Supelcowax 10 column

Tea leaves (containing one bud and three leaves) were placed in one of the following solutions: (1) water (as control); (2) 12 mM l a,b,b,2,3,4,5,6-²H₈]Phe (l-[²H₈]Phe, deuterium atom ≥ 98%, Cambridge Isotope Laboratories, Inc.). After 18 h treatment, to identify endogenous volatile products, finely powdered floral tissues (500 mg fresh weight) were extracted with 2 mL of hexane:ethyl acetate (1:1) for 7 h in the dark. The extract was then filtered through a short plug of anhydrous sodium sulfate and 1 μL of the filtrate was subjected to GC-MS analysis. The injector temperature was 230 °C, splitless mode was used with a splitless time of 1 min, and helium was the carrier gas with a velocity 1.6 mL/min. The SUPELCOWAX^TM 10 column (Supelco Inc., 30 m × 0.25 mm × 0.25 μm) was used with an initial temperature of 60 °C, a ramp of 20 °C/min to 180 °C, and then 10 °C/min to 240 °C, and hold at 240 °C for 20 min. The MS analyses were carried out in full scan mode (mass range m/z 44–200).

The sample preparation and analysis were previously described in detail [59 ]. Briefly, each urine sample was run in triplicate; three 1 mL aliquots were pipetted into 10 mL clean glass headspace vials along with ACS grade sodium chloride to saturation, and 153 ng of naphthalene-d8 as an internal standard for normalizing peak areas between samples.
Samples were analyzed by headspace solid-phase microextraction (SPME) on a 7890A-5975C gas chromatograph-mass spectrometer (Agilent Technologies, Santa Clara, CA) fitted with a CombiPAL robotic sampling preparation and injection system (Autosampler Guys, Alexandria, VA). The CombiPAL heated and agitated the sample at 37°C for 30 min to equilibrate the sample with the headspace, then for 45 min to equilibrate the headspace with a 1 cm 50/30 μm divinyl benzene-carboxen-poly(dimethylsiloxane) (DVB/CAR/PDMS) stable-flex SPME fiber (Sigma-Aldrich, St. Louis, MO). The SPME fiber and column were heated to the maximum temperature between samples to prevent carry-over. The instrument and autosampler system were controlled using MSD Chemstation software ver E.02.02 (Agilent Technologies, Santa Clara, CA). Chromatographic separation was obtained with a 0.25 mm ID by 30 m long SUPELCOWAX 10 column with a 0.25 μm film (Sigma-Aldrich, St. Louis, MO). The mass spectrometer operated in the full scan mode with a range from 40 to 350 m/z.

Volatile compounds from kernel and oil were extracted by solid-phase microextraction (SPME) [13 (link)]. Briefly, 3.0 g of grounded kernel flour or extracted oil was mixed with 2.5 mg/kg of 4-methyl-2-pentanol (internal standard), stirred for 10 min at 40 °C until equilibration in a 20 mL glass vial with a PTFE/silicone septum (Agilent Technologies, Palo alto, CA, USA). Next, a solid phase microextraction (SPME) fiber (DVB/CAR/PDMS, Sigma-Aldrich, St. Louis, MO, USA) was exposed to the sample headspace for 40 min. Next, the volatile compounds were analyzed with a GC system (Agilent Technologies) comprising an autosampler (Agilent PAL RSI 85), gas chromatograph (GC Agilent 7820A) and mass spectrometer (Agilent 5977B) designed with an electron impact source and quadrupole analyzer. Volatile compounds were separated with a Supelcowax 10 column (30 m × 0.25 mm × 0.25 µm, Sigma-Aldrich, USA) using helium as a carrier gas at a flow rate of 1 mL/min. GC oven temperature was started at 40 °C and increased by 3 °C/min after 10 min, until it reached a final temperature of 200 °C. Volatiles were identified by two methods, including the use of NIST 08 Mass Spectral Library and comparison of their retention time and mass spectrum to their respective standards. Results were expressed as µg of internal standard per kg of kernel or oil sample.

Fatty acids were isolated and methylated using a modified Folch method as previously described [4 (link),26 (link)]. Fatty acid in plasma samples were quantified by gas chromatography–mass spectroscopy (GC-MS) with a Supelcowax-10 column (Sigma-Aldrich, Saint Luis, MO, USA). Peak identification was based upon comparison of both retention time and mass spectra of the unknown peak to that of known standards. Fatty acid methylated ester (FAME) mass was determined by comparing areas of unknown FAMEs to that of a fixed concentration of 17:0 internal standard. Response factors were determined for each individual FAME to correct for GC-MS total ion chromatogram discrepancies in quantification. These factors were determined through the use of a GLC reference standard which contained known masses of FAMEs ranging from 14–24 °C. The response ratio of each FAME is corrected to a fixed amount ratio for each FAME relative to 17:0. Individual FA is expressed as a percent of the total FA mass (mol%).