Ultimate 3000 system

Chromatography was carried out on a Dionex UltiMate 3000 system equipped with a Zorbax SB-C18 column (50 × 4.6 mm, 1.8 µm, Agilent, Santa Clara, CA, USA). The following gradient employing solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.3 mL/min was applied: 0–10 min, 1–30% B; 10–15 min, 30–70% B; 15–20 min, 1% B. Injection volume was 10 µL. The ABSciex 4000 QTRAP mass spectrometer used for detection was operated in positive MRM mode. A list of transitions with retention times and collision energies (CEs) of analytes is shown in Table 1. Global instrument parameters: curtain gas: 20 (arbitrary units); collision gas: high; ion spray voltage: 4500 V; temperature: 450 °C; ion source gas: 1 and 2: 25 (arbitrary units) and 40 (arbitrary units); declustering potential: 50 V; and entrance potential: 10 V. As GSH–NEM is a diastereomer [9 (link),25 (link)], all three GSH–NEM analytes (GSH–NEM, GSH-d₅-NEM, and ¹³C₂, ¹⁵N-GSH-d₅-NEM) elute as twin peaks. The sum of areas under both peaks was used for integration and quantification. We also detected two peaks for the transition for NEM, but only the peak with the higher retention time showed the expected UV absorbance at 305 nm, suggesting that the other peak may resemble a hydrolysis product of NEM (Suppl. Figure S7).

High-performance liquid chromatography (HPLC) was performed to support the stability and quality of the ZGYD extract. The following chromatographic conditions were used: a Thermo Fisher Ultimate 3000 system with an Agilent 5 HC-C18 column (250 nm × 4.6 nm; 5 μm), a column temperature of 30°C, a flow rate of 1 mL·min⁻¹, a wavelength of 250 nm, and 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) gradient elution described as follows: 0-10 min, 95-93% (A); 10-20 min, 93-89% (A); 20-35 min, 89-85% (A); 35-55 min, 85-82% (A), 55-63 min, 82-72% (A); 63-73 min, 72-5% (A); and 73-78 min, 5-5% (A).