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.

JOURNAL OF COSMETIC SCIENCE 148 QUANTIFICATION OF SAFROLE CONCENTRATIONS IN ESSENTIAL OIL SAMPLES Twenty to fi fty mg of different essential oil samples were mixed with 5 ml of IS solution (DA total 5 mg). A volume of 0.1 μl of the mixture was directly injected into a gas chro- matograph for analysis. The levels of safrole in the essential oil samples were calculated according to the following equation (2): Safrole content (mg / g essential oil) = (AS / AIS) × (WIS / RRF) × 1/W. (2) Here, W is the weight of t he sample. Analysis of each sample was performed in triplicate. THE AOAC METHOD (19,23,24) Safrole in the samples was isolated by steam distillation, followed by chloroform extrac- tion. The chloroform-extracted samples were then analyzed by gas chromatography. LOWEST QUANTITATIVELY DETERMINABLE CONCENTRATION OF SAFROLE The safrole stock solution (1,000 μg/ml) was diluted with methanol to concentrations of 50, 25, 10, 5, 2.5, and 1 μg/ml. One milliliter of each diluted solution was mixed separately with 1 ml of DA IS solution. The mixtures were injected directly into a gas chromato- graph for analysis in triplicate to calculate the lowest quantitatively determinable concen- tration of safrole. The coeffi cient of variation (CV%) for α-terpineol recovery was set at 15%. RECOVERY OF SAMPLES FORTIFIED WITH SAFROLE Safrole at 2 or 10 mg was mixed with 1 g of essential oil prepared from Stout Camphor wood in the laboratory as described previously, or safrole at 1 or 10 mg was mixed with 1 g of the upper layer of Small-fl ower Camphor (C. micranthum Hayat) essential oil. A control sample was also prepared without the addition of safrole. The samples were then mixed with 0.5–5 ml of DA IS solution (100–1,000 μg/ml), and 0.1 μl of each fi nal mixture was injected into a gas chromatograph to calculate the recovery of safrole in the sample. Analysis of each sample was performed in triplicate. GAS CHROMATOGRAPH CONDITIONS A gas chromatograph (GL Sciences 390B, Tokyo, Japan) equipped with a fl ame ionization detector (FID) was used with the H2 fl ow rate at 30 ml/min and the air fl ow rate at 300 ml/min in this study. The temperatures of the injection port and detector were 245°C and 315°C, respectively. The fl ow rate of the carrier gas (N2) was set at 5 ml/min. A CP-Sil 8 CB column (30 m × 0.53 mm i.d./1.0 μm Chrompack, the Netherlands) was used. The oven temperature was programmed to initiate at 80°C and hold for 6 min. The temperature was raised to 120°C at a rate of 6°C/min. Finally, the temperature was in- creased to 300°C at a rate of 35°C/min, and held for 10 min. The injection volume was 0.1 μl in the direct injection mode.