DETERMINATE SAFROLE IN COMMERCIAL ESSENTIAL OILS 153 We also explored the reproducibility of the method. Taking six different aliquots pre- pared from the same commercial source of stout camphor essential oil, our method deter- mined the safrole concentrations in these six samples to be 136.73, 135.28, 131.19, 139.54, 136.05, and 135.92 mg/g (mean = 135.79 mg/g, SD = 2.70 and CV = 1.99%). The results indicated that this method had a high accuracy. We also tested the reproduc- ibility of our method by analyzing three replicates of each of the samples on each of 3 d and found that our method resulted in reproducibility standard deviations of 1.8% and 2.1% for the measurement of safrole. These results indicated that the method was stable and reproducible. The recovery rate test showed that the recovery rates were all in the range of 96–104%, with CVs all below 5.6%. Our method used direct injection of safrole samples into a gas chromatograph, and the fi ndings demonstrated a higher accuracy of determination of safrole concentration than that of the AOAC method. In addition, we performed experiments using different small amounts of a safrole control standard mixed with IS DA at different ratios and found that the safrole–IS plot showing the peak area ratio (Y axis) to the IS (X axis) had a linear regression R2 value above 0.99 in the linear range of 0.05 to 10.10 mg/g (Figure 4). The results indicated that this method had good linearity in the test range. Table IV Safrole Content in Stout Camphor Essential Oil (S1) and Natural Peppermint Oil Analyzed Using Direct Injection GC and AOAC Methods Safroleb mg/gb (cv%)c Methoda Stout Camphor essential oil (S1) Natural peppermint oil AOAC 36.23 (5.89%) 34.01 (6.11%) Direct injection 37.65 (3.62%) 35.28 (2.73%) a Direct injection method = method developed in this study AOAC method = steam distillation, chloroform extraction and analysis by GC (4). b Average of triplicate analyses. c Coeffi cient of variation (cv%). Table III Recoveries of Spiked Safrole from Stout Camphor Essential Oil (Extracted with Ether from C. kanehirai Hayata) and Small-Flower Camphor (C. micranthum Hayat) Essential Oil (Upper Layer) by the Direct Injection Method Sample Blanka (mg) (A) Amount added (mg) (B) Amount found (mg)b (C) Recovery (%)c CV (%)d Antrodia essential oil 32.73 10.36 43.25 101.54 2.41 32.73 2.07 34.75 97.58 5.61 Small-fl ower Camphor essential oil (upper layer) 0.00 10.36 10.72 103.50 3.49 0.00 1.03 0.99 96.12 4.24 a Safrole in 1 g Stout Camphor essential oil. b Average of triplicate analyses. c Recovery (%) = (C - A)/B × 100%. d Coeffi cient of variation (cv%).

JOURNAL OF COSMETIC SCIENCE 154 DETERMINATION OF SAFROLE LEVELS IN ESSENTIAL OIL SAMPLES FROM MARKETS We used Stout Camphor essential oil prepared by ether extraction from Stout Camphor wood as the positive control and determined the safrole contents in 15 commercially available Stout Camphor essential oil samples, Small-fl ower Camphor essential oil (upper layer and lower layer) samples, and one sample each of cypress oil, natural peppermint oil, China oil, Pak Fah Yeow, Green oil and Cardamom fl ower oil (23 samples in total). Using the method developed in this study, safrole was detected in 21 of the 23 samples. All 15 Stout Camphor essential oil samples purchased from markets were found to contain safrole, in levels ranging from 37.65 to 355.07 mg/g. The upper layer of Small-fl ower Camphor essential oil did not have a detectable level of safrole, whereas the lower layer of the essential oil had up to 642.98 mg/g of safrole. The safrole levels in the remaining essential oil sam- ples were as follows: cypress oil, 129.11 mg/g natural peppermint oil, 35.28 mg/g China oil, 109.56 mg/g Pak Fah Yeow, 33.37 mg/g Green oil, 0 mg/g and Cardamom fl ower oil, 22.06 mg/g. The results indicated that many of the Stout Camphor essential oils and other essential oils available on the market have high levels of the carcinogenic agent safrole. CONCLUSION In this study, we developed a fast and simple gas chromatographic method to quantify saf- role in essential oils. The method only requires the addition of an IS DA methanol solution, Table V Safrole Contents of Various Commercially Available Essential Oils Sample Content (mg/g)a Stout Camphor essential oil (extracted by ether) 32.73 Small-fl ower Camphor essential oil (upper layer) ND Small-fl ower Camphor essential oil (Lower layer) 642.98 Commercial Stout Camphor essential oil (S1) 37.65 Commercial Stout Camphor essential oil (S2) 306.52 Commercial Stout Camphor essential oil (S3) 326.98 Commercial Stout Camphor essential oil (S4) 256.18 Commercial Stout Camphor essential oil (S5) 281.60 Commercial Stout Camphor essential oil (S6) 196.48 Commercial Stout Camphor essential oil (S7) 136.46 Commercial Stout Camphor essential oil (S8) 176.85 Commercial Stout Camphor essential oil (S9) 301.65 Commercial Stout Camphor essential oil (S10) 355.07 Commercial Stout Camphor essential oil (S11) 269.70 Commercial Stout Camphor essential oil (S12) 227.09 Commercial Stout Camphor essential oil (S13) 304.91 Commercial Stout Camphor essential oil (S14) 199.76 Commercial Stout Camphor essential oil (S15) 278.37 Cypress oil 129.11 Natural peppermint oil 35.28 China oil 109.56 Pak Fah Yeow 33.37 Green oil ND Cardamom fl ower oil 22.06 a Average of duplicate analyses. (24) ND: not detected.