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Spectrophotometric Determination of Terbutaline Sulphate and Tetracycline Hydrochloride via ion pair Complex Formation Using Eosin Y.

Byline: Mohamed Y. Dhamra Theia'a N. Al-Sabha and Thabit S. Al-Ghabsha

Abstract

A simple sensitive and rapid spectrophotometric method was developed and validated for the determination of terbutaline sulphate and tetracycline hydrochloride drugs in pure form and pharmaceutical formulations. The proposed method is based on the formation of binary complexes between these drugs and eosin Y in aqueous acetate buffered medium. Under the optimum conditions the binary complexes showed absorption maxima at 545 nm. Beer's law was rectilinear over concentration range of 0.5-10 and 5-45 g/mL R2 were 0.9984 and 0.9988 RSD were = 0.72 and = 0.19 (n=5) with average recovery % 101.42 % and 100.08 % and the average recovery values of pharmaceutical formulations 101.48 and 98.01 for above drugs respectively. The limit of detection (LOD) were 0.030 and 0.613 g/mL and limit of quantitation (LOQ) were 0.103 and 2.00 g/mL with molar absorptivity values 3.169 103 and 6.347 103 l. mol-1. cm-1 and the relative standard deviation values =0.720 and = 0.19 for both drugs respectively.

No interference was observed from the excipients that are commonly present in pharmaceutical formulations. The proposed method was successfully applied to the analysis of terbutaline sulphate tablet and tetracycline hydrochloride capsule in their dosage forms.

Keywords: Ion association; Spectrophotometry; Terbutaline; Eosin Y.

Introduction

Terbutaline sulphate [2-(tert-Butylamino)- 1-(35-dihydroxyphenyl)ethanol sulphate] [1] is a short-acting bronchorelaxant which can be given orally [2]. It is readily metabolized in the gut wall and liver when given orally. It has a short duration of action [3]. It has the following chemical structure: Equation

Terbutaline sulphate is widely used as an effective bronco dilator in the management of asthma. This is used as prophylactic drug as well as to prevent acute exacerbations of asthma chronic bronchitis emphysema and other lung diseases. It relaxes and opens air passage in the lungs making it easier to breathe [34].

Tetracycline hydrochloride [(4S 4aS 5aS 6S 12aS) 4 - Dimethylamino-1 4 4a 5 5a 6 11 12a-octahydro - 3 6 10 12 12apentahydroxy-6- methyl 1 11 -dioxonaphthacene-2-carboxamide hydrochloride] [1] is an effective antibiotic in treating infections [5] its absorption is reduced by anti-acids and milk because it can form insoluble complexes with Ca Mg Al and Fe [6] . It has the following chemical structure: Equation

Tetracycline is an antibiotic with a broad antibacterial spectrum and bacteriostatic activity having a good activity against acute disease caused by gram-positive and gram-negative bacteria including the species Spirochete Actinomyces Ricketsia and Mycoplasma [7].

Different analytical techniques have been developed for determination of terbutaline sulfate and tetracycline including HPLC [89] LCMS [10] CE [11] CEMS [12] and voltammetry [13]. Usually HPLC and CE are applied to the determination of terbutaline sulfate in real samples.

Fluorimetry [1314] electrochemical method [1516] liquid chromatography [1718] capillary electrophoresis [19] and chemiluminescence [2021] have been reported for determination of tetracycline. These methods are often time- consuming expensive and cumbersome. Spectrophotometry continues to be very popular because of its simplicity versatility and low cost. Several spectrophotometric methods using different reagents have been reported for determination of terbutaline sulphate. 4- Aminoantipyrine in the presence of potassium ferricyanaide [22] Fe (III) in the presence of potassium ferricyanide [23] sodium periodate in the presence of acetylacetone [24] p-aminophenol- molecular oxygen [25] 3- methyl-2- benzothiozolone hydrazone hydrochloride in the presence of ferric chloride [26] and phenanthro[910-d]imidazole-2-N-chloroimide [27] reagents were used for determination of terbutaline sulphate.

Chloramine-T [28] ammonium vanadate in sulphuric acid medium [29] p-NN-dimethyl phenylenediamine [30] hydroxylamine in the presence of ferric ions chloranil in the alkaline solution and Au (III) and Hg (II) ions [31] reagents were used for determination of tetracycline.

Hence we report a new simple sensitive rapid and precise spectrophotometric method for determination of terbutaline sulphate in pure form and pharmaceutical formulations.

