GC/MS analysis of cuticular hydrocarbons

Identification and quantifications of CHCs were performed by using a GC/MS instrument (7890B-7000D, Agilent, USA), equipped with a HP-5MS column (Agilent, 30 m × 0.25 mm i.d., 0.25μm film thickness, 5% phenyl methyl siloxane stationary phase). The GC/MS conditions were as follows: the GC injection port temperature was maintained at 300°C and the samples were injected in the split-less mode. The oven temperature was programmed from an initial temperature of 80°C held for 2min, followed by a ramp at 20°C/min to 200°C, and a second ramp at 5°C/min to 290°C and held in that temperature for 19 min. The transfer line between GC and MS was set at 290°C, and the temperatures of the MS source were 230°C and operated under electron ionization mode. The scan mode was used for monitoring the targeted compounds. The carrier gas was helium (purity > 99.999%) at a constant flow rate of 1.0 mL/min. Data acquisition and evaluation were carried out using Agilent MassHunter Data Acquisition, Quantitative Analysis and Qualitative Analysis programs (version B07.00, Agilent Technologies, CA), respectively. The peaks were identified based upon comparison with standards and the National Institute of Standards and Technology library (NIST, version 17). The peak area of each CHC component was calculated as a proportion of the total area of all detected peaks for each samples.