ethambutol in bulk and pharmaceutical formulations using triphenyl methane dyes viz., bromocresol green (BCG), bromocresol purple (BCP) and bromophenol blue (BPB) in acidic medium. The developed methods involve formation of coloured chloroform extractable ion-pair complexes of the drug with dyes in acidic medium. The proposed methods have the advantages of speed and simplicity besides being accurate and precise, and can be adopted by the pharmaceutical laboratories for industrial quantitative analysis of Ethambutol.

MATERIALS AND METHODS:

Ethambutol was procured from Srini Pharmaceuticals Limited, Hyderabad as a gift sample. The dyestuffs viz., BCG, BCP and BPB (AR grade) supplied by SD Fine Chemicals Ltd. Mumbai, were used without any further purification.  The dyestuffs were used as 0.025% solutions in doubly distilled water. Sodium acetate-hydrochloric acid buffers of pH 2.5, 3.0 and 3.5 were prepared by mixing 50ml of 1.0M sodium acetate solution with calculated volume 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. Chloroform (HPLC grade) supplied by SD Fine Chemicals Ltd. Mumbai was used throughout the work.

The spectra (Fig. 2-4) of ion-pair complexes have been recorded on Elico double beam SL 210 spectrophotometer using quartz cells of 10 mm path length. An Elico model Li-120 pH meter was used for pH measurement.

fig-2

FIG. 2: ABSORPTION SPECTRUM OF ETHAMBUTOL-BROMOCRESOL GREEN (BCG) COMPLEX EXTRACTED INTO 10 ML CHLOROFORM

[drug] = 25 mg ml-1 + 5 ml of 0.025% BCG + 5 ml of pH 2.5 buffer

fig-3

FIG. 3:  ABSORPTION SPECTRUM OF ETHAMBUTOL- BROMOCRESOL PURPLE (BCP) COMPLEX EXTRACTED INTO 10 ML CHLOROFORM                                                                                                                          

[drug] = 25 mg ml-1 + 5 ml of 0.025% BTB + 5 ml of pH 3.0 buffer

fig-4

FIG. 4:  ABSORPTION SPECTRUM OF ETHAMBUTOL-BROMOPHENOL BLUE (BPB) COMPLEX EXTRACTED INTO 10 ML CHLOROFORM                                                                                                                             

[drug] = 25 mg ml-1 + 5 ml of 0.025% BCP + 5 ml of pH 3.5 buffer

Calibration curve:

Different aliquots of drug solution were transferred into 125 ml separating funnel. To this 5 ml of buffer (pH 2.5, 3.0 and 3.5), 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 atleast for 3 hrs is measured at 420nm 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.5) are linear over the concentration ranges and are within the permissible range. The optical characteristics and statistical data for the regression equation of the proposed methods are presented in Table 1.

fig-5

FIG. 5:  CALIBRATION GRAPHS FOR DRUG-BCG, BCP & BPB ION-PAIR COMPLEXES

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

 

Parameters

 Extraction methods withb
BCG BCP BPB
λmax (nm) 420 419 415
Beer’s law limit (μg ml-1) 2.0-25 3.0-30 4.0-40
Molar absorptivity (L mol-1 cm-1) 18532 20237 21590
Formation constant, K, M-1 1.28x106 1.53x106 1.65x106
Sandell sensitivity (μg cm-2) 0.0249 0.0152 0.0137
Slope (specific absorptivity),  b 0.0401 0.0655 0.0728
Intercept (a) 0.0212 0.0221 0.0325
Correlation coefficient (r) 0.999 0.998 0.995
Standard deviation of intercepts (% n=6) 0.0062 0.0071 0.0102
Limit of detection, μgml-1 0.5102 0.3577 0.4623
Limit of quantification, μgml-1 1.5461 1.0839 1.4010
Regression equationa Y=0.0401C± 0.0212 Y=0.0655C± 0.0221 Y=0.0728C± 0.0325

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

bSix replicate samples

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 2: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF ETHAMBUTOL IN PURE FORM

Taken

(μg ml-1)

 Proposed methods                                      Reference method

 

Found (μg ml-1)                               Recovery %                Recovery %
  BCG BCP BPB BCG BCP BPB  
4 4.09 3.98 4.11 101.25 99.5 102.75 101.25
8 7.96 7.91 8.18 99.5 98.87 102.25 99.82
12 12.06 11.95 12.10 100.5 99.58 100.83 101.0
16 16.05 16.01 15.96 100.3 100.06 99.75 101.02
20 20.03 20.10 19.98 100.15 100.5 99.9 101.15
100.06
101.12
99.98
RSD (%) 0.6296 0.6160 1.343 0.6019
Mean±SD 100.34

±0.6318

 99.70

±0.6149

 101.09

±1.3583

 100.67

±0.6060
t-test 0.0785 0.0405 1.1632
F-test 0.9200 0.9711 0.1990

Procedure for the assay of dosage forms:

Ten tablets of Combutol 10 mg each 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.

