Cuticular wax analysis by GC-MS

Cuticular waxes were extracted from 1 g of leaves. Each sample was dipped in 10 ml of chloroform for 30 s, and then 2 μg of C15:0 was included as an internal standard (Li et al., 2008). The solvent was evaporated under nitrogen gas at 40 °C and the wax mixtures were treated with 300 μl of n-hexane to dissolve the sample. After adding 2 ml of 5% methanol sulfuric acid, the mixture was heated in a water bath at 85 ° C for 1.5 h. Then, 750 μl of 1% KCl was added to the sample, washed three times, and the supernatant was collected and dried under vacuum. The sample was dissolved with 300 μl of n-hexane, and was analyzed by GC-MS (Agilent 7890A/5975C). A 1 μl aliquot of solution was injected using an HP 7683 auto sampler (Agilent) and introduced in split mode (1:20) for the quantitative element analysis, applying the following parameters: oven 2 min at 50 °C, raised by 40 °C min⁻¹ to 200 °C, injection at 220 °C for 2 min, an increase of 3 °C min⁻¹ to 320 °C, maintained for 30 min at 320 °C, and He carrier gas inlet pressure was programmed for a constant flow of 1.4 ml min⁻¹. Products were identified using the National Institute of Standards and Technology (NIST) search engine, version 2.0f (Agilent). The quantitative analyses of wax mixtures were performed by comparing GC-FID peak areas against the internal standard and dividing by the injection volume determined for the corresponding sample.