Spectrophotometric determination of thymol in pharmaceuticals with Gibb's reagent

A simple and rapid spectrophotometric method for the determination of thymol in pure form and in pharmaceutical preparations is described .The method is based on the color reaction between thymol and 2,6dichloroquinone-4chloroimide (Gibb's reagent) in borate buffer medium (pH 10) to form a blue indophenol dye with maximum absorption 605 nm. Beer's law is obeyed over the concentration range of 0.04–16 μg. ml −1 with a molar absorptivity of 18430 l. mol.cm −1 and sandell sensitivity index of 0.008 Spectrophotometric determination of thymol in pharmaceuticals with ... 18 μg.cm. The results obtained were both precise (RSD) better than 1% and accurate (average recovery) 100.50%. The method does not resort to temperature control or to solvent extraction .The method has been applied further, successfully to the determination of thymol in mouthwashes and the results obtained are comparable with those given by the standard 4aminoantipyrine method. Introduction Thymol is a component of thyme essential oil, which has been reported to possess interesting antimicrobial effects on various microorganisms, hence thymol is used in the case of female urogenital infections, bacterial vaginosis, urinary tract infections and vaginitis (1). Thymol has also many uses, including perfumes, food flavoring, mouthwashes, cosmetics and also as stabilizer to several therapeutic agents including halothane (2,3). Thymol has been determined spectrophotometrically via oxidative coupling with N,N-diethylphenylenediamine in the presence of Nbromosuccinamide in alkaline medium (4) and p-phenylenediamine in the presence of sodium metaperiodate in alkaline medium(5) or by coupling with diazotized 2,4,6-trimethylaniline reagent in borate buffer solution (6) and diazotized p-nitroaniline in basic medium (7), in addition of its spectrophotometric determination by reacting with sodium nitroprusside and hydroxylamine hydrochloride in phosphate buffer solution (8). Also, several analytical methods have been reported for the estimation of thymol including high–performance liquid chromatography (9) and gas chromatography (10, 11). Gibb's reagent (2,6-dichloroquinone-4chloroimide) has numerous applications as analytical reagent .It has been used for the determination of hallogenated derivatives of 8-hydroxyquinoline (12), some adrenergic drugs (13), aminosalicylate sodium (14), salbutamol (15). The aim of this work is to develop a new simple spectrophotometric procedure for accurate and rapid analysis of thymol using 2,6dichloroquinone-4chloroimide as a coloring reagent. Apparatus Absorption spectra and absorbances were measured in a Shimadzu UV 150-02 double beam spectrophotometer using 1-cm glass cells. Reagents All chemicals used were of analytical reagent grade. Usra. I. S. Al-Neaimy 19 Thymol solution, 100 μg.ml -1 This solution was prepared by dissolving 0.01 g of thymol (BDH) in 5ml of absolute ethanol and then the volume was completed to 100ml with distilled water. The solution was stable at least for two weeks. Gibb's reagent, 5 x 10 -3 M The solution was prepared freshly by dissolving 0.1052 g (Fluka) of 2,6-dichloroquinone -4chloroimide (DCQ) in ethanol and diluted to 100ml in volumetric flask with the same solvent and kept in a dark bottle. Borate buffer solution (pH 10) This solution was prepared by mixing 50ml of 0.025M Na2B4O7 .10 H2O with 18.3ml of 0.1M sodium hydroxide then the volume was completed to 100ml with distilled water. Procedure for calibration To a series of 25ml calibration flasks, increasing volumes of thymol working standard solution were transferred to cover the range (0.04–16)μg.ml -1 in final dilution. 