Determination Thymol in Thyme extract and its Pharmaceutical forms by using Gas Chromatography method

 

Raghad Helaliy¹, Fadi Alrouh², Saleh Trefi¹, Yaser Bitar¹

¹Department of Pharmaceutical Chemistry and Quality Control-Faculty of Pharmacy,

University of Aleppo-Syria.

²Department of Organic Chemistry -Faculty of Medicine-University of Al-Hawash-Syria.

 

ABSTRACT:

Objective: The objective of this study was developed and validated of a simple, rapid and accurate GC method for quantitative determination of thymol which considers as a chemical marker in thyme extract and in the final formulation by GC-FID chromatography. Method: thymol analysis was done by using menthol as internal standard, chromatographic conditions were TRB-5-625 capillary column and its specification 60m_˟0.25mm i.d0.25µm film thickness and nitrogen as a carrier gas at a flow rate of 0.6mL/min. The oven temperature was programmed between 185℃ and 235℃ for 16min. The injector and detector temperatures were maintained 290℃. Detection was done by using flame ionization detector FID. Results: the developed method was validated according to ICH guidelines. The developed method was given accepted linearity which R>0.99 in the concentration range of 1-40mg/10ml of thymol, accuracy with mean recovery values between 98%-102%, precision with relative standard deviation of the calculated concentration was less than 2%. Conclusion: The results showed that the developed method was effective in determination of thymol in pharmaceutical forms and routine analysis in quality control laboratories simply.

 

 

INTRODUCTION:

In recent years, plant derived products are increasingly being sought out as medicinal products. According to an estimate of the World Health Organization (WHO), about 80% of the world population still uses herbs for their primary health care needs^[1]. Herbal drugs are finished labeled products that contain active ingredients such as aerial or underground parts of plant or other plant material or combination^[2-3].

 

Generally, the quality control of plant materials and phytomedicines relies on the identification and quantitation of chemical markers^[4], chemical marker considered active principles of plants used in traditional plants, as well as in the formulation of herbal medicines^[5]. Genus Thymus contains about 300-400 species, the most commonly used is thymus vulgaris^[6], Thymus has changed from a traditional herb to a serious drug^[7].

 

Thymol is the major constituent of essential oils and thyme extract of thymus vulgaris^[8], so it considers as a biological marker^[9], beside thymol another thirty components in the plant have been identified like example carvacol, p-cymene, linalool, myrecen, a-peinine…etc.^[6-10-11].

 

Thymol (2-isopropyl-5-methyphenol) is a white crystalline monoterpene phenol with an aromatic odor, its density at 25℃ is 0.96g/cm³ with melting point ranging from 49℃ to 51℃^[12-13]. It is highly soluble in alcohols, other organic solvent and it is slightly soluble in water^[14]. The chemical structure of Thymol is shown in Fig.1   

 

Fig.1: Chemical structure of thymol

 

Thymol use antimicrobial, preservative^[12], antiseptic^[15], in dentistry for toothache^[16], antioxidant, anti-inflammatory, antispasmodic and antitumor activities^[14] and Gastro-intestinal disorders^[9]. Thymol is a broncholytic and secretomotoric effects. Its activity on β₂ receptors as a possible mechanism of action was demonstrated^[17]. Because thymol is a major component therefore, used for standardization in German pharmacopeia (0.03% phenols calculated as thymol)^[17-18]

 

The quantification of thymol in drug formulation and/or biological samples was presented in several reports. Analytical methods in these articles involved the use of high-performance thin layer chromatographic HPTLC ^[19], HPLC with uv/vis detection^[6], HPLC-PAD detection^[20], thin layer chromatography^[21], Spectrophotometric analysis by diazotization reaction ^[12], flow injection spectrophotometric detection^[22] and titrimetric methods^[23-24]. Gas chromatography is well-established analytical technique for analysis of volatile oils due to the availability of mass spectrometer detector principle constituents are identified^[25-26], the interested components can be quantized by the flame ionization detector FID^[27]. In the other site, another technique such as reverse phase RP-HPLC is a wide using in the field of pharmaceutical analysis identified^[28-38]. This work describes a new simple, direct and selective capillary GC/FID method for the determination of thymol which considers as a chemical marker at thyme extract and thyme pharmaceutical formulation. This method could be an additional analytical procedure in the quality control of raw materials, active pharmaceutical ingredients and pharmaceutical formulations.

