**7. Extraction, identification and quantification of oleocanthal**

The method used for the extraction, identification and quantification of oleocanthal described herein, is selective for oleocanthal and was developed by Beauchamp and coworkers (Beauchamp et al., 2005). More recently, this method was adapted and involves the quantification of oleocanthal using an internal standard (ISTD), 3,5 dimethoxyphenol (Beauchamp et al., 2005, Cicerale et al., 2009b).

The extraction of oleocanthal from the oil matrix involves liquid-liquid partitioning using both hexane and acetonitrile, whereby the phenolic fraction partitions into the acetonitrile phase. Acetonitrile is then removed and the dried down extract is dissolved in methanol:water and analyzed by HPLC. Separation of the oleocanthal phenolic compound is carried out using a HPLC system with a diode array detector set to 278 nm. A reverse phase-C18 column (250 mm × 4.6 mm ID, 5 μm) is used for the separation at a constant temperature of 25°C using the gradients listed in Table 1. A flow rate of 1 ml/min is used, and the injection volume is 20 μl. See Figure 6 for HPLC chromatogram.

Fig. 6. HPLC chromatogram of olive oil phenolic extract containing oleocanthal and 3,5 dimethoxyphenol (ISTD) (Cicerale et al., 2009b).

Via mass spectrometry (6210 MSDTOF), oleocanthal is further identified under the following conditions: drying gas, nitrogen (N2) (7 mL-1, 350°C); nebulizer gas, N2 (15 psi); capillary voltage 4.0 kV; vaporizer temperature, 350°C; and cone voltage, 60 V. Figure 7 displays the negative ion mass spectrum of oleocanthal with the characteristic [M-H]- ion at *m/z* 303.12 highlighted.


Table 1. Mobile phase gradient for oleocanthal separation.

364 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

The method used for the extraction, identification and quantification of oleocanthal described herein, is selective for oleocanthal and was developed by Beauchamp and coworkers (Beauchamp et al., 2005). More recently, this method was adapted and involves the quantification of oleocanthal using an internal standard (ISTD), 3,5 dimethoxyphenol

The extraction of oleocanthal from the oil matrix involves liquid-liquid partitioning using both hexane and acetonitrile, whereby the phenolic fraction partitions into the acetonitrile phase. Acetonitrile is then removed and the dried down extract is dissolved in methanol:water and analyzed by HPLC. Separation of the oleocanthal phenolic compound is carried out using a HPLC system with a diode array detector set to 278 nm. A reverse phase-C18 column (250 mm × 4.6 mm ID, 5 μm) is used for the separation at a constant temperature of 25°C using the gradients listed in Table 1. A flow rate of 1 ml/min is used,

Fig. 6. HPLC chromatogram of olive oil phenolic extract containing oleocanthal and 3,5

Time (min)

Via mass spectrometry (6210 MSDTOF), oleocanthal is further identified under the following conditions: drying gas, nitrogen (N2) (7 mL-1, 350°C); nebulizer gas, N2 (15 psi); capillary voltage 4.0 kV; vaporizer temperature, 350°C; and cone voltage, 60 V. Figure 7 displays the negative ion mass spectrum of oleocanthal with the characteristic [M-H]- ion at

Oleocanthal

dimethoxyphenol (ISTD) (Cicerale et al., 2009b).

*m/z* 303.12 highlighted.

mAU

**7. Extraction, identification and quantification of oleocanthal** 

and the injection volume is 20 μl. See Figure 6 for HPLC chromatogram.

ISTD

(Beauchamp et al., 2005, Cicerale et al., 2009b).

Fig. 7. Negative ion mass spectrum of oleocanthal with the characteristic [M-H] ion at *m/z* 303.12 (Cicerale et al., 2009b).
