Spectrophotometric Determination of Thymol in Pharmaceutical Preparation via Diazotization Reaction with 4-aminoacetophenone

A simple and sensitive spectrophotometric method is proposed for the determination of thymol. The method is based on the coupling reaction of the drug with diazotised 4aminoacetophenone reagent in alkaline medium to produce an intense red coloured water soluble and stable azo dye which exhibits a maximum absorption at 502 nm. Beer’s low is obeyed over the concentration range 0.2 – 16 μg/ml with a molar absorptivity of 2.89×10 L.mol.cm. The limit of detection is 0.0216 μg/ml and the limit of quantitation is 0.072 μg/ml, average recovery is 101.6%, and RSD is less than 2.4%. The proposed method is applied successfully to the determination of thymol in pharmaceutical preparation.

The objective of investigation reported in this paper is to evaluate a sensitive and an accurate method for the assay of thymol in pharmaceutical preparations. This method is based on coupling of the drug with diazotized 4-aminoacetophenone to form a stable azo dye product.

EXPERIMENTAL Apparatus
A Shimadzu model 1650 computerized spectrophotometer provided with 1.0-cm matched quartz cells was used for all absorbance measurements.

Reagents
All chemicals used were analytical grade and obtained from Fluka and BDH companies.
Thymol standard solution (100 µg/ml). This solution is prepared by dissolving 0.01 g of pure thymol in 5 ml of ethanol and the volume completed to the mark with distilled water in a 100-ml volumetric flask. 4-aminoacetophenone reagent solution (0.1%). This solution is prepared by dissolving 0.1 g of the compound in distilled water and the volume is completed to the mark in a 100-ml volumetric flask with distilled water. Hydrochloric acid solution (1N). This solution is prepared by diluting 8.4 ml of 11.8 N concentrated acid to the mark in a 100-ml volumetric flask with distilled water. Sodium nitrite solution (1%). This solution is prepared by dissolving 1g of sodium nitrite in distilled water and diluted to the mark in a 100-ml volumetric flask with distilled water. Sulphamic acid solution (2%). This solution is prepared by dissolving 2 g of sulphamic acid in distilled water and the volume is completed to the mark in a 100-ml volumetric flask.

Sodium hydroxide solution (1N)
. This solution is prepared by dissolving 4 g of sodium hydroxide in distilled water and the volume is completed to the mark in a 100 ml volumetric flask. Interference solution (1000 µg/ml). An amount of 0.1 g of each foreign compound is dissolved and completed to 100 ml with distilled water. cetyl trimethyl ammonium bromide(CTAB) surfactant solution (0.1%). This solution is prepared by dissolving 0.1 g of cetyl trimethyl ammonium bromide in distilled water and diluted to the mark in a 100-ml volumetric flask with distilled water.

Recommended procedure and calibration graph
Employing the established optimum condition, the calibration graph was constructed as follows: To a series of 25-ml volumetric flask, 0.5 ml of 0.1% of 4-aminoacetophenone, 0.2 ml of 1N HCl and 0.5 ml of 1 % of NaNO 2 are transferred. The reaction mixture is shaken and left for 5 minutes then 0.5ml of 2 % sulphamic acid is added with shaking and left for 5 minutes. After that an aliquot of aqueous solution containing (5-700) µg of thymol is added, 3 ml of 0.1% CTAB solution and 1.0 ml of 1N of NaOH are also added, and the flasks are diluted with distilled water and after leaving for 10 minutes as a reaction time, the absorbance of coloured azo dye was measured versus reagent blank at 502 nm.
The calibration graph shown in Fig. 1 was linear over the range of 0.2-16 µg/ml of thymol. The higher concentration shows a negative deviation from Beer's law. The apparent molar absorptivity has been found to be 2.89×10 4 L.mol -1 .cm -1 . The limit of detection is 0.0216 µg/ml and limit of quantitation is 0.072 µg/ml. Procedures for assay of thymol in pharmaceutical preparations: Mouthwash: 25 ml of mouthwash (0.06% thymol) solution was transferred into 50 ml volumetric flask and diluted to the mark with distilled water, from this solution, the suitable volume was to obtain 100 µg/ml of thymol. An aliquots of this solution containing 2, 8, 12 µg/ml was treated as described under the recommended procedure for determination of thymol.

RESULTS AND DISCUSSION Principle of the colour reaction
4-Aminoacetophenone was reacted with excess nitrite in acidic medium to form the corresponding diazonium salt, as follows :.
The residual nitrite as nitrous acid which was undesirable due to its side reaction was removed by sulphamic acid as in the following reaction.
Diazotized 4-aminoacetophenone is coupled with thymol in basic medium to form red azo dye as in the following reaction:

Study of the optimum reaction condition
The various parameters affecting the colour intensity of the dye have been studied and optimum conditions are selected.

