Hp 5ms gc column

To quantify α-humulene production, 200 µL samples were taken from the n-dodecane phase of the cultivation. Samples are centrifuged at 16,000× g for 3 min and stored at −20 °C until analysis. Prior to analysis, the samples were diluted with acetone 1:10 including 50 mg/L zerumbone as an internal standard. A standard curve was made with α-humulene diluted with n-dodecane. Prior to analysis, the standards were diluted 1:10 with acetone including zerumbone like the samples. 1 μL of samples and standards were applied to a GC-MS system with an HP-5MS GC column (Agilent, Santa Clara, CA, USA).

Fifty-three OC congeners were selected as target analytes. These included dichlorodiphenyltrichloroethanes (∑DDTs): o,p', p,p'-DDD, -DDE, -DDT; hexachlorocyclohexanes (∑HCHs): α, β, γ, δ-HCH; ∑Drins (aldrin, endrin, dieldrin, endrin aldehyde, endrin ketone);∑CHLs: (trans-chlordane (TC), cis-chlordane (CC), heptachlor, heptachlor endo-epoxide, heptachlor exo-epoxide), endosulfan: (α-, β-endosulfan, and endosulfan sulfate); p,p'-Methoxychlor (MXC); hexachlorobenzene chlordanes (HCB); and PCBs (numbers 8, 18, 28, 44, 52, 66, 77, 81, 101, 105, 114, 118, 123, 126, 128, 138, 153, 156, 157,167, 169, 170, 180, 187, 189, 195, 206 and 209). The basic physicochemical properties of these OCs are summarized in Supplementary Table S2.
The target OCs were quantitatively analyzed using an Agilent 7890B gas chromatograph-7000C tandem mass spectrometry (GC–MS/MS) with electron impact ionization (EI). An HP-5MS GC column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies Inc.) was used to separate the analytes. The GC oven temperature was programmed as follows: 80 °C (5 min) → 20 °C/min → 160 °C (0 min) → 4 °C/min → 240 °C (0 min) → 10 °C/min → 295 °C (2 min). Helium was used as the carrier gas. One μL of sample was injected at constant port temperature of 250 °C. Multi-reaction monitoring (MRM) model was used for mass spectrometry.

The composition of SEL was analyzed according to a previously reported method [36 (link)]. In short, 100 mg of SEL was mixed with 2 mL of 4% methanol and sulfuric acid and heated in a water bath at 65 °C for 1 h in order to obtain fatty acid methyl esters (FAMEs). Afterward, 1 mL of both n-hexane and deionized water was added to FAMEs, and the upper organic phase was taken after shaking. Subsequently, the organic solvent in the liquid was thoroughly blown dry to obtain methyl-esterified fatty acids (MEFs). MEFs were finally dissolved in dichloromethane (500 μL) for gas chromatography–mass spectrometry (GC-MS) (Agilent, Santa Clara, CA, USA) analysis. An HP-5MS GC column (30.0 m × 250 µm, Agilent, Santa Clara, CA, USA) was used to analyze FAMEs. In this study, 1 µL of each FAMEs sample was injected into the column. The constant pressure mode was used, and the shunt ratio was 10:1, with helium as a carrier gas. The column temperature increased from 60 °C to 180 °C at a constant rate of 25 °C per min and then increased to 240 °C at a rate of 3 °C per min, remaining at 240 °C for 1 min and finally increasing to 250 °C at a constant rate of 5 °C per min. Mass spectrometry was performed using full-scan mode detection. Fatty acids were identified using the mass spectrometry library of the National Institute of Standards and Technology (NIST).