Hp 1 column

The headspace of sulfur volatile-exposed and non-exposed orange plants was collected on HayeSep Q adsorbent using dynamic headspace sampling in a climate-controlled cabinet for 6 h [14 (link)]. The traps were extracted with 500 μL hexane containing nonyl acetate (1.75 ng μL⁻¹) as the internal standard, and concentrated to 100 µL for GC-MS analysis. The samples were injected on an Agilent 8890 GC-5977B MSD equipped with a HP-5MS column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, Santa Clara, USA) with spitless mode (helium, 1.0 mL min⁻¹). Column temperatures were programmed from 40 °C for 1 min, raised to 200 °C at 8 °C min⁻¹, isotherm of 1 min, then raised to 280 °C at 20 °C min⁻¹, and isotherm of 5 min. Injector and detector temperatures were 250 °C and 280 °C, respectively. Volatile compounds were identified according to their RIs (calculated using C7−C30) and mass spectra stored in the NIST library, and were identified by co-injection of authentic standards. The quantification of identified volatiles was conducted on an Agilent 7890B GC-FID equipped with an HP-1 column (30 m × 0.32 mm × 0.25 μm), using the same temperature programs as in the GC-MS analysis. Six independent plant replicates were used for each treatment.

A modified solid phase microextraction (SPME) procedure was used to enable analysis by GC–MS (Plutowska and Wardencki, 2008 (link)). The reaction mixture was 125 μm acyl-CoA substrate, 5% ethanol and 560 nm Eht1 in 50 mm sodium phosphate buffer, pH 7.4. After 30 min of incubation at room temperature, 200 µl of the reaction mixture was transferred into a 10 ml glass vial and a conditioned polydimethylsiloxane (PDMS) fibre (Supelco; 100 µm) was introduced into the vial headspace. After 30 min at 30 °C, the fibre was removed from the vial and immediately inserted into the injection port of a ThermoQuest TraceMS instrument fitted with a 50 m × 0.32 mm × 0.17 mm HP1 column (Agilent Technologies). The injector was maintained at a constant 240 °C. The oven temperature was initially held at 40 °C for 1 min before being increased to 180 °C at a rate of 5 °C/min, then to 240 °C at a rate of 10 °C/min with a final hold time of 13 min. Samples were analysed under electron ionization conditions, with the MS scanning between m/z 50–650. Ethyl esters were identified based upon their characteristic mass spectra and related to a library of known alkyl esters (Christie, 2014 ).

For wax extraction, a second leaflet from the same leaf used for terpene analysis was collected. Leaflet areas were scanned prior to being stored at −80 °C. Cuticular wax extraction was performed as per previous protocols^{42 (link)}. Leaflets were dipped in 10 mL of chloroform containing 1 μg/μL of the internal standard tetracosane (Agilent Technologies). Separation was achieved by injecting 1 μL into a GC-FID equipped with a HP-1 column (30 m × 0.32 mm × 1.00 µm; Agilent) using the following temperature profile: 50 °C for 2 min; 45 °C/min to 200 °C with 1 min hold time; 4 °C/min to 300 °C with 10 min hold time. Peaks were identified as described above and peak areas were normalized to the internal standard and leaflet surface area.

The steroids were chromatographically separated on a HP1 column (16 m × 0.20 mm, 0.11 μm film), Agilent Technologies, Santa Clara, CA, USA). A 7890A GC with a 7000 Triple Quadrupole Detector (Agilent Technologies, Santa Clara, CA, USA) was used for separation and detection using electron impact (70 eV) and selective reaction monitoring (SRM). Nitrogen was used as collision gas and helium as carrier gas (1 ml/min, constant flow mode). The injection temperature was 290°C, with the MS source at 310°C. Chromatography was performed using a temperature program for optimal separation: 0.2 min 100°C, ramp 70°C/min until 180°C, ramp 3°C/min until 210°C, ramp 5°C/min until 228°C, and finally ramp 65°C/min until 310°C.
The specific gravity of the samples was analyzed by a digital refractometer (UG‐1 from Atago).
All urinary steroid concentrations are expressed as the unconjugated plus the glucuronide conjugated fraction. The effect of the urine dilution was adjusted for by normalizing the concentrations to a specific gravity of 1.020.