Experimental Apparatus

Shimadzu UV-1650 PC UV-Visible spectrophotometer equipped with a 1.0-cm path length silica cell Philips PW (9421) pH-meter with a combined glass electrode was used for pH measurements. All calculations in the computing process were performed in Microsoft Excel for Windows.

Reagents

All reagents were of analytical-reagent grade which were provided by BDH and Fluka companies. Stock solutions of terbutaline sulphate and tetracycline hydrochloride drugs were prepared in concentration of 100 g/mL by dissolving 0.01 g of each in distilled water and making the volume up to 100 mL in volumetric flask. The solutions were kept in refrigerator. Working standard solutions for calibration graph were prepared by suitable dilution in 10 mL volumetric flasks.

The solution of eosin Y (410-3M) was prepared by dissolving 1.3837 g in distilled water mixing well and making the volume up to 500 mL in volumetric flask.

The acetate buffer solution was prepared with pH 3.2 and 3.5 by mixing sodium acetate and acetic acid solutions of 0.1 M and adjusted by pH meter.

General procedure

Into two series of 10mL volumetric flasks aliquots of solution containing 5-45 and 0.5-10 g/mL terbutaline sulphate and tetracycline hydrochloride respectively were added separately. 0.5 ml of 410-3M and 3.0 mL of 810-3M eosin Y followed by addition 0.6 mL of pH 3.2 and 0.3 mL of pH 3.5 acetate buffer solution to terbutaline and tetracycline respectively. The solutions were diluted to the mark with distilled water and the absorbance was measured at 545nm for terbutaline and tetracycline at room temperature respectively.

Procedure for pharmaceutical formulations Terbutaline tablet

Twenty terbutaline tablets (each tablet contains 5 mg) were accurately weighed and pulverized. A portion of the fine and homogenized powder equivalent to one tablet was accurately weighed and dissolved in distilled water mixed well and filtered with Whatman filter paper No.1. The filtrate was diluted to the 100 mL with distilled water in a volumetric flask to obtain 50 g/mL-1. A suitable volume was diluted and the general procedure was followed.

Tetracycline chloride capsule

Ten tetracycline hydrochloride capsules content (each capsule contains 250 mg) were accurately weighed and pulverized. A portion of the fine and homogenized powder equivalent to one capsule was accurately weighed and dissolved in distilled water mixed well and filtered with Whatman filter paper No.1. The filtrate was diluted to the 250 ml with distilled water in a volumetric flask to obtain 1000 gml-1. A suitable volume was diluted and the general procedure was followed.

Results and Discussion

Terbutaline and tetracycline react with eosin Y through an ion-pair salt formation forming a reddish orange complex in acidic medium with max at 545 (Fig. 1). These complexes were probably formed via electrostatic interaction between the most basic center in the drug molecule (amino group) and the carboxylate anion of the dye. This primarily occurs in an acidic solution increasing the electron delocalization of eosin and producing a bathochromic shift of the dye about 30 nm [32].

Optimization of reaction conditions

The influence of different parameters on the color development was studied to determine optimum conditions for the assay procedure.

Effect of eosin Y concentration

Various concentrations of eosin Y were added to the studied drugs (110-4-110-3 M) as shown in (Fig. 2) it was found that the ion-pair formation was optimized using 0.5 mL of 410-3 M and 3 ml of 810-4 M eosin Y for terbutaline sulphate and tetracycline HCl respectively. These concentrations were used in subsequent experiments.

Effect of acid pH and buffer solution

As mentioned above the complexes were formed in acidic medium therefore different acids were tested to obtain high sensitivity. As shown in (Fig. 3) acetic acid gave highest absorbance value for both drugs. It was found that these complexes are formed at pH 3.2 and 3.5 in the presence of 0.5 and 0.3 ml of 2 M acetic acid for terbutaline and tetracycline in final dilution respectively. However; different buffers with above pHs were prepared and tested. Acetate buffer gave stable complexes and considered as the optimum for both drugs with amounts of 0.6 and 0.3 mL for terbutaline and tetracycline respectively (Fig. 4).

Effect of temperature and developing time

The optimum reaction time was determined by following the color development at ambient temperature (261C) and 401C. It was found that both complexes are formed with maximum absorption immediately at room temperature and remain constant for more than 24 hr. However; the absorbance of both complexes were decreased gradually at 40C indicating the destruction of ion pair complexes (Fig. 5). Then room temperature was considered the optimum and used in all subsequent experiments.