3ml of borate buffer solution (pH 10) followed by 3ml of DCQ reagent solution were added. The mixture was well mixed, allowed to stand for 5 min at room temperature and the absorbance was measured at 605 nm. A reagent blank was run simultaneously. Assay procedure for a drug Determination of thymol in listerine antiseptic original mouthwash : Thymol was prepared in the concentration 256μg.ml -1 by diluting 20ml of Listerine (from Warner – Lambert Pharmaceutical Co. which was certified to contain 64mg thymol/100ml) to 50ml with distilled water and 100 μg.ml -1 of thymol solution was prepared from this above solution, then different volumes used from this solution containing the concentration range 0.04 – 16μg.ml -1 . After that the calibration procedure described above was carried out. Determination of thymol in septica effervescent tablets : Septica is effervescent tablets from avicenna labs. Damascus. Each tablet contains 4 mg thymol. Twenty tablets were weighed, powdered, mixed and an amount of the powder equivalent to five tablets was transferred into 100ml calibrated flask, dissolved in distilled water and the volume was completed with the same solvent, then 100μg.ml -1 was prepared from this above solution and the calibration procedure was carried out. Spectrophotometric determination of thymol in pharmaceuticals with ... 20 Results and discussion Absorption spectra Thymol was reacted with DCQ in basic medium producing a blue colored product with maximum absorption at 605 nm, while the reagent blank shows no absorption at this wavelength (Fig 1). Fig (1) : Absorption spectra : A : Thymol (6 μg . ml -1 ) – DCQ (5x10 -3 ) product versus reagent blank. B : Reagent blank versus D.W. Effect of pH and buffers The effect of pH on the sensitivity of the colored reaction product was investigated in the range of 4-13. The results obtained showed that the optimum pH value was 10. Therefore different buffer solutions of pH 10 were prepared. The results shown in Table 1 and 2 indicate that borate buffer solution gives clear blue color with maximum intensity and it was found that the optimum amount of borate buffer was 3ml. Usra. I. S. Al-Neaimy 21 Table (1): Effect of buffer solution on the absorbance Table (2): Effect of borate buffer amount on the absorbance Effect of temperature and reaction time The reaction time was determined by following the color development at room temperature and in a thermostatically controlled water bath adjusted at 0,40 and 50C°. The absorbance was measured at 5min intervals against reagent blank treated similarly. It was observed that the absorbance reached maximum after 5 min at room temperature and remained constant for at least 3 hours and the absorbance decreased slowly thereafter. Hence, room temperature and reaction time (5 min) were chosen for color development (Table 3) . Table (3): Effect of temperature and reaction time Effect of 2,6-dichloroquinone-4-chlorimide (DCQ) concentration The effect of various DCQ concentrations on the absorbance of solution containing 6 μg.ml -1 thymol was studied, it is evident that the absorbance increases with increasing DCQ concentration and reached maximum on using 3ml of 5x10 -3 M DCQ .Therefore, this volume was used in all subsequent work (Table 4). Buffer solution pH 10 Phosphate Borate Carbonate Absorbance 0.485 0.516 0.443 pH 1 2 3 4 5 Absorbance 0.513 0.540 0.601 0.552 0.536 Temp (C°) Absorbance / min standing time 0 5 10 20 30 40 50 60 90 180 240 0 -` 0.581 0.579 0.575 0.578 0.574 0.568 0.568 0.563 0.543 0.540 Room temp 0.600 0.603 0.603 0.603 0.603 0.603 0.603 0.603 0.603 0.603 0.580 40 0.587 0.579 0.575 0.570 0.569 0.568 0.560 0.554 0.532 0.501 50 0.586 0.570 0.571 0.570 0.569 0.560 0.549 0.540 0.530 0.513 Spectrophotometric determination of thymol in pharmaceuticals with ... 22 Table (4): Effect of DCQ concentration Effect of surfactant The effect of different types of surfactants were used for the improvement of the absorption but the results shown in Table 5 confirm that there is no improvement in the absorption, therefore they were excluded. Table (5): Effect of surfactant Surfactant* Absorbance Cetyltrimethylammonium bromide (0.1 %) 0.692 Sodium dodecyl sulphate (0.1%) 0.686 Triton X-100 (1.0 %) 0.699 Without surfactant 0.731 * 1 ml of surfactant Analytical data Under the proposed experimental conditions linear relation between the absorbance and the concentration of thymol was observed over the concentration range 0.04-16μg.ml -1 (Fig 2) with a correlation coefficient of 0.9996 and intercept of 0.015. A negative deviation from Beer , s law was observed above 16 μg.ml -1 concentration of thymol .The molar absorptivity was 18430 l.mol -1. cm -1 . y = 0.1228x + 0.0152


