Zhang Qianli, Zhou Jianke, Han Kang, Zhang Li
(Research Center of Physics and Chemistry Analysis, Hebei University; Hebei Province Key Laboratory of Analytical Science and Technology, Baoding 071002 China)

Received on Aug. 29, 2005

Abstract The separation and simultaneous quantitative determination of salicylic acid, phenol and resorcinol in Compound Fluocinnonide Tincture using reversed-phase ion-pair high-performance liquid chromatography on a C₁₈（150*4.6mm i.d.,5）column was investigated. A mixture of methanol-acetonitrile-water10:25:65(v/v/v) containing 1.0mmol/L tetrabutyl ammonium bromide was used as the mobile phase and the detection wavelength was 225nm.The average spiked recoveries were all above 85.0% and the relative standard deviations were between 0.48% and 3.34%.The calibration curve was linear between0.014g/ml to 5.40g/mL, and the linear relativity were 0.9971, 0.9997 and 0.9977. The limits of detection were 0.014g/ml,0.027g/ml,0.019g/ml, respectively.

The Compound Fluocinonide Tincture can kill germs and diminish inflammation. It is widely used for the treatment of neurodermatitis and is also effective in curing psoriasis in a sense.It consists of many components, such as fluocinonide, salicylic acid, resorcinol, borneolum syntheticun, ethanol et al .During the course of production and transportation, salicylic acid would probably be degradable to phenol with the existence of violet^[1],and resorcinol is easily oxidated , which make the color of the Tincture solution deeper and deeper, and the effect of the Tincture decreased. Establishing an effective and quick method to monitor the quality of the Tincture is of great importance. Currently a number of methods have been described for the determination of salicylic acid or resorcinol, which include neutralization titration^［］, ultraviolet spectrophotometry，HPLC^［3-5］,capillary zone electrophoresis^[6] ,gas chromatography ^[7]et al. But some of these methods could not simultaneously detect them. The goal of this article was to develop an alternative method for the separation and simultaneous determination of salicylic acid, phenol and resorcinol. The method here was proved simple, direct, valid and sensitive for the detection of these three phenolic compounds and the results were satisfactory.

1. EXPERIMENTAL
1.1 Apparatus
The HPLC system consisted of LC-10AT solvent delivery pump, 7725i injector and SPD-10A UV-VIS detector from Shimadzu Corporation(Japan).TheN-2000 Double-Channel Chromatograph Data Operation Station and Software were from ZheJiang University Intelligent Information Engineering Research Institute. The UV-265 spectrophotometer was from Shimadzu Corporation (Japan).
1.2 Reagents and standards
Salicylic acid, phenol, resorcinol andtetrabutyl ammonium bromide were of analytical reagent grade and were used without further purification. HPLC grade solvents such as methanol and acetonitrile were used to prepare the mobile phase. Compound Fluocinonide Tincture was from a Shanghai Pharmaceutical Company，with the batch number 050401.Water used was distilled twice by passing through SZ-93 pure water system.
Stock solutions were fresh weekly prepared individually by dissolving 156.5mg Salicylic acid, 149.1mg phenol and 64.7mg resorcinol with the mobile phase in 100ml volumetric flasks. All stock solutions were stored at 4C in a refrigerator and would be diluted to the required concentrations before being injected directly into the chromatograph.
1.3 Chromatographic conditions
Separation was performed using Kromasil C₁₈（150*4.6mm i.d., 5μ）(Nobel Eka ,Sweden).The temperature of the column was kept at room temperature. The mobile phase consisted of methanol-acetonitrile-water10:25:65 (v/v/v) containing 1.0mmol/l tetrabutyl ammonium bromide. The detection was carried out at the wavelength of 225 nm.The flow rate was 1.0ml/min and the injection volume was 5.0l.
1.4 Sample preparation
The Compound Fluocinonide Tincture (with the batch number 050401) was transferred accurately 2.0ml with a pipet It was extracted with 1.0 ml petroleum ether, and the supernatant was discarded. Then the extracted layer was extracted again with 1.0 ml petroleum ether. Again the supernatant was discarded and 1.0ml distilled water was added to the extracted layer. The solution stratified, and the supernatant was discarded. The extracted layer was filtered with a syringe filter, diluted to the final concentration with mobile phase and injected into the chromatography system.

2. RESULTS AND DISCUSSION
2.1 Detection wavelength
The UV spectra of these three phenolic compounds were recorded from 200nm to 400 nm, and the wavelengths corresponding to UV maximums of these standard compounds were: salicylic acid (225 nm), phenol (265 nm and 221 nm),resorcinol (278 nm and 227.5nm) respectively. In order to achieve a better sensitivity and less interference, the detection was performed at 225nm in sensitivity range 0.01 AUFS.
2.2Selection of mobile phase
To obtain satisfactory resolution and to avoid peak tailing of the compounds , an optimization of the proposed method was carried out using different mobile phase. Mobile phases of various compositions of methanol and water were tested. Using the system of methanol-water 20:70 and 40:60 (v/v ) the peaks of resorcinol and phenol were not well separated, and the retention time of resorcinol was too short. With an increase of the volume of acetonitrile added into the mobile phase, all the compounds could be separated but the retention time of resorcinol was still too short. Since these phenolic compounds were all weak acids and could ionize to a certain extent, using the ion-pair chromatography could improve the retention and separation.Different concentration of tetrabutyl ammonium bromide were added into the mobile phase, and the effect on elution behavior was investigated. The results indicated that with the increasing concentration of the tetrabutyl ammonium bromide, the retention time delayed accordingly. But when the concentration was 2.0 mmol/l or more , the retention time of resorcinol was shortened. The best results were obtained using the mobile phase methanol-acetonitrile-water 10:25:65(v/v/v) containing 1.0mmol/L tetrabutyl ammonium bromide, and the analysis could be finished within 8 minutes. The chromatogram of the standard compounds is shown in Fig 1.

