Fourteen 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 A. cinnamomea. α -terpineol and vanillin at a purity > 99% were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan).

Stout Camphor wood (50 g) was ground into small pieces with a Chinese medicine grinder (Model 6022; Shin-Jen Co., Taichung, Taiwan) to a size of 1~2 mm, and was placed in a glass bottle. After adding 250 mL of ether, the sample was sonicated for 60 mins with a sonicator (DC-600H; DELTA). The solution was filtrated with qualitative filter papers (Grade No. 5A; ADVANTEC TOYO, Tokyo, Japan), and the solvent was removed by heating at 45 ℃ in a water bath. The resulting liquid was the pure essential oil used as the positive control in this study.

α -terpineol (100 mg) or vanillin (100 mg) was placed into a 100-mL volumetric flask and dissolved in isopropanol to 100 mL. The solutions used were stock solutions of α -terpineol standard (S) solution (1 000 μ g· mL^-1) and vanillin internal standard (IS) solution (1 000 μ g· mL^-1).

The relative response factor (RRF) of the α -terpineol standard (S) to the vanillin internal standard (IS) was measured using the following method. The stock S and IS solutions were mixed together at a serial ratio (S: IS) of 10: 1, 5: 1, 2: 1, 1: 1, 1: 2, 1: 5 and 1: 10, and the mixtures were subjected to chromatographic analysis. The response factor of a sample was defined as the peak area of the sample in gas chromatographic analysis divided by the concentration of the sample. Thus, RRF can be obtained by Equation (1).

RRF=(AS/WS)/(AIS/WIS)(1)

where A_S is the peak area of α -terpineol and A_IS is the peak area of vanillin. W_S is the weight of α -terpineol, and W_IS is the weight of vanillin.

The α -terpineol stock solution (1 000 μ g· mL^-1) was diluted with isopropanol to concentrations of 50, 25, 10, 5, 1 and 0.5 μ g· mL^-1. One mL of each diluted solution was mixed separately with 1 mL of internal standard solution (vanillin). The mixtures were injected directly into a gas chromatograph in triplicate for analysis. The coefficient of variation (CV%) for α -terpineol recovery was set at 15%; the lowest concentration of α -terpineol obtained was the lowest quantitatively-determinable concentration obtained by gas chromatography.

Twenty-five or fifty mg of each stout camphor essential oil sample were mixed with 5 mL of internal standard solution (vanillin; 1 000 μ g· mL^-1). A volume of 0.1 μ L of the mixture was injected into a gas chromatograph for analysis. The recovery of each sample was measured in triplicate. The levels of α -terpineol in the essential oil samples were calculated by Equation (2):

α⁃terpineolcontent(mg·g-1essentialoil)=(AS/AIS)×(WIS/RRF)×1/W(2)

where W is the weight of the sample.

In a 20-mL vial, α -terpineol (10 or 1 mg) was mixed with 25 mg of essential oil samples extracted from Small-flower Camphor (C. micranthum Hayat; sample S14) or stout camphor (sample S2). A blank sample was prepared without addition of α -terpineol. After adding 5 mL of internal standard solution (vanillin; 1 000 μ g· mL^-1), 0.1 μ L of each mixture was injected into a gas chromatograph. The recovery level of each was measured in triplicate.

A gas chromatograph (GL Sciences 390B, Tokyo, Japan) equipped with a flame ionization detector (FID) was used with the H2 flow rate at 30 mL· min^-1 and the air flow rate at 300 mL· min^-1 in this study. The temperatures of the injection port and detector wese 250 and 310 ℃, respectively. The flow rate of the carrier gas (N₂) was set at 5 mL· min^-1. A CP-Sil 8 CB column (30 m× 0.53 mm i.d./1.0 μ m; Chrompack, Netherlands) was used.

The oven temperature was qrogrammed to initiate at 80 ℃ and hold for 3 min. The temperature was raised to 150 ℃ at a rate of 6 ℃· min^-1, and hold for 1 min. Finally, the temperature was increased to 300 ℃ at a rate of 30 ℃· min^-1, and hold for 10 min. The injection volume was 0.1 μ L in the direct injection mode.