Abstract
A simple and rapid spectrophotometric method for the determination of thymol in pure form and in pharmaceutical preparations is described .The method is based on the color reaction between thymol and 2,6dichloroquinone-4-chloroimide (Gibb's reagent) in borate buffer medium (pH 10) to form a blue indophenol dye with maximum absorption 605 nm. Beer's law is obeyed over the concentration range of 0.04-16 μg. ml −1 with a molar absorptivity of 18430 l. mol.cm −1 and sandell sensitivity index of 0.008 μg.cm. The results obtained were both precise (RSD) better than 1% and

Introduction
Thymol is a component of thyme essential oil, which has been reported to possess interesting antimicrobial effects on various microorganisms, hence thymol is used in the case of female urogenital infections, bacterial vaginosis, urinary tract infections and vaginitis (1). Thymol has also many uses, including perfumes, food flavoring, mouthwashes, cosmetics and also as stabilizer to several therapeutic agents including halothane (2,3).
Thymol has been determined spectrophotometrically via oxidative coupling with N,N-diethylphenylenediamine in the presence of Nbromosuccinamide in alkaline medium (4) and p-phenylenediamine in the presence of sodium metaperiodate in alkaline medium (5) or by coupling with diazotized 2,4,6-trimethylaniline reagent in borate buffer solution (6) and diazotized p-nitroaniline in basic medium (7), in addition of its spectrophotometric determination by reacting with sodium nitroprusside and hydroxylamine hydrochloride in phosphate buffer solution (8). Also, several analytical methods have been reported for the estimation of thymol including high-performance liquid chromatography (9) and gas chromatography (10,11).
The aim of this work is to develop a new simple spectrophotometric procedure for accurate and rapid analysis of thymol using 2,6dichloroquinone-4-chloroimide as a coloring reagent.

Apparatus
Absorption spectra and absorbances were measured in a Shimadzu UV 150-02 double beam spectrophotometer using 1-cm glass cells.

Reagents
All chemicals used were of analytical reagent grade.

Thymol solution, 100 μg.ml -1
This solution was prepared by dissolving 0.01 g of thymol (BDH) in 5ml of absolute ethanol and then the volume was completed to 100ml with distilled water. The solution was stable at least for two weeks.

Gibb's reagent, 5 x 10 -3 M
The solution was prepared freshly by dissolving 0.1052 g (Fluka) of 2,6-dichloroquinone -4-chloroimide (DCQ) in ethanol and diluted to 100ml in volumetric flask with the same solvent and kept in a dark bottle.

Borate buffer solution (pH 10)
This solution was prepared by mixing 50ml of 0.025M Na 2 B 4 O 7 .10 H 2 O with 18.3ml of 0.1M sodium hydroxide then the volume was completed to 100ml with distilled water.

Procedure for calibration
To a series of 25ml calibration flasks, increasing volumes of thymol working standard solution were transferred to cover the range (0.04-16)μg.ml -1 in final dilution. 3ml of borate buffer solution (pH 10) followed by 3ml of DCQ reagent solution were added. The mixture was well mixed, allowed to stand for 5 min at room temperature and the absorbance was measured at 605 nm. A reagent blank was run simultaneously.

Assay procedure for a drug Determination of thymol in listerine antiseptic original mouthwash :
Thymol was prepared in the concentration 256μg.ml -1 by diluting 20ml of Listerine (from Warner -Lambert Pharmaceutical Co. which was certified to contain 64mg thymol/100ml) to 50ml with distilled water and 100 μg.ml -1 of thymol solution was prepared from this above solution, then different volumes used from this solution containing the concentration range 0.04 -16μg.ml -1 . After that the calibration procedure described above was carried out.

