4.5. GC-MS-Based Assays

The enzyme assays were performed essentially as previously described [13] using 20 mM MOPS (pH = 7.0) as a buffer. The protein concentration was set to 0.05 mg/mL. The substrate concentration was 0.03 mM. The NADPH concentration was 0.4 mM, and the final assay volume was 1 mL. The reaction was terminated after 1 h by adding 1 mL of dichloromethane at 25 °C. The reaction products were extracted, and the organic phase was evaporated at 25 °C. For the GC-MS analysis (Agilent, Palo Alto, CA, USA), the samples were dissolved in 100 μL of dichloromethane. 8-Oxogeranial was purchased from Toronto Research Chemicals (TRC) (Toronto Research Chemicals, Toronto, ON, Canada).

GC-MS analysis was performed in an Agilent 7890 A System coupled to an Agilent 5975 C MS detector (Agilent, Palo Alto, CA, USA). A J & W GC column (30 m × 0.25 mm × 0.25 μm) and helium gas (1.2 mL/min) were used. The program started at 60 °C, and the temperature was increased 5 °C/min to 150 °C, 20 °C/min to 240 °C, 20 °C/min to 290 °C and then maintained for 5 min at 290 °C.