 

MATERIALS AND METHODS:

Chemicals and reagents:

Working standard of thymol crystals and thyme extract are given by Asia Industries, Aleppo-Syria. Chloroform (Surechem Products Ltd- England) and working standard of menthol is given by Asia Industries, Aleppo-Syria. The syrup samples brand names are Hustagil each 5ml contain 480mg of standardized thyme liquid extract produced by Asia Industries Aleppo, Syria, Kanabron each5ml contain 480mg of standardized thyme liquid extract produced by Kanawati industries Damascus, Syria Thymex each 5ml contain 480mg of standardized thyme liquid extract produced by Seapharma industries Tartous, Syria

 

Apparatus:

GC-2010 gas chromatograph equipped with a spilt/spilt less injector and a flame ionization detector from Shimadzu Technologies Inc. the column type was TRB-5-625 capillary column and its specifications 60m×0.25mm i. d0.25µm film thickness was used in this study. Nitrogen ultrapure was obtained from G1010E nitrogen generator Peak scientific and used as carrier gas. 5µl gas-tight syringe SGE Analytical science were used.

 

Standard solution preparation:

The standard stock solution of thymol was prepared by weight 200mg thymol and transferred to volumetric flask, then dissolved in chloroform to a final volume 10ml so was reached to final concentration 200mg/10ml, the standard stock solution was used to prepare all diluted solution.

 

Internal standard solution preparation:

The internal standard solution was prepared by weight 20mg menthol and transferred to volumetric flask then dissolved in chloroform to a final volume 10ml and the final concentration reached to 20mg/10ml.

 

Standard Solutions:

The working standard solutions of thymol were prepared by taking suitable aliquots of drug solution from the standard stock solution then added 1ml of internal standard solution and the volume reached to 10ml with chloroform to get concentrations between 1-40mg/10ml. three replicates 3µl of each working standard solution were injected immediately after preparation.

 

Sample preparation of syrups:

For standard thyme extract 50g of this extract was weighted and transferred into 500ml conical flask, extraction of thymol content from the extract was done by using two portion of chloroform each 10ml. For syrups the content of one bottle of each syrups were mixed well then transferred into 500ml conical flask, extraction of thymol content of the syrup was done using two portion of chloroform each 10ml. Then 9ml from extraction process was transferred to volumetric flask and added 1ml from internal standard solution to a final volume 10ml.

 

RESULTS AND DISCUSSION:

GC analysis:

 

Fig.2: GC chromatogram of the standard reference solution of thymol using menthol as an internal standard and by adjusting the thermal programmed from 185℃ for a minute then raised at rate of 5℃/min to reach 235℃ and remain constant for 5min.

 

Chromatogram appeared the top of solvent, A=menthol and B for standard thymol.

A specific and sensitive capillary column gas chromatographic method was developed to determine thymol by using on a TRB-5-625 as capillary column. Three replicates 3µl of each working standard solution were injected immediately after preparation into the injector at 290℃ .in the mode at a split ratio of 0.5:10 sharp and symmetrical peaks were achieved by programming the oven temperature at 185℃ for 1min, then programmed to 235℃ at a rate 5℃ min and maintained for 5min. the retention times of menthol is 7.2min and the retention time for thymol 7.8 and the detector was 290℃ the Fig.2 shows the chromatogram for experiment. 

 

Analytical method validation:

Method validation was done according to International Conference on Harmonization (ICH) recommended test conditions^[39-42].

 

Linearity and Rang:

The linearity of analytical procedure is its ability (within a given range) to obtain results which are directly proportional to the concentration of an analyte in the sample^[39-42]. The linearity between the concentration and the area resulting from the proportion of the analyte on the internal standard. The linearity was evaluated by linear regression analysis. The linear equation obtained by the least squares method was y=0.3791x+0.5827 Fig.3. The correlation coefficient allowing estimating the quality of curve was R²=0.9998 this value of R greater than 0.99 indicates a satisfactory linearity. The linearity was obtained at the range between 1-40mg/10ml.

 

Fig.3: linearity of thymol.