TABLE 3: APPLICATION OF PROPOSED METHODS FOR THE ANALYSIS OF ETHAMBUTOL IN PHARMACEUTICAL FORM

Taken

(μg ml-1)

   Proposed methods                                      Reference Method
Combutol

10 mg

 Found (μg ml-1) Recovery ( %)
BPB BTB BCP   BPB BTB BCP         Recovery(%)
5 4.98 5.08 4.97 99.6 101.6 99.4 98.98
10 10.05 9.98 10.12 100.5 99.8 101.2 101.25
15 14.98 15.05 15.15 99.86 100.33 100 101.24
20 20.02 20.15 19.98 100.1 100.75 99.9 99.88
25 25.02 24.98 24.95 100.08 99.84 99.8 100.25
100.25
99.96
101.34
99.82
RSD (%) 0.3223 0.7415 0.6762 0.7976
Mean±SD 100.03

±0.3224

 100.46

±0.7450

 100.06

±0.6767

 100.38

±0.8007
t-test 1.5775 0.1351 0.1584
F-test 1.2376 1.1550 1.4000

RESULTS AND DISCUSSION:

Formation of Ion-pair complexes:

Ethambutol forms ion-pair complexes in acidic buffer with dyestuffs such as bromocresol green (BCG), bromocresol purple (BCP) and bromophenol blue (BPB) and these complexes are quantitatively extracted into chloroform.  Ion-pair complexes of drug with BCG, BCP and BPB absorbed maximally at 420, 419 and 415 nm   respectively (Fig. 2-4). The reagent blank under similar conditions showed no absorption. Ethambutol contains secondary amine groups which are protonated in acid medium, while sulphonic acid group present in BPB, BTB and BCP undergoes dissociation in the pH range 1-5. The colour of the ion-pair complex is attributed to opening of lactoid ring and subsequent formation of quinoid group in dye moiety. It is appropriate to mention 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 Ethambutol forms ion-pairs with dyestuffs which are quantitatively extracted into chloroform. Fig. 6 shows the possible structures of ion-pair complexes.

fig-6

FIG. 6: ETHAMBUTOL-DYE ION-PAIR COMPLEXES

Stoichiometry:

In order to establish molar ratio between Ethambutol and dyestuffs used, the Job’s method 28 of continuous variation 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. 7). This measurement showed that 1:1 complex was formed with each dyestuff. The formation constants29,30  were also estimated and found to be 1.39x 106, 1.61x 106 and 1.71x 106 K M-1 for complexes with BCG, BCP and BPB respectively.

fig-7

FIG. 7:  CONTINUOUS-VARIATIONS STUDY OF DRUG-DYE SYSTEMS

[drug] = [dye] = 8x10-5 M

 Optimization of the factors affecting the absorbance:

The influence of pH on the ion-pair formation of Ethambutol with various dyestuffs has been studied using sodium acetate-hydrochloric acid buffer. The results are shown in Fig.8. It is evident that absorbance of complexes with BCG, BCP and BPB was found to be constant within the pH ranges 2.0-3.0, 2.5-3.5 and 3.0-4.0 respectively. Thus, all the absorbance measurements were made at pH 2.5, 3.0 and 3.5 with BCG, BCP and BPB respectively.

 fig-8

FIG. 8: EFFECT OF pH

[Drug] = 8µg 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 Ethambutol (8 µg ml-1). It is apparent from Fig. 9 that the maximum absorbance, in each case, was found with 3.5 ml of dyestuff, beyond which absorbance was constant. Thus, 5 ml of each dyestuff was used for ion-pair formation throughout the experiment.

fig-9

FIG. 9: INFLUENCE OF THE VOLUME OF 0.025% DYE

[Drug] = [8µg ml-1]

A systematic study of the effect of foreign species present along with Ethambutol on the determination of Ethambutol at 8 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 an Ethambutol solution of 8 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

Sl.no Excipients

 

 Tolerance limit

(μg ml-1)
1 Microcrystalline cellulose 88
2 Starch 155
3 Lactose 132
4 Silicon dioxide 68
5

6

 Titanium dioxide

Magnesium stearate

 36

32

Validation of the proposed method:

All the three  proposed methods have been validated in terms of guideline proposed by International  Conference on Harmonization 31  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 10µg ml-1 of Ethambutol 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 Ethambutol in pharmaceutical preparations. The performance order of the proposed methods is BCG>BCP>BPB. The results obtained and shown in Table 2 and Table 3 were compared to those obtained by a reference method 31 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.

CONCLUSION: Ethambutol forms ion-pair complexes with acidic triphenylmethane viz., bromocresol green, bromocresol purple and bromophenol blue in 1:1 proportion. These complexes are extractable into chloroform and their maximum absorbances offer a basis for assay of the drug. The developed methods are simple, sensitive, reproducible and can be used for routine analysis of Ethambutol in pure and pharmaceutical dosage forms.

 ACKNOWLEDGEMENTS: The authors are grateful to Prof. G. Venkateshwarlu, Department of Chemistry, Osmania University, Hyderabad  for helpful discussion  and to Sri M. Ravindra Reddy, Chairman, Managing Committee & Dr. D. Dathatreya Reddy, Principal of  SAP College, Vikarabad for providing laboratory facilities.  The authors are thankful to the UGC for financial assistance under Major Research Project.

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    How to cite this article:

    Sayanna, Jyothi M, Veeraiah T and Reddy CH.VR: Spectrophotometric Determination of Ethambutol in Pure and Pharmaceutical forms Using Triphenyl Methane Dyes. Int J Pharm Sci Res 2016; 7(10): 4191-99.doi: 10.13040/IJPSR.0975-8232.7(10).4191-99.

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

     

Article Information

35

4191-99

516

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English

IJPSR

Sayanna , M. Jyothi, T. Veeraiah * and CH. Venkata Ramana Reddy

Department of Chemistry, SAP College, Vikarabad, Ranga Reddy Dist., Telangana, India

tadooru_veeraiah@rediffmail.com

16 May, 2016

24 June, 2016

29 June, 2016

10.13040/IJPSR.0975-8232.7(10).4191-99

01 October 2016

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