Instrumental analysis of kinetic in vitro samples was performed on an Agilent GC-QToF 7890B/7200 (Agilent Technologies, Milano, Italy), equipped with an Agilent HP1 column (length: 17 m; diameter: 0.2 mm; film-thickness: 0.11 µm) with helium as carrier gas and a flow of 0.8 mL/min. Injection was performed in split mode with a 1:10 ratio at 280 °C. The oven program had the following heating rates: 188 °C hold for 2.5 min, 3 °C/min to 211 °C and hold for 2 min, 10 °C/min to 238 °C, 40 °C/min to 320 °C and hold for 3.2 min. The coupled QToF was operated in full scan with an EI source and ionization energy of 70 eV. Ions were detected from m/z 50 to m/z 750.
Calibration was performed with 9 different concentrations of 5βAdiol with fixed concentrations of MeT. Each calibration level was measured twice. Calibration was tested for linearity applying the Mandel Test, tested for outliers (no residuals >3 standard deviations) and weighted with a factor 1/x. A signal to noise ratio of 10 to 1 was regarded as LOQ. Samples with signals below the LOQ were regarded as not containing any analyte.

Gas chromatographic-mass spectrometric (GC–MS) analysis of the samples was performed on an Agilent 7890A gas chromatographic system coupled to an Agilent 5975 C inert mass selective detector equipped with an Agilent HP1 column (17 m, 0.2 mm id, 0.11 μm film thickness). The following parameters were used for the analysis of products: carrier gas: helium, oven program: 183 °C, +3 °C/min to 232 °C (rate 1), +40 °C/min to 310 °C (rate 2), hold for 2 min, injection volume: 2 μL, split 16:1, injection temperature: 300 °C, electron ionization (EI): 70 eV, full scan mode from m/z 40 to m/z 1000. Prior to GC–MS analysis the dried residues were pertrimethylsilylated (TMS derivatives) by reaction with 100 μL TMIS reagent (MSTFA/ammonium iodide/ethanethiol, 1000:2:3, v:w:v) at 75 °C for 20 min.

Dopamine isotopic enrichment was determined as native dopamine and as the n-propyl derivative [27] (link) (diester, amide) by LC-MS/MS (Shimazu UFLCXR, QTRAP 6500) by negative electrospray ionization. Three parent-daughter ion pairs were monitored to confirm peak identification as dopamine (154/137, 154/119, and 154/91), or as its n-propyl derivative (322/137, 322/210, and 322/266). A gradient elution Solvent A: aqueous ammonium acetate 12 mM, Solvent B: acetonitrile) using a C8 reverse phase HPLC column (on a Phenomenex Luna 5u C8, 100A, 150 × 4.6 mm, 5 microns) was used for dopamine (gradient: 5–20% solvent B) and the dopamine n-propyl derivative (gradient: 50–70% solvent B). Isotopic enrichments were determined by monitoring parent-daughter ion pairs of dopamine (and the n-propyl derivative) formed from the labeled precursor: dopamine m+3 from [2, 5, 6-D3] L-Dopa, dopamine m+6 from ring [13C3]tyrosine, and dopamine m+9 from [13C9, 15N]tyrosine. Tyrosine isotopic enrichment was determined following derivatization as the n-trifluoracetyl-n-butyl ester [28] by Chemical ionization GC-MS (Agilent: HP-1 column, G1530A GC, and 5975C MS) using isobutane as the reagent gas, He gas at a flow rate of 1 ml/min was the carrier, and a temperature program ramped from 100 to 200 °C.