Determination of thymol in septica effervescent tablets :
Septica is effervescent tablets from avicenna labs. Damascus. Each tablet contains 4 mg thymol. Twenty tablets were weighed, powdered, mixed and an amount of the powder equivalent to five tablets was transferred into 100ml calibrated flask, dissolved in distilled water and the volume was completed with the same solvent, then 100μg.ml -1 was prepared from this above solution and the calibration procedure was carried out.

Absorption spectra
Thymol was reacted with DCQ in basic medium producing a blue colored product with maximum absorption at 605 nm, while the reagent blank shows no absorption at this wavelength (Fig 1).

Effect of pH and buffers
The effect of pH on the sensitivity of the colored reaction product was investigated in the range of 4-13. The results obtained showed that the optimum pH value was 10. Therefore different buffer solutions of pH 10 were prepared. The results shown in Table 1 and 2 indicate that borate buffer solution gives clear blue color with maximum intensity and it was found that the optimum amount of borate buffer was 3ml.

Effect of temperature and reaction time
The reaction time was determined by following the color development at room temperature and in a thermostatically controlled water bath adjusted at 0,40 and 50C°. The absorbance was measured at 5min intervals against reagent blank treated similarly. It was observed that the absorbance reached maximum after 5 min at room temperature and remained constant for at least 3 hours and the absorbance decreased slowly thereafter. Hence, room temperature and reaction time (5 min) were chosen for color development (Table 3) .

Effect of 2,6-dichloroquinone-4-chlorimide (DCQ) concentration
The effect of various DCQ concentrations on the absorbance of solution containing 6 μg.ml -1 thymol was studied, it is evident that the absorbance increases with increasing DCQ concentration and reached maximum on using 3ml of 5x10 -3 M DCQ .Therefore, this volume was used in all subsequent work (Table 4).

Effect of surfactant
The effect of different types of surfactants were used for the improvement of the absorption but the results shown in Table 5 confirm that there is no improvement in the absorption, therefore they were excluded.

Analytical data
Under the proposed experimental conditions linear relation between the absorbance and the concentration of thymol was observed over the concentration range 0.04-16μg.ml -1 (Fig 2)

Accuracy and precision
To determine the accuracy and precision of the calibration graph, thymol was determined at three different concentrations. The results shown in Table 6 indicate a satisfactory precision and accuracy.

Nature of product and reaction mechanism
To establish the composition (ratio of thymol to DCQ reagent) of the blue indophenol dye formed, Job , s method of continuous variations and mole-ratio method have been used. The resulting data show that 1:1 thymol to reagent are reacted and the color reaction does not require any oxidant (16). Therefore, the formation of the product may be illustrated as follows: NCl O The stability constant of the product was estimated and found to be 5x10 6 l.mol -1 .

Effect of interferences
In order to assess the possible analytical application of the proposed method, the effect of some common excipients usually present with drug in pharmaceutical formulation was studied by analyzing synthetic sample solution containing 100μg of thymol and (10-fold excess) of each excipient, none of these substances interfered seriously (Table 7). The proposed method was applied to the determination of thymol in pharmaceutical formulations. Good recovery was obtained and the results compared with the standard 4-aminoantipyrine (17) ( Table 8 and Table 9). The experimental t-test found to be 1.55 and 0.24 for listerine and septica, respectively. While the tabulated value is 2.776 at the 95% confidence limit for four degrees of freedom, which indicate that this method is reliable for application.

Table (8): Assay of thymol in mouthwash Listerine
Certified value 64 mg / 100 ml * Average for three determinations  Table 10 gives the comparison between the present method and another spectrophotometric method.

CONCLUTION
DCQ is a sutiable chromogenic reagent for the determination of thymol in pure form or in its pharmaceutical preparations. The suggested method is simple, time saving, sensitive and reproducible.