*2.4. LC–MS/MS Analysis*

A HPLC (LC-20A, Shimadzu Corporation, Japan) coupled with a Sciex Qtrap 5500 tandem quadrupole mass spectrometer (Danaher Corporation, Washington, DC, USA) with an electrospray ionization (ESI) source was used for the detection of ENR in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on a C18 column (Agilent poreshell 120 EC-C18, 100 × 4.6 mm i.d., 2.7 μm, Agilent Technologies, Santa Clara, CA, USA) under a flow rate of 0.8 mL/min at 40 ◦C, with an injection volume of 2 μL [38]. Mobile phase A water (containing 1 mL/L of formic acid and 2 mmol/L ammonium acetate) and mobile phase B methanol were used for chromatography separation with the following gradient elution procedure: 15% B for 1.0 min, 15% B to 30% B over 2.5 min, 1.0 min at 30% B, ramping to 95% B over 2.0 min, held for 1.7 min at 95% B, followed by a return to 15% B within 0.1 min and 1.2 min at 15% B for re-equilibration.

The parameters for mass spectrometry include a spray voltage of 4500 V, a CUR pressure of 42 psi, GS1 and GS2 pressures of 55 psi, an ion source temperature at 550 ◦C, and CAD of the medium. MS/MS parameters for ENR, ENR-*d5,* and ENR-*d3* are summarized in Table 1.


**Table 1.** MS/MS parameters and standard curves with two isotope surrogates.

\* Quantitative ion. CE, collision energy; *r***2**, coefficient of determination.
