JOURNAL OF COSMETIC SCIENCE 146 differences in the main component of essential oils obtained from their leaves, camphor trees can be subclassifi ed into fi ve subgroups: (a) camphor tree (C. camphora subsp. formosana var. oxidentalis), in which the main component is camphor (b) linalool tree (C. camphora CT linalool), in which the main component is linalool (c) cineole tree (C. camphora (Linn.) Presl var. camphora), in which the main component is cineole (d) sesquiterpene tree, in which the main component is nerolidol and (e) safrole tree, in which the main component is safrole. Although it is not easy to distinguish them according to their morphology, the main component in their leaf essential oil can help to classify these trees. A study of cam- phor tree essential oils (Fujita et al., [9]) showed that oils extracted from the leaves and fruits of C. camphora Sieb contain 85% and 30–43% camphor, respectively, and oils extracted from the fruits also contain 40–59% safrole. Safrole is a known carcinogenic substance that causes liver tumors (10–12). The metabolites of safrole, including 1-hydroxysafrole, isosafrole, and dihydrosafrole, are all carcinogens (11). A study has also shown that orally administered safrole in humans resulted in four major metabolites, of which safrole 2′,3′-oxide (SFO) is a reactive electrophilic metabolite and a high concentration of safrole (0.5%) increased the incidence of cancer in a mouse model (12). Exposure to SFO caused an increase in micronuclei in mouse red blood cells and signifi cantly induced DNA strand breaks, indicating that the toxicity of safrole is due to its metabolites having carcinogenic effects (13–15). At present, methods for the analysis of safrole include gas chromatographic methods (16–20) and HPLC methods (6,7,21,22). Among these methods, the Association of Offi cial Analytical Chemists (AOAC) method (19,23,24) uses steam to distill safrole, followed by chloroform extraction and subsequent analysis using gas chromatography. However, the protocol is tedious and time-consuming, and the yield of safrole is low because losses occur during the multiple steps of the process. In addition, chloroform is a toxic and controlled reagent. Therefore, the method is complicated and has many limitations. Currently, aro- matic therapy is becoming popular, and many essential oils are used in body sprays, evapo- rative aromatherapy, and even for oral administration. If essential oils have high safrole levels, serious negative health effects will result. Therefore, the development of a simple and rapid quantitative method is required for screening the safrole contents of essential oils on the market. This study aimed to develop a simple and reliable method to quantify safrole that only requires the addition of an internal standard (IS) to essential oil samples without pretreat- ment, and the sample can be directly analyzed using gas chromatography. METHODS AND MATERIALS MATERIALS Essential oil samples were purchased from the market, including 15 Stout Camphor essen- tial oil (prepared from C. kanehirai Hayata) samples from four different manufacturers in Taiwan (Joben Bio-Medical co., Pingtung I Chuan Bio-Tech Corp., Tainan Golden Dapu Biotech Crop., Chaiyi and Yu-jang Biotech Co., Taoyuan), one sample each of the upper layer and the lower layer of Small-fl ower camphor essential oil, and a cypress oil sample (from Joben Bio-Medical co.), all of which were included in the analyses in this study.

DETERMINATE SAFROLE IN COMMERCIAL ESSENTIAL OILS 147 Stout camphor essential oil samples of different brands were purchased from markets, and Stout Camphor wood (C. kanehirai Hayata) was provided by a company that cultivates Antrodia cinnamomea (Taoyuan, Taiwan). α-terpineol and vanillin at a purity 99% were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Other essential oils that are widely used in several Asian countries, including one sample each of natural peppermint oil (Li Ping Co., New Taipei City), China oil (derived from the blossoming, above-ground parts of Menthae ×piperitae L. by distillation Bio-Diaet-Berlin GmbH ), Pak Fah Yeow (also called White Flower Analgesic Oil, which is made from a blend of aromatic herbs, contains 6% camphor How Hin Pak Fah Yeow Manufactory Limited, Taipei, Taiwan), Green oil (a popular oil in China that helps to relieve minor body aches and pains in the muscles and joints and contains 3% camphor Hsin Wan Jen Pharmaceutical Co., Taichung, Taiwan), and cardamom fl ower oil (Cheong Kim Chuan (CKC) co., Penang, Malaysia), were purchased from Tainan Pharmacy, and the essential oils were prepared by steam distillation. Safrole, n-decanol (or n-decyl alcohol, DA), and methanol of analytical grade at a purity 99% were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). PREPARATION OF ESSENTIAL OIL FROM STOUT CAMPHOR WOOD Stout Camphor wood (50 g) was ground into small pieces using a Chinese medicine grinder (Model 6022 Shin-Jen Co., Taichung, Taiwan) to a size of 1–2 mm and then placed in a glass bottle. After adding 500 ml of ether, the sample was sonicated for 40 min with a sonicator (DC-600H DELTA, New Taipei City, Taiwan). The solution was fi ltrated with qualitative fi lter papers (Grade No. 5A Advantec Toyo, Tokyo, Japan), and the solvent was removed by heating at 45°C in a water bath. The resulting liquid was pure essential oil, which was used as the positive control in this study. PREPARATION OF SAFROLE STANDARD AND DA IS SOLUTIONS Safrole (100 mg) or DA (100 mg) was placed into a 100-ml volumetric fl ask and dis- solved in methanol to 100 ml. The solutions subsequently obtained were used as stock solutions (w/v) of safrole standard solution (1,000 μg/ml) and DA standard solution (1,000 μg/ml). RELATIVE RESPONSE FACTOR OF SAFROLE TO DA The stock safrole standard (S) and DA IS solutions were mixed together at serial ratios of 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10 in methanol, and the mixtures were subjected to chromatographic analysis. The relative response factor (RRF) of a sample was defi ned as the peak area of the sample in gas chromatographic analysis divided by the concentration of the sample, and, therefore, the RRF of safrole to DA can be calculated based on the following equation (1): RRF = (AS/WS) / (AIS/WIS), (1) where AS is the peak are a of safrole and AIS is the peak area of DA. WS is the weight of safrole and WIS is the weight of DA.