17a gas chromatograph

Terpenoids from organic extractions were analyzed on a Shimadzu 17A gas chromatograph coupled to a Shimadzu QP5050A quadrupole mass selective detector. Separation was performed on a Restek SHR5XLB column (30 m × 0.25 mm i.d. × 0.25 mm thickness) under the following conditions: helium was the carrier gas (flow rate of 5 mL min⁻¹), a splitless injection (injection injector temperature = 250 °C) was used, and a temperature gradient of 5 °C /min from 40 °C (3 min hold) to 240 °C was applied. Terpenoid products were identified using the National Institute of Standards and Technology (NIST) mass spectral database and by comparison of retention times and mass spectra with authentic reference compounds purchased from Sigma-Aldrich (St. Louis, MO, USA). Quantification was performed as previously reported [26 (link),32 (link)]. Representative single-ion peaks of each compound were integrated and compared with the equivalent response of the internal standard (single-ion method).

Total fatty acid composition of the milk was determined by the direct trans-esterification method of Lepage and Roy [43 (link)] as previously described [34 (link)] with n = 6–7 dams per group. Briefly, 50 to 100 µL of milk was placed into a screw-capped Teflon-lined tube containing C23:0 as an internal standard. After the 1-h trans-esterification procedure and recovery of the fatty acid methyl esters (FAMEs) in the benzene phase, the FAMEs were analyzed by capillary gas liquid chromatography. FAMEs were then separated and measured on a Shimadzu (17A) gas chromatograph with flame ionization detection. A 50 mm × 0.25 mm BPX-70 fused silica capillary column (SGE Scientific; Ringwood, VIC, Australia) with a film thickness of 0.25 µm was used in conjunction with a Shimadzu on-column auto-injector. Ultrahigh purity hydrogen was used as a carrier gas at a flow rate of 2 mL/min. A temperature gradient program was used with an initial temperature of 170 °C, increasing at 3 °C/min to 218 °C. Identification of the FAMEs was made by comparison with the retention times of chromatography reference standard mixtures (Nu-Chek Prep; Elysian, MN, United States of America) [44 (link)].