Effect of order of addition of reactants

The effect of variation of the order of addition of reactants on the absorption of both complexes was not observed.

Composition and stability constant of the ion-pair complexes

The composition of the ion-pair was studied by Job's of continuous variation and mole ratio methods [33] and was found to be 1:2 drug: eosin Y in both complexes (Fig. 6).

The apparent stability constant was estimated by comparing the absorbance of a solution containing stoichiometric amounts of the drug and eosin Y (As) to one containing an excessive amount of eosin Y reagent (Am). The average conditional stability constant of the complexes were calculated according to the 1:1 ratio by the following equation:

where Kc is the stability constant (l .mol) the dissociation degree and C the concentration of the complex which is equal to the concentration of drug. However; the average of stability constant for three different concentration was found 4A-109 and 1.2A-10 l.mol for terbutaline and tetracycline respectively indicating the good stabilities.

Mechanism

The proposed method is based on a binary complex formation between the studied drugs and eosin Y. These complexes were probably formed via electrostatic interaction between the most basic center in the drug molecule (amino group) and the carboxylate anion of the dye. This primarily occurs in an acidic solution increasing the electron delocalization of eosin Y and producing a bathochromic shift of the dye about 30 nm [32] (Fig. 1) Applying Job's and mole ratio methods (Fig. 6) it was found that the reaction proceeds in the ratio of 1:2 of drug to eosin for both drugs as seen in their chemical structures they have two basic centers the proposed mechanism of the reaction pathway is shown in (Fig. 7).

Quantitation

Under the described experimental conditions standard calibration curves for terbutaline sulphate and tetracycline HCl with eosin Y were constructed by plotting absorbance against concentration (Fig. 8). The Beer's law limits and molar absorptivity values were evaluated and given in Table1 which indicated that the method is sensitive. The linearity was represented by the regression equation and the corresponding correlation coefficient for drugs determined by the proposed method represents excellent linearity. The relative standard deviation (RSD) and accuracy (average recovery %) for the analysis of five replicates of each three different concentrations for terbutaline sulphate and tetracycline HCl indicated that the method is precise and accurate. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated according to the following equations:

LOD = 3.3s/b and LOQ = 10s/b

where s is the standard deviation of five reagent blank determinations and b is the slope of the calibration curve. The results obtained are in the accepted range below the lower limit of Beer's law range (Table 1).

Table 1. Summary of optical characteristics and statistical data for the proposed method.

Parameter###Terbutaline###Tetracycline

max (nm)###545###545

Linear range (g/mL)###0.5-10###5-45

Molar absorptivity (l.mol . cm ) -1###-1

###3.16910###3

###6.347 103

LOD (g/mL)###0.030###0.613

LOQ (g/mL)###0.103###2.00

Average recovery (%)###101.42###100.08

Correlation coefficient###0.9984###0.9988

Regression equation ( Y)

Slope a###0.1698###0.0134

Intercept b###0.0623###0.2726

RSD###0.72###0.19

Specificity

The specificity of the method was investigated by observing any interference encountered from the common excipients of the pharmaceutical formulations by measuring the absorbance of solutions containing 2 g/mL-1 terbutaline sulphate and 20 g/mL-1 tetracycline HCl separately and various amounts of diverse species up to 100 fold excess in a final volume of 5 mL. It was found that the studied excipients did not interfere seriously (Table 2).

Table 2. Effect of excipients for assay of terbutaline sulphate and tetracycline HCl.

###Recovery %

###Excipient

###2 g ml-1 of terbutaline###20 g ml-1of tetracycline

###sulphate per fold###HCl per fold excess

###excess foreign added###foreign added

###10###50###100###10###50###100

###Glucose###98.50###98.01###98.00###99.20###98.56###97.64

###Lactose 100.01###98.42###97.87###99.22###98.33###96.50

###Acacia###99.70###98.21###95.01###97.30###95.63###94.22

###Starch###97.31###95.09###93.21###96.33###95.06###95.11

###NaCl###99.70###98.88###98.42###98.10###97.71###95.54

###Urea###98.82###98.80###97.80###98.91###99.90###98.81

###MgCl2###97.55###95.50###94.94###98.01###96.00###96.53

###Na2SO4###97.7###95.53###93.94###98.10###96.51###96.72

Method validation and applications

To evaluate the analytical applicability of the proposed method it was successfully applied to determine terbutaline sulphate and tetracycline HCl in some pharmaceutical preparations. The obtained recovery % values cited in Table 3 indicated high accuracy and there is no serious interference in the determination of above drugs in such samples. However; standard addition procedure was applied for determination of terbutaline sulphate in tablets (Fig. 9). The results cited in Table 3 indicated good recovery. The results obtained by the proposed method were compared with British Pharmacopoeia (BP) method (Table 3) by applying the F-test and the t- test at 95% confidence level. The calculated values for F and t tests for proposed method did not exceed the theoretical values.

