The extractions were carried out in triplicate, and each sample was injected three times, with an injection volume of 1 μL. The isolated sample was chromatographically separated on a capillary column DB-Wax (60 m × 0.25 mm × 0.25 μm) from Varian (Walnut Creek, CA, USA) using an Agilent
Technology 6890N GC-FID system (Palo Alto, CA, USA). The following conditions were used: injector temperature, 250 °C; detector temperature, 250 °C; carrier gas flow (He), 1 mL/min. The injections were made in split mode (split ratio 1:25) and the oven temperature was maintained at 40 °C for 3 min, then increased from 40 to 200 °C at 6 °C/min and eventually held for 6 min. Quantification of the limonene was selleck inhibitor carried out by the internal buy SCH772984 standard method (propyl benzene). Linearity of responses was acceptable (r2 > 0.998) across a standard concentration range that exceeded that of the samples (10–2500 μg/g). The particle size of the suspended pulp in the orange juice was analysed using a laser diffraction particle size analyser (LS 13 320, Beckman Coulter, High Wycombe, UK), capable of measuring particle diameters in the range 0.4 μm–2000 μm. Samples (1 mL) were introduced into the universal liquid module, and obscuration was maintained
at 5% by dilution with deionised water (White, Fisk, Makkhun, & Gray, 2009). Orange juices with pulp concentrations (0, 10, 20, and 100 g/100 g) were observed at the optical microscope Leitz Diaplan Microscope, (Wild Leitz, Heerbrugg, Switzerland) (×10) (Iwanaga et al., 2007). Images analysis was performed O-methylated flavonoid using Pixelink Software 2004 (Ottawa, Canada). A Micromass LCZ mass spectrometer was used, fitted with an MS Nose interface (Micromass, Manchester, U.K.) to sample the headspace above the solutions
(Linforth & Taylor, 1999). Selected ion monitoring (SIM) analysis was used for all experiments. Cone voltage and ion monitored was 15 V and 137 m/z, respectively. MassLynx software (Micromass, Manchester, UK) was used for all data extraction. Orange juice (30 mL) samples (OJ) were placed in Duran graduated laboratory bottles (nominal size = 100 mL, real volume = 123 mL) (Sigma–Aldrich, Poole, U.K.) fitted with a one port lid that allowed headspace sampling. Samples were equilibrated at room temperature and the headspace was sampled through the port into the APCI-MS via a heated transfer line (120 °C), with a gas flow rate of 2.5 mL/min. Each sample was measured in triplicate following a fully randomised design. Resultant detector responses (mV) were converted to Aqueous Standard Equivalents (ASE) by comparing the headspace to that of limonene control samples as analysed by static headspace (APCI-MS). Solutions with different concentration of limonene were measured and coefficient of determination was calculated (r2 = 0.985). The air–water partition coefficient of limonene has previously been documented by Falk, Gullstrand, Löf and Wigaeus-Hjelm (1990) as 0.
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