Thymol occurs as colorless crystals or white crystalline masses. It has an aromatic odor, and has a burning taste. It is very soluble in acetic acid , freely soluble in ethanol (95%) and in diethyl ether, and slightly soluble in water. Thymol is 5-methyl-2- (methylethyl) phenol Figure [1] that is extensively used as preservative and antiseptic in medicine and as antioxidant, perfumery flavoring and synthetic menthol in industry [1-3].

C₁₀H₁₄O: 150.225-Methyl-2-(1-methylethyl)phenol

Figure 1: Chemical structure of Thymol.

Thymol is an active ingredient in pesticide products registered for use as animal repellents, fungicides fungi stats, medical disinfectants. Thymol has also many non-pesticides uses, including use in perfumes, food flavoring, mouth washes, pharmaceutical preparations, cosmetics and also as a stabilizer to several therapeutic agents, including halothane [4,5]. Therefore, Spectrophotometry is a very valuable technique which has proven its worth in the field of pharmaceuticals analysis over the years. It is particularly popular because of its features like ruggedness, economical, suitable for wide range of pharmaceuticals by using different reagents [6-9]. Many assay methods have been reported for the determination of thymol. The official titrimetric method [2]. high performance liquid chromatography [10] ;differential pulse voltammetry [11] and gas chromatography [12-13]. In this paper we describe a simple, selective and precise method for spectrophotometric determination of Thymol in pharmaceutical formulations. The method is based on the p-aminophenol, which reacts with thymol in alkaline medium to yield a color compound shows maximum absorbance at 600 nm. And determination of thymol in pure form, pharmaceutical formulations and in industrial wastewater samples.

Apparatus

Shimadzu UV- 1700 pharm spec (double beam) spectrophotometer with 1.0 cm quartz cells was used for absorption measurement, and Jenway 3310 pH meter was used.

Reagents

All chemical used were of analytical or pharmaceutical grade and Thymol standard material was provided from AL-hokamaa company for pharmaceutical industries (HPI) Mosul- Iraq.

Thymol solution: (100µg/ml) (6.65x10-4M)

This solution was prepared by dissolving 0.1 gm of thymol (Merck) in 100ml ethanol and diluting to 1Lwith distilled water in a volumetric flask. This solution is then transferred to a dark bottle where it is stable for at least 1 month.

P- aminophenol::(1.835x10^-3M ) this solution was prepared by dissolving 0. 02 gm of reagent in 100ml distilled water. This solution is kept in a dark bottle.

KOH (0.5 M):This solution was prepared by dissolving2.8 gm of KOH in 100ml distilled water.

An aliquot of Thymol standard solution containing (2-28µg/ml) of Thymol was transferred into a series of 25 ml volumetric flasks, .Add 1ml of KOH (0.5 M) and 10ml of P- aminophenol: (1.835x10-3M ). Dilute to the mark with distilled water .The blue color, which developed instantaneously. The absorbance was measured at.600 nm against a reagent blank prepared in the same manner without Thymol, and a calibration graph was constructed.

Procedures for smicks ointment:- Prepare a composite sample by mixing the contents of five containers and accurately weigh a sample equivalent to 1.0 gm. of smicks ointment Dissolve in hot ethanol(70-80)mL , mixed well for 20 min and then cool in freezer and filtered using filter paper .The filtrate was made up to 100mL with ethanol .pipette 10 ml of (1.835x10^-3M ) P- aminophenol into a 25ml calibrated flask, then add3 ml of (0.5 M) KOH solution and 3ml of extract solution , dilute with water to the mark and allow to stand for 5 minute. Measure the absorbance at 600nm against reagent blank.

Procedure for Spiked Industrial Wastewater

To demonstrate the practical applicability of the proposed method, industrial waste water sample from AL-hokamaa Company (HPI) Mosul- Iraq. Were analyzed, since industrial waste water sample was found to be free from thymol, synthetic samples were prepared by spiked known amount of thymol with the concentrations ranging from 4-20 µg.ml^-1 and aliquot of this solution was treated as described above for recommended procedure.

The absorption spectrum of the resulting colored product shown in figure [2].The maximum absorbance of the blue product at 600nm against blank and this wavelength recommended for determination.

Figure 2: Absorption spectra of 10µg/ml Thymol against reagent blank.

Study of the Optimum Reaction Conditions

Effect of Amount of p-Aminophenol Reagent Solution:

It was observed that the addition of more than 9ml of (1.835x10^-3M) reagent solution was required to obtain a maximum and reproducible absorbance for 0.3mg of thymol. Therefore 10mlof reagent solution were used throughout the study.

Effect of amount of KOH solution: The preliminary examination of color reaction of thymol with P-Aminophenol indicated that a characteristic blue color of the product was formed only in alkaline solution .The maximum constant blue color intensity was reached when using 3ml of 0.1N and remained constant up to 5ml.However3ml of KOH solution was selected for subsequent work.

Effect of time: The minimum time for complete color development of the product was found to be 5 minutes at room temperature. The absorbance was then stable for at least 24 hour.

Order of the addition of reagents: To test the effect of order of the addition of the reagents on the absorbance, different order were tested. The selected order was the thymolL p-amino phenol, followed by the KOH solution, because of high absorbance value.

Beer ?s law

Employing the conditions described in the general procedure a linear calibration graph of Thymol was obtained figure [2], which shows that Beer’s law was obeyed over the concentration range 2-28µg/ml with correlation coefficient of (R²= 0.9992, intercept of 0.058 and slope of 0.0499 .The conditional molar absorptivity of the product formed and sandell? s sensitivity were found to be 0.613x10⁴ L/ mol .cm and 24.66 ng/cm² respectively. The limit of detection and quantification [14-15] were evaluated as: LOD = Intercept /Slopex10 And LOQ = 3.3LOD. The limit of detection was 0.0300 ng/ml and the limit of quantification 0. 099 ng/ml as the lowest standard concentration which could be determine with acceptable accuracy.

Figure 2: Calibration curve for Thymol.

Accuracy and Precision

The accuracy and precision of the method was established by analyzing the pure drug solution at three different levels. Each determination being repeated ten times. The average recovery which is a measure of accuracy is 100 ± 0.95 revealing high accuracy of the method. The relative standard deviation (RSD), which is an indicator of precision is less than 1%. The results are compiled in Table [1].

 Table 1: Optical characteristics and statistical data for regression equation of the proposed method.

In this work, a simple, rapid, precise and accurate spectrophotometric method was developed and validated for the determination of Thymol in pharmaceutical preparations and industrial waste water samples. The method free from such experimental variables as heating or solvent extraction step. The method rely on the use of simple and cheap chemicals and techniques and can be used for rapid routine determination and quality control of Thymol in pure form, bulk sample ,pharmaceutical preparations and real industrial waste water sample.

Acknowledgments

The first author (Dr. Nief Rahman Ahmad) wishes to express gratitude to Al-Hokama company for pharmaceutical industry (HPI) (Nineveh – Iraq.) for providing gift samples of Thymol standard material and for permission and facilities to carry out the research work.

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