We performed a test to select a suitable gas chromatography column and appropriate analytical conditions. In terms of gas chromatography column selection, a high-polar column, CP-Wax (30 m× 0.53 mm), a non-polar column, CP SIL 200 (30 m× 0.53 mm), and a weak polar column, CP-SIL 8CB (30 m× 0.53 mm), were tested. The results indicated that the weak polar column (CP-SIL 8CB) was suitable for the analysis of terpineol due to the high polarity of terpineol. Samples were applied using the direct injection mode under column conditions of initiation at 80 ℃ followed by holding for 3 min. The temperature was raised to 150 ℃ at a rate of 6 ℃· min^-1, and held for 1 min. It was finally increased to 300 ℃ at a rate of 30 ℃· min^-1, and held for 10 min. Under these conditions, compounds other than α -terpineol and vanillin were eluted earlier. The retention time for α -terpineol and vanillin was 8.14 and 13.46 min, respectively (Figure 1). The results of the gas chromatographic analyses of the ether-extracted stout camphor sample and the commercial essential oil samples were as shown in Figures 2 and 3.

Fig.1

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	Fig.1 Gas chromatograph of α -terpineol and vanillin (IS, internal standard) authentic standard. Peak 1=α -terpineol; Peak 2=vanillin (IS)

Fig.2

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	Fig.2 Gas chromatograph of the ether extract of stout camphor tree camphorate wood essential oil (stout camphor tree essential oil (A) commercial camphor oil). Peak 1=α -terpineol; Peak 2=vanillin (IS)

Fig.3

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	Fig.3 Gas chromatograph of commercial stout camphor tree camphorate wood essential oil. Peak 1=α -terpineol; Peak 2=vanillin (IS)

In order to accurately quantify the content of α -terpineol in the stout camphor essential oil, this study used water-soluble vanillin as an internal standard. The process first determined the RRF of α -terpineol to vanillin, and then, using the RRF value, the content of α -terpineol in each essential oil sample could be calculated according to Equation [2]. Figure 4 shows a plot of the peak area ratios (Y axis: α -terpineol/vanillin) against the concentration ratios (X axis: α -terpineol/vanillin), which demonstrates that the coefficient of determination (R²) for the linear regression model was > 0.999 and the RRF was 2.907 4 (Figure 4 and Table 1).

Fig.4

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	Fig.4 The calibration curve of α -terpineol to vanillin internal standard

Table 1

Table 1

Table 1 Relative response factor (RRF) and gas chromatographic retention time (RT) of α -terpineol to vanillin Compound RRFa RTb Vanillin (IS)c 1.000 0 13.457 α -Terpineol 2.907 4 8.137 a RRF of α -terpineol to vanillin; b A CP-SIL 8CB column (0.53 mm× 30 m, DF=1.0 μ m) was used; c Internal standard

Table 1 Relative response factor (RRF) and gas chromatographic retention time (RT) of α -terpineol to vanillin

Following the addition of 50 μ L of internal standard solution (=5 μ g vanillin) to each serially-diluted standard (50, 25, 10, 5, 1 and 0.5 μ g· mL^-1), the mixtures were directly injected into the gas chromatograph equipped with a FID under the conditions described in the Materials and Methods section. The result showed that when the CV% for α -terpineol recovery was set at 15%^{[20, 21]}, the lowest quantitatively-determinable concentration of α -terpineol was 5 μ g· mL^-1 (Table 2).

Table 2

Table 2

Table 2 Lowest quantitatively-determinable concentration of α -terpineol by gas chromatography with a flame ionization detector Compound Concentration /(μ g· mL-1) Detectability Recoverya /% RSDb /% α -Terpineol 50.0 Yes 99.7 4.8 25.0 Yes 104.5 3.9 10.0 Yes 102.1 5.7 5.0 Yes 108.7 10.4 2.5 No 117.8 17.5 1.0 No 151.9 29.7 aAverage of triplicate analyses; bCoefficient of variation (CV%)

Table 2 Lowest quantitatively-determinable concentration of α -terpineol by gas chromatography with a flame ionization detector

