DETERMINATE SAFROLE IN COMMERCIAL ESSENTIAL OILS 149 RESULTS AND DISCUSSION GAS CHROMATOGRAPHY CONDITIONS Tests using different gas chromatography columns and conditions were performed. The results indicated that a weak polar column, CP-SIL 8CB (30 m × 0.53 mm), resulted in a better outcome than a CP-Wax column (30 m × 0.53 mm) for safrole analysis. In terms of the gas chromatography conditions, with methanol dilution, the samples were directly injected into the gas chromatograph for analysis under the column conditions described in the Materials and Methods section. Other major components could be eluted earlier than safrole, and the retention time (RT) for safrole was 13.90 min. In terms of selection of theIS, we tested 1,5-pentanediol, 1,6-hexanediol and DA. Adding a small amount of these reagents resulted in RTs of 5.83, 8.09, and 11.38 min, respectively. Comparing the chromatograms of these reagents and those of the essential oil samples, only DA showed no overlap with any peak of the components of the essential oil samples. Therefore, DA was used as the IS for gas chromatographic analysis for quantifi cation of the safrole level in the essential oil samples. The gas chromatographs of safrole and DA are shown in Figure 1. Figures 2 and 3 present the results of the gas chromatographic analyses of the ether-extracted Stout Camphor sample (Figure 2) and the upper and lower layers of Small-fl ower Camphor essential oil (Figure 3). DETERMINATION OF THE RRF OF SAFROLE TO DA DA was used in this study, which has a similar RT to that of safrole. To accurately quan- tify the contents of safrole in the essential oil samples, the RRF of safrole to DA had to be determined. With the RRF value, the content of safrole in each essential oil sample could be calculated according to equation (2). Figure 4 shows a plot of the peak area ratios of safrole to DA (Y axis) against the concentration ratios of safrole to DA (X axis), which demonstrates that the coeffi cient of determination (R2) for the linear regression model was 0.997 and the RRF was 1.2327 (Figure 4 and Table I). Figure 1. Gas c hromatogram of DA (IS) authentic standard. Peak 1 = DA Peak 2 = safrole.

JOURNAL OF COSMETIC SCIENCE 150 MEASUREMENT OF THE LOWEST QUANTITATIVELY DETERMINABLE CONCENTRATION OF SAFROLE The stock safrole standard solution (1,000 μg/ml) was serially diluted to 50, 25, 10, 5, 2.5, and 1 μg/ml. Following addition of DA IS solution (1 mg each), the diluted standard solution containing DA was directly injected into the gas chromatograph equipped with an FID under the conditions described in the Materials and Methods section, with settings of FID range = 2 and attenuation = 2. Under these conditions, the results showed that when the CV% for safrole recovery was set at 15%, the lowest quantitatively determinable concentration of α-terpineol was approximately 5 μg/ml (Table II). RECOVERY RATES IN SAMPLES FORTIFIED WITH SAFROLE When 2 or 10 mg of safrole were added to 1 g of Stout Camphor essential oil prepared in our laboratory, the recoveries of safrole from the samples were 97.6 to 101.5%, with a CV% of 5.6% or lower (Table III). When 1 or 10 mg of safrole were added to 1 g of the upper layer of Small-fl ower Camphor essential oil, the recoveries of safrole from the sam- ples were 96.1 to 103.5%, with a CV% of 4.2 or lower (Table III). The results indicated that with high or low safrole contents in the essential oil samples, the recovery rates were not only high, but also accurate using our quantitative method. Figure 2. Gas ch romatogram of (A) Stout Camphor essential oil (extracted with ether from C. kanehirai Hayata) and (B) commercial Stout Camphor essential oil. Peak 1 = DA Peak 2 = safrole.