 

Accuracy:

The accuracy of an analytical method: expresses the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. For the quantitative approaches, at least nine determinations across the specified range should be obtained ^[39-42].

 

Table.1: accuracy of the developed method.

*n=3

 

Table.2: Repeatability precision of developed method.

*mean, n=6    

Table.3: Intermediate precision of developed method:

 

Accuracy study was done by injection three replicates of three concentrations of thymol at the range of linearity. Recovery results in table.1 for thymol was 99.95%-101.45% which was at accepted range of accuracy 98%-102% and this indicates that the developed procedure is accurate and applicable to determination of thymol.

 

Precision:

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions^[39-42].

 

Repeatability precision was proved by injection the standard solution with concentration 30mg/10ml six times N=6 the data's show that the RSD was less than 1%. Intermediate precision expresses within laboratories variations for example different days, different analysts, and etc ^[28] was done by three replicate injections of three concentrations during a period of two days. RSD was less than 2% for two days and this result indicated that the intermediate precision of this method was acceptable for thymol and the results for precision are showed in table 2 and 3.

 

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage ^[39-42]. To prove that the developed method is robustness a small different variations were changed like oven programmed where under normal conditions the temperature raised by 5℃/min, but to prove that the method was robustness a change in the rate of hyperthermia by 5℃±1, temperature of detector and injector changed in the set temperature by 290℃±5, flow rate was changed by 0.6±0.05ml/min.

 

The summit between peak height thymol of menthol were calculated for normal conditions and for variation condition and the RSD were under 5% and this indicate that the developed method was robustness. Results are summarized in table 4, 5 and 6.

 

 

Table.4: different variation of programmed oven temperature.

 

Table.5: slight changes in flow rate.

 

Table.6: slight changes in temperature of the detector and injector

 

 

From above it was found that all RSD results were less than 5% for all changes and therefor the method is robustness.

 

Sensitivity:

LOD and LOQ were calculated to evaluate the sensitivity of the developed method. The calculated LOD and LOQ for thymol were 16.5ɲg/1ml and 50ɲg/1ml respectively.

 

Specificity:

To prove the specificity of developed method, reference solution was exposed to sunlight for 60 days at room temperature. The chromatogram shows that the reference solution was stable in 60days in these conditions Fig.4.

 

Fig.4: Chromatogram of the standard reference solution degradation of thymol.

Chromatogram appeared the top of solvent and B for standard thymol.

 

 

Table.7: results of thymol syrups assay by developed method.

 

Table. 8: result of thyme extract assay by developed method.

Application of developed method on pharmaceutical formulation:

We have applied the developed method on commercial products in the Syrian market, the quantitative results showed that all studied products were concentrated in accordance with the specifications prescribed by the German Pharmacopeia^[18] which states that the minimum thymol in 100g extract is 30mg, at syrups formulation each 100ml syrup contain 9.6g of thyme extract which contain not less than 2.88mg thymol. The results showed at table 7 that all the formulation contains more than 2.88mg so all samples were approved.

 

For thyme extract it should contain 15mg thymol at least according to the extract weight which was given, depending on germane pharmacopeia (contain at least 0.03%) the result is shown at the table 8.

 

Depending on the German Pharmacopeia which specifies the standard method for extracting the thyme plant which is Standardized thyme liquid extract (1: 2-2.5) with min. 0.03% thymol extraction means: liquid ammonia 10% (m/m) of NH3: 85% glycerol: ethanol 90% (V / V) water (1:20, 70: 109) (m/m).

 

Depending on all thyme preparation prepared according to this method extract, we have found that all of them were in conformity with the constitutional specifications.

 

In literature based on Grigore et al^[43] thymol was determined by GC-FID but at hexane extract thymol appeared at RT=27.5 min so our method determined it with less time.

 

There aren't many studies that have identified thymol within extracts and syrups and only one article has been found according Bishr et al^[44] but relied on GC-MS and the RT for thymol was 12 min.

 

CONCLUSION:

Modern GC-FID method was developed for determine thymol which is considered the chemical marker of thyme extract. The developed method has proved specific, precise and accurate for assaying thymol. This method could be an additional analytical procedure in the quality control of raw materials, active pharmaceutical ingredients and pharmaceutical formulations.

 

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Accepted on 21.02.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4055-4060.