CML and CEL

According to Jiao et al. (10) and Yu et al. (11), 150-μl reaction sample was spiked with 150 μL of internal standard solution which containing 71.2 ng of d₄-CML and 66.0 ng of d₄-CEL. Chromatographic separation was performed on a Waters 2695 liuid chromatograph (LC) module coupled with a Waters X-Bridge C18 column (2.1 × 100 mm, 3.5 μm, Milford, MA, USA). The mobile phase A was acetonitrile and mobile phase B was NFPA (5 mM). The flow rate was 0.3 mL/min, the injection volume was 5 μL and the column temperature was set to 40°C. The gradient elution was performed as follows: 0–0.1 min, 5% A; 0.1–5 min, 5–60% A; 5–8 min, 60%−100% A; 8–10 min, 100% A; 10–12 min, 100–5% A; 12–20 min, 5% A.

The LC was interfaced with a Micromass Quattro Micro triple quadrupole mass spectrometer (Manchester, UK). The mass spectrometer (MS) was operated in an electrospray ionization (ESI) positive mode with multiple reaction monitoring (MRM). The capillary voltage was 3.6 kV. Temperatures of the ionization source and the desolvation gas were 110 and 400°C, respectively. The flow rates of the cone gas and desolvation were 50 and 600 L/h, respectively. Argon gas was used as the collision gas in the collision cell. For CML, d₄-CM, CEL, and d₄-CEL, the fragments m/z 205 → 84, m/z 209 → 88, m/z 219 → 84, and m/z 223 → 88, respectively, were used for quantification.