Fig 1 Chromatogram of standard compounds 1-Resorcinol;2-Phenol;3-Salicylic acid	Fig 2 Chromatogram of the sample a-Unknown;1-Resorcinol;2-Phenol;3-Salicylic acid

2.3System suitability
System suitability tests were performed and chromatographic parameters were calculated from experimental data, such as the number of theoretical plate and the resolution. The number of theoretical plate for resorcinol was 2500 and the resolution was 3.3 between resorcinol and the unknown compound according to the chromatogram of the sample.
2.4 Optimization of the sample preparation
Some components such as fluocinonide and borneolum syntheticum in the sample also had ultraviolet absorption at 225nm and it interfered with the detection of the phenolic compounds .To eliminate the interferences in the detection, the sample preparation was optimized. The sample solution was initially only extracted with 2.0ml petroleum ether and filtered with a syringe filter before detection . It was observed that although the sample preparation step was simple, some components that interfered with the detection of the phenolic compounds could not be eliminated and the reliability of the method could not be checked. When 1.0ml distilled water was added into the solution that purified by petroleum ether, the solution stratified evidently. The supernatant was discarded and the extracted layer filtered. Before injected into the chromatographic system, the solutions were diluted with mobile phase. Under the optimal chromatographic conditions, the components that interfered with the detection were eliminated, and the concentrations of salicylic acid, phenol and resorcinol were detected. The mechanism is likely that these phenolic compounds are water-soluble compounds but other major components in the sample can only be dissolved in water with the presence of other organic solvents and the addition of water broke their capacity of dissolving in water. Thus the interferences of fluocinonide , borneolum syntheticum and other components in the sample solution could be eliminated.
2.5Linear equations, correlation coefficients and detection limits
A series of working solutions were prepared by diluting the stock solutions with mobile phase to the final concentrations and they were detected under the optimized chromatographic conditions. The calibration graphs of the standard compounds were found to be linear over the concentration range studied. The linear equations and the detection limits of the three compounds are listed in Table 1. (Y-peak area; X-the concentration of the detected compound)

Table 1 Linearity of the determination of three phenolic compounds

Components	Linear equations	Correlation coefficients	Linear ranges g/ml	Detection limits g/ml
Salicylic acid Phenol Resorcinol	Y=976.15+5930.42X Y=37.22+3792.90X Y=24.85+6571.71X	0.9971 0.9997 0.9977	0.019-3.90 0.027-5.40 0.014-1.40	0.019 0.027 0.014

2.6Recoveries and precision
The recoveries of the method were studied . The stock solutions of different concentrations were added in the Compound Fluocinonide Tincture solution . Then the sample solutions were prepared with the same method as described in 1.4 and diluted with mobile phase before detection.The concentrations of the phenolic compounds were examined and the recoveries could be calculated according to the linear equation. The intra-day precision of the method was determined by preparing the sample with the same method as described in 1.4 for each analysis and the values were detected by 5 repeated analysis in a single day. The sample solutions were fresh daily prepared for the determination of the inter-day precision of the method which was checked by determining each phenolic compound during 5 days. The results are given in Table 2.
2.7The determination of thesample
The applicability of the method for the simultaneous determination of salicylic acid, phenol and resorcinol was verified by the determination of them in the Compound Fluocinonide Tincture. The sample solution was prepared with the same method as described in1.4. Then the sample solution was diluted 40,000 times, 40 times, 200,000 times respectively with mobile phase before detection and the concentrations of salicylic acid, phenol and resorcinol were determined by 5 repeated analysis. The results are given in Table 3. The chromatogram of the sample is shown in Fig 2.

Table 2 Recoveries and assay precision

Components	Added contents ml	Determined contents ml	Recoveries /%	Mean /%	Intra-day precision RSD /%	Inter-day precision RSD /%
Salicylic acid	1.25	1.14	91.29
1.57	1.45	92.25	92.09	3.34	2.76
1.88	1.74	92.75
Phenol	1.49	1.39	93.57
1.86	1.73	93.16	92.84	0.48	0.77
2.24	2.06	91.79
Resorcinol	0.52	0.47	93.65
0.65	0.61	91.54	93.03	1.01	0.88
0.78	0.74	94.97

Table 3 Results of the determination of the sample

Components	Marked content ／	Average determined content ／
Salicylic acid Phenol Resorcinol	－ － －	4.54 0.054 12.35

3. CONCLUSION
The proposed method above was simple and effective as for the preparation course of the sample .Some other compounds in the sample such as fluocinonide, borneolum syntheticum and ethanol can also diminish inflammation and kill germs. But they did not interfere with the separation and detection of salicylic acid, phenol and resorcinol. Phenol was detected in the sample solution although it is known as skin irritant. So during the course of production and transportation, the Compound Fluocinonide Tincture should kept in proper condition in order not to be deteriorated. The validity of the method was established through the study of linearity, sensitivity , precision and accuracy and the results were satisfactory , which proved the method as an alternative and useful one to control the quality of the Compound Fluocinonide Tincture.