Triplus rsh

Carbon isotope ratios were determined via compound-specific stable isotope analyses using GC-isotope ratio mass spectrometry (IRMS). The organic mixture was separated by gas chromatography and baseline-separated compounds were chemically converted into the analyte gas CO₂ for ¹³C/¹²C analysis (Elsner et al., 2012 (link)). Carbon isotope fractionation was determined in triplicate by injection into the gas chromatograph (Agilent 6890, Palo Alto, CA, United States) using a CP-PoraBOND column (50 m × 0.32 mm, 5 μm inner diameter, J&W Scientific, Germany). One mL of the reaction mixture was transferred to a He-flushed 10 mL glass vial wherefrom 1 mL headspace sample was injected via autosampler (TriPlus RSH, Thermo Scientific, Germany) with a split ratio of 1:5. For chromatographic separation a temperature gradient program was used, starting from 30°C (held for 10 min) followed by a 20°C ⋅ min^–1 gradient to 250°C (held for 5 min) with 2 mL ⋅ min^–1 flow and an injector temperature of 280°C (Franke et al., 2017 ). Carbon isotope ratios were determined at the IRMS (MAT 235, Thermo Scientific, Germany) relative to the laboratory reference gas [CO₂, calibrated against Vienna Pee Dee Belemnite standard V-PDB, IAEA Vienna, Austria (Coplen et al., 2006 (link))]. The overall analytical uncertainty was <0.5%.

Freeze–dried kale powder (50 mg) was suspended in 1 mL distilled water in a 2 mL microcentrifuge tube (Fisher Scientific, Waltham, MA, USA). Hydrolysis products were generated naturally by endogenous myrosinase in the absence of light at room temperature for 24 h. After adding 1 mL of dichloromethane, the samples were centrifuged at 12,000× g for 2 min, and the lower organic layer was collected. A gas chromatograph (Trace 1310 GC, Thermo Fisher Scientific, Waltham, MA, USA) coupled to an MS detector system (ISQ QD, Thermo Fisher Scientific, Waltham, MA, USA) and an autosampler (Triplus RSH, Thermo Fisher Scientific, Waltham, MA, USA). A capillary column (DB-5MS, Agilent Technologies, Santa Clara, CA, USA; 30 m × 0.25 mm × 0.25 µm capillary column) was used to determine GS hydrolysis products. A 1 μL sample of the dichloromethane extract was injected into the GC–MS with a split ratio of 1:1. After an initial temperature held at 40°C for 2 min, the oven temperature was increased to 260°C at 10°C/min and held for 10 min [42 (link),82 (link)]. The injector and detector temperatures were set to 200°C and 280°C, respectively. The flow rate of the helium carrier gas was set to 1.1 mL/min. The peaks were identified using information from a previous publication [42 (link)] or by comparison with data in the National Institute of Standards and Technology (NIST) library.