These confirming that there are no significant differences between the proposed method with BP method for terbutaline sulphate [3334] and tetracycline HCl.

Table 3. Statistical analysis of results obtained by the proposed eosin method compared with the official BP and standard addition

methods.

###Drug###Drug

###Average###Certified

###Pharmaceutical###amount###Recoverya###content

###Procedure applied###recovery###value

###preparation###present###(%)###found

###(mg)###(mg)

###( g/mL)###(mg)

###3###99.99###4.99

###Samabutalineb###5.06

###6###103.33###5.16###5

###tablet###(0.507.16)c

###8###101.12###5.05

###Proposed eosin method

###15###97.12###242.80

###Samacyclineb###245.02

###25###98.33###245.82###250

###capsule###(1.051.02)

###35###98.59###246.45

###Bricanyl d

###15mg###99.21###14.88mg###-###15

###tablet

###British Pharmacopoeiae

###Samacycline

###250mg###97.92###244.80###-###250

###capsule

###1###96.39###4.82

###Samabutaline

###Standard addition method###2###101.20###5.06###4.99###5

###tablet

###4###101.80###5.09

Comparison of the proposed method with reported methods

The proposed method compared favorably with other reported spectrophotometric methods.

As shown in Table (4) the present method is simpler than other methods as no need an extraction step and have stability period but less sensitive than some methods.

Table 4. Comparison of the proposed method with other spectrophotometric methods.

Table 4. Comparison of the proposed method with other spectrophotometric methods.

Analytical###Tetracycline###Terbutaline###Tetracycline###Terbutaline

parameters

Reagent###Eosin Y###Au (III)###Hg (II)###DMPD a###PI-NClb###p-Aminophenol###L-amino

###antipyrine

max

###625###525###590###550

(nm)###545###425###320

###Alkali

pH###3.5###3.2###4###2-4###7.0###8.0###9.5

###(0.1 N NaOH)

###Water and

Solvent###Water###ethyl acetate###n-butanol###50% Ethanol###Water###Water

Temp.

###RTc###75###RT###40###RT###RT

(C)

Development time

###Immediately###15###30###20###Immediately###Immediately

(min)

Stability period

###greater than 24 hrs###-###120###50###5###3

(min)

Beer's law

###5-45###0.5-10###570###5-50###0.7-270###20-160###4-20

(g/mL)

Molar absorptivity

###6120###73800###14600###11905

(l.mol-1.cm-1)###6347###3169###3482###6965

Recovery###98.50 -

###98.01###101.48###100.78###100.95###99.04###100.97###99.87

(%)###98.90

RSD

###0.19###0.72###0.961###2.20###0.62###1.23###0.937

(%)

###Tablet###Tablet

Applications###Capsule###Tablet###Capsule###Tablet syrup###Tablet

###Capsule###injection

Disadvantages###Less sensitive than some###The methods need###Tedious###Using of###No stability for###No stability for

###cited methods###extraction heating###and need###mixed###the product and###the product

###and complexes###extraction###solvent###measurements###measurements

###have no###sensitivity###must be carried###must be carried

###stability period###and stability###within 5 min.###within 3 min

###affected by###and dilution

###pH###with buffer

Reference###Present method###[31]###[30]###[27]###[25]###[22]

Conclusion

A simple sensitive fast accurate and precise spectrophotometric method was developed for the determination of terbutaline sulphate and tetracycline HCl in some of their pharmaceutical formulations. The statistical parameters and the recovery test data indicated the high reproducibility and accuracy of the proposed

method. Analysis of authentic samples containing the studied drugs showed no interference from common additives and auxiliary substances in general. The advantage of the method being less time consuming and do not require various elaborate treatments and tedious extraction procedures. In addition to the satisfactory sensitivity and reproducibility method is convenient and simple as well.

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