Next, we studied the recoveries of α -terpineol in two different types of essential oil samples fortified with additional α -terpineol. When 10 or 1 mg of α -terpineol was added to Small-flower Camphor essential oil (a camphor species known to contain no α -terpineol; sample S14), the recovery rates were 102.7% and 98.4%, respectively, with a CV% of 5.24% or lower (Table 3). On the other hand, when 10 or 1 mg of α -terpineol was added to stout camphor essential oil (sample S2), the recovery rates were 103.4% and 98.3%, respectively, with a CV% of 10.8% or lower (Table 4). Based on the aforementioned results, we demonstrated that our method only requires the addition of isopropanol solution that contains a known concentration of internal standard, and the mixed samples can be analyzed by gas chromatography using the direct injection mode. The analysis took only 30 min for one sample, which is fast and simple.

Table 3

Table 3

Table 3 Recovery of spiked α -terpineol from small-flower camphor essential oil by the direct injection GC method Compound Blanka /mg (A) Amount added/mg (B) Amount foundb/mg (C) Recoveryc /% CVd /% α -Terpineol 0.00 10.00 10.27 102.70 3.87 0.00 1.00 0.98 98.42 5.24 a α -terpineol in stout camphor Tree essential oil; b Average of triplicate analyses; c Recovery(%)=(C-A)/B× 100%; d Coefficient of variation (CV%)

Table 3 Recovery of spiked α -terpineol from small-flower camphor essential oil by the direct injection GC method

Table 4

Table 4

Table 4 Recovery of spiked α -terpineol from stout camphor tree essential oil (S2) by the direct injection GC method Compound Blanka /mg (A) Amount added/mg (B) Amount foundb/mg (C) Recoveryc /% CVd /% α -Terpineol 213.56 10.00 223.90 103.40 6.28 213.56 1.00 214.54 98.33 10.79 a α -terpineol in stout camphor tree essential oil; b Average of triplicate analyses; c Recovery(%)=(C-A)/B× 100%; d Coefficient of variation (CV%)

Table 4 Recovery of spiked α -terpineol from stout camphor tree essential oil (S2) by the direct injection GC method

We used stout camphor essential oil prepared by ether extraction from stout camphor wood as the positive control, and determined the α -terpineol contents in the sample prepared from stout camphor wood, in commercially-available stout camphor essential oil samples, and in Small-flower Camphor essential oil (upper layer). Using the method developed in this study, the α -terpineol contents in the 14 essential oil samples were found to range from 213 to 533 mg· g^-1 (Table 5). This finding indicated that the α -terpineol levels in different samples varied, and a difference of 2.5-fold was observed between the highest and the lowest contents. This is in agreement with several previous studies^{[20, 21, 22, 23]}, which reported that the components of stout camphor essential oil might vary depending on the which parts of the tree are used as the material to prepare the essential oil. In addition, the age of the tree may also affect the quality of the essential oil. This study also confirmed that essential oil extracted from the small-flower camphor tree did not contain α -terpineol.

Table 5

Table 5

Table 5 α -terpineol content in some commercial Stout Camphor Tree essential oil ample α -Terpineol content/ (mg· g-1) Stout Camphor Tree essential oil (Extracted by ether) 516.27 Commercial Stout Camphor Tree essential oil (S1) 512.13 Commercial Stout Camphor Tree essential oil (S2) 213.56 Commercial Stout Camphor Tree essential oil (S3) 228.65 Commercial Stout Camphor Tree essential oil (S4) 304.29 Commercial Stout Camphor Tree essential oil (S5) 352.17 Commercial Stout Camphor Tree essential oil (S6) 265.94 Commercial Stout Camphor Tree essential oil (S7) 365.47 Commercial Stout Camphor Tree essential oil (S8) 412.09 Commercial Stout Camphor Tree essential oil (S9) 333.28 Commercial Stout Camphor Tree essential oil (S10) 279.34 Commercial Stout Camphor Tree essential oil (S11) 394.12 Commercial Stout Camphor Tree essential oil (S12) 410.58 Commercial Stout Camphor Tree essential oil (S13) 275.96 Small-flower Camphor essential oil (S14) ND a Average of duplicate analyses; b ND=not detected

Table 5 α -terpineol content in some commercial Stout Camphor Tree essential oil