5977a series gc msd

Analysis was carried out using a 7890B GC and 5977A Series GC/MSD (Agilent, Santa Clara, CA, USA). Separation was achieved on a 30 m × 0.25 mm × 0.25 μm VF-WAXms capillary column (Agilent J&W, Santa Clara, CA, USA). The injection volume was 1 μL, with a split ratio of 10:1. The carrier gas was helium maintained at a 1.5 mL/min constant flow. The injector temperature was 280 °C and the detector temperature 350 °C. The temperature program was as follows: initial column temperature set at 50 °C for 1 min then programmed to 170 °C at 15 °C/min and held for 5 min, then from 170 °C to 200 °C at 3 °C/min, held 5 min, and from 200 °C to 230 °C at 5 °C/min, and held 17 min.
A standard Supelco 37 component FAME Mix in dichloromethane (Bellefonte, PA, USA) was used for identification and quantification. Compounds were identified based on a comparison of retention times with authentic compounds. With each set of samples, blank samples and the FAME Mix standard were analyzed to verify the stability of the analytical system. The results are expressed as the weight percent of an individual fatty acid to the total fatty acid (TFA) content calculated from the peak area using the appropriate correction factors [30 ].

Amino acid analysis was performed using a 7890B GC and 5977A Series GC/MSD (Agilent, Santa Clara, CA, USA). Separation was achieved on a 10 m × 0.25 mm × 0.15 mm ZB-AAA GC column provided in the EZ:faast kit together with a FocusLiner^®. The split ratio was 15:1, and the injector temperature was 250 °C. The injection volume was 1.5 μL. Helium was used as the carrier gas at a 1.5 mL/min flow rate. The temperature program was 110 °C to 320 °C at 30 °C/min. The detector temperature was set to 310 °C. Calibration curves were prepared for individual amino acids at concentrations of 50, 100 and 200 nmol/mL using the standard mixture (SD) provided. The amino acid standard mixture consisted of 200 nmoles/mL of each amino acid: alanine (ALA), glutamic acid (GLU), hydroxylysine (HLY), leucine (LEU), phenylalanine (PHE), threonine (THR), valine (VAL), aspartic acid (ASP), glycine (GLY), hydroxyproline (HYP), lysine (LYS), proline (PRO), tryptophan (TRP), cystine (C-C), histidine (HIS), isoleucine (ILE), methionine (Met), serine (SER) and tyrosine (TYR). Each calibrant was prepared in triplicate. From then on, the SD solutions were treated following the same procedure as the samples. Individual amino acids were identified by comparing peak retention times with known amino acids in the standard. The amino acid content results are expressed in mg/g of sample dry weight (dwt).

Fatty acid methyl esters (FAME) were extracted as per previous protocols⁴³ , with minor modifications. Briefly, frass and leaf samples were ground to powder with liquid nitrogen and 40 mg weighed in glass vials. Each frass and leaf sample had three technical replicates. To each vial, 2 mL of a 1.5 M H₂SO₄ solution containing 5 μg/mL nonadecanoic acid (internal standard; Agilent Technologies) was added, then heated to 85°C for 1.5 h with periodic mixing. After cooling, pentane and 0.9% NaCl (1:1) was added to separate methyl esters. The organic phase was analyzed by an Agilent 5977A Series GC-MSD (GC-mass spectrophotometer detector) fitted with a 30 m × 0.25 mm × 0.25 μm HP-5 column using the following temperature program: 8 °C/min from 100 to 250 °C with 10 min hold time. Compounds were identified by comparing their spectra against the NIST library, and peak areas were integrated using MassHunter Quantitative Analysis (Agilent Technologies). Areas were normalized to the internal standard and ground weight.