Effect of acid on diazotization formation
The effect of quality and quantity of acid on the intensity of the coloured dye is examined. Different volumes (0.1 -1.0 ml) of 1 N of different acid solutions are transferred to volumetric flasks containing 1ml of 0.1% of reagent then 0.25 ml of (0.1%) NaNO 2 are added and shaken for 5 min, then 0.25 ml of (0.2 %) sulphamic acid was added and shaken for 5 min, after that the 1 ml of (100 ppm) thymol and 1 ml of (1N)NaOH are also added. The intensities of absorption are read against the reagent blank at 484 nm after diluting by distilled water to the mark. The results are shown in Table (1). The results in Table 1 indicate that 0.2 ml of 1N HCl is considered as an optimum value therefore it is recommended for subsequent experiments.

Effect of nitrite amount and developing time
Different amounts of the 1% NaNO 2 solution are added and the time needed to complete the diazotization of 4-aminoacetophenone is studied by standing the solution after adding sodium nitrite solution for different time, the colored dye reached its maximum intensity when using 0.5 ml of 1% of NaNO 2 solution after 5 minutes as reaction standing time [ Table ( 2)].

Effect of the reagent amount
The effect of 4-aminoacetophenone reagent amount on the maximum formation of the coloured product was investigated. The results shown in Table 4 indicated that 0.5 ml of 0.1% 4-aminoacetophenone solution is the optimum amount due to the higher colour intensity.

Effect of base type and its amount
Preliminary experiments have shown that coupling reaction of diazotized 4aminoacetophenone with thymol occurs in basic medium; therefore several bases (strong and weak) have been examined at different amounts The results in Table (5) indicate that 1.0 ml of sodium hydroxide gives maximum absorbance so it was chosen in this method.

Effect of surfactants
The effect of surfactants was studied by the addition of 1 ml of various types of surfactants (cationic, anionic and neutral) to the medium of reaction with two different orders.
The results in Table (6) indicate that cationic cetyl trimethyl ammonium bromide (CTAB) causes a red bathocromic shift of the absorption maxima wavelength of the produce dye from 484 nm to 502 nm, and also causes an increase in the colour intensity therefore this surfactant has been used for the subsequent experiments.

R) +Nitrite (N)+ Sulphamic acid(A) + Thymol(D) +surfactant (S) +Base (B) ** R+N+A+D+B+S
In order to determine the optimum amount of CTAB, volumes from 0.5 -5 ml of 0.1% of CTAB were examined, the results in Table (7) indicate that 3 ml of (0.1%) CTAB is the best for colour intensity so it was chosen for further studies.

Effect of time and temperature
The effect of time with different temperatures in the range (0-60 o C) has been investigated for maximum intensity of dye and the stability period. The results in Table (9) indicate that maximum absorbance reading is given after 10 min from mixing the components of the reaction and remains constant for at least 60 min.

Absorption spectra
Absorption spectra of the coloured dye formed by coupling of diazotized 4-aminoacetophenone with thymol in basic medium in presence of CTAB surfactant under the established optimum amounts show a maximum absorption at 502 nm in contrast to the reagent blank which shows no absorption in the visible region (Fig. 2)

Fig. 2: Absorption spectra of (A) 50 µg thymol against reagent blank, (B) reagent blank against distilled water
Wavelength A B

Accuracy and Precision
To evaluate the accuracy (recovery %) and precision (RSD) of the calibration graph, a pure thymol was analyzed at three different concentrations in four replicates. The results shown in Table 10 indicated that a satisfactory accuracy and precision could be attended by using the proposed method.

Nature of the dye
Applying Job's method of continuous variations and mole-ratio method (Harvey, 2000) showed that the formed dye has the composition of 1:1, thymol: diazotized 4aminoacetophenone as shown in (Fig. 3), therefore, the formed dye may be written as follows:  The apparent conditional stability constant of the produced azo dye described under the recommended procedure has been estimated to be 3.0 ×10 5 L . mol -1 .

Interferences
The effect of common excipients used in the pharmaceutical preparations was studied by analyzing a synthetic sample solution containing the quantity of drugs as mentioned in Table ( 11), in presence of different amounts of excipients. It was found that excipients show no significant interference.

Analytical applications
The proposed method was successfully applied to the determination of thymol in its pharmaceutical preparations as in Table (12). The obtained results were compared statistically by a student's t-test for accuracy and variance ratio F-test for precision with standard method (American P.H.A., 1975).
At the 95% confidence level with six degrees of freedom as cited in Table ( 13). The results showed that the t-test and F-test were less than the theoretical value (t=2.306, F= 6.39), indicating that there was no significant difference between the proposed method and the standard method.   (Table 14) shows a comparison between the present method and another spectrophotometric method. The present method is more sensitive than the recently-published method for thymol (Mohammad, 2005).

Comparison of methods
CONCLUSION A spectrophotometric method for the determination of thymol in an aqueous solution is described. The method is based on the azo-coupling reaction of thymol with diazotized 4aminoacetophenone reagent in an alkaline medium. It is considered to be simple (it does not need heating and extraction), rapid (10 minutes development time), accurate (average recovery 101.6 %) and precise (RSD < 2 %), and the results suggested that there is no interference from excipients which are present in commercial dosage forms.