Model 5973

Analysis of amino acids was performed using the Phenomenex EZ:Faast kit for free amino acids (www.phenomenex.com). Six replicate samples for each nectar type were collected as described previously. Due to low volume of nectar produced by the foliar nectary, these nectar samples were pooled from a maximum of 90 nectaries, collected from six separate plants. Each sample (20 µL nectar per extraction) was subjected to solid phase extraction and derivatized according to the manufacturer’s instructions, with one adjustment: after addition of the norvaline internal standard (5 nmol) to each sample, 125 µL of 10% propanol (v/v)/20 mM HCl was added to acidify the sample. Following derivatization, samples were concentrated by evaporation under a stream of nitrogen gas before amino acids were analyzed using an Agilent Technologies model 6890 gas chromatograph with a ZB-AAA 10 m × 0.25 mm amino acid analysis column coupled to a model 5973 mass selective detector capable of electrical ionization (EI). The GC–MS instrument settings followed the manufacturer’s recommendations.

Total lipids were extracted from K. olearia grown at 40 °C to early stationary phase and 65 °C to mid-log, early stationary, late stationary and death phase using methanol–chloroform (1:1 v/v). Fatty acid methyl esters (FAME) were prepared from total lipids extracts using mild alkaline methanolysis (Guckert et al. 1985 (link)). Dried FAME samples were re-dissolved in 300 µL chloroform (HPLC grade, Fisher Scientific) and analyzed by gas chromatography with mass spectrometry (GC–MS) on an Agilent 6890N gas chromatograph with a model 5973 inert mass selective detector (Agilent) fitted with an Agilent HP-5MS capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness; J + W Scientific). Helium was used as the carrier gas with a temperature program of 130 °C increasing at 3 °C min⁻¹ to 290 °C and held for 2 min. Sample peaks were identified by comparison to Bacterial Acid Methyl Ester Mix standards (Supelco, Sigma Aldrich) or on the basis of mass spectra and expressed as a percentage of the total FAME quantified in each sample.

A Model 6850 gas chromatograph equipped with a Model 5973 mass spectrometer with dimensions of 30 m × 0.25 mm and a 0.25-μm nonpolar capillary column HP-5MS (5%-phenyl)-methylpolysiloxane (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the synthetic 2AP, including its intermediate, byproducts and reaction product with Cr(CO)₆. The GC carrier gas used was purified helium (99.99%) at a constant flow rate of 1 mL min⁻¹. The oven temperature was initially held at 45 °C and then increased at a rate of 3 °C min⁻¹ to a final temperature of 150 °C. To analyze the rice sample extracts, the initial oven temperature was set at 45 °C, held for 5 min and increased to 200 °C at a rate of 4 °C min⁻¹. The injector temperature was 230 °C. The sample was injected with a split ratio of 10:1. The mass spectrometer was operated in electron impact (EI) mode with an electron energy of 70 eV. The ion source and quadrupole temperatures were set at 230 °C and 150 °C, respectively. The mass range (m/z) was 29–550 with a scan rate of 0.68 s. The GC–MS transfer line was set to 280 °C. 2AP and Cr(CO)₆ were identified by matching their mass spectra with synthetic and standard compound reference spectra in the Wiley 7n Mass Spectral Library (Revision C.00.00). The NIST 17 Mass Spectral Library (Revision D.01.00/1.6d.) was also utilized.