*3.5. UHPLC Analysis*

We analyzed the diluted spiked glycerin without solid-phase extraction step using the QTOF6500 HRMS mass spectrometer coupled to the UHPLC system. A liquid chromatography system 1290 (Agilent Technologies, Santa Clara, CA, USA) with a BEHC18 column (2.0 mm × 100 mm) with 1.8 μm particles (Waters, Milford, MA, USA) at 30 ◦C was used for HPLC separation. Mobile phase A consisted of 92:8 methanol/water with 0.05% formic acid; mobile phase B consisted of 98:2 isopropanol/water with 0.05% formic acid. The flow rate was 300 μL/min, and the gradient used was as follows: from 0–3 min, mobile phase B 0–30%; from 3–10 min, mobile phase B 30–70%, followed by holding at 70% B for 5 min and returning to 0% B in 2 min. The first 1 min of the LC flow was diverted to the waste to prevent the glycerin matrix from entering the mass spectrometer.

#### *3.6. HRMS Analysis*

The Agilent 6550 QTOF instrument (Agilent Technologies, Santa Clara, CA, USA) is equipped with an Agilent Jet Stream Technology Dual Spray ESI source and an iFunnel. The instrument was calibrated in the extended dynamic range (2 GHz, High Res Mode) and lower mass range (*m/z* < 1700) in the positive ion mode. Data were collected in centroid format. Reference masses at *m/z* 121.0509 and *m/z* 922.0089 were continually introduced via a second sprayer for accurate mass calibration. The reference ions used were purine (C5H4N4) at *m/z* 121.0509 and HP-921 (hexakis-(*1H*,*1H*,*3H*-tetrafluoropentoxy)phosphazene (C18H18O6N3P3F24)) at *m/z* 922.0089 for positive mode. The source conditions were as follows: sheath gas flow, 11 L min−1; sheath gas temperature, 350 ◦C; nebulizer pressure, 40 psi; drying gas temperature, 150 ◦C; drying gas flow, 15 L min−1; nozzle voltage, 380 V; fragmentor voltage, 360 V; capillary voltage, 3500 V. Nitrogen was used as source gas and as collision gas. Full scan MS were acquired over the mass range *m*/*z* 100–1000 at a scan speed of 2 scans/s. Agilent Mass Hunter workstation software version B.05.00 was used for data acquisition, and B.06.00 qualitative analysis was used for processing. Mass calibration was performed prior to analysis.

#### **4. Conclusions**

We accomplished our goal in this study by successfully developing a UHPLC/Q-TOF-based screening method to detect a variety of GEs and MCPDEs in glycerin samples (both crude and refined grades). It is a dilute-and-shoot method with minimal sample preparation that can be adapted to other matrices. The screening method by FbF against our in-house library produced many false positives for GEs, which were eliminated by comparing the retention times with standards. There were no hits for MCPDEs in the limited glycerin samples we analyzed.

**Author Contributions:** L.G., K.H., and H.J. conceptualized the study and planned the laboratory work; L.G. executed LCMS method development; L.G. and H.J. worked with the Mass Hunter software and FbF algorithm for data processing; L.G., H.J., and K.H. prepared the original draft. All the authors (L.G., K.H., H.E., R.R., O.C., and H.J.) reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Philip J. Kijak for administrative support and Matthew Bell for assistance in the graphical design of figures.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are available from the authors.
