GC–FID analytical methods.

All GC analysis was performed on an Agilent 6850 (Agilent Technologies) equipped with an FID. An Agilent DB-WAXetr capillary column (30 m × 0.56 mm × 1 μm) was used for separation. The inlet and detector were held at 250 and 260 °C, respectively. The GC was operated in constant pressure mode with a pressure of 3.66 psi. Helium was used as the carrier gas. Air and hydrogen were supplied to the FID at 350 and 40 ml min⁻¹, respectively. All gasses were purchased from Airgas and 5 μl of sample was injected with a split ratio of 2:1.

For analysis of citral and its reduction product citronellal, the oven was initially held at 150 °C for 10 min and then ramped at a rate of 45 °C min⁻¹ to 240 °C. Citral and citronellal eluted at 9.32 and 4.50 min, respectively. Octanol was used as an internal standard.

For analysis of trans-2-hexen-1-al and its reduction product trans-2-hexan-1-al, the oven was initially held at 50 °C for 1 min, the oven was ramped at 15 °C min⁻¹ to 120 °C, then ramped at 20 °C min⁻¹ to 230 °C and held for 3 min. Trans-2-hexen-1-al and trans-2-hexan-1-al eluted at 6.41 and 4.78 min, respectively. Octanol was used as an internal standard.

For analysis of 4-phenyl-3-butyn-2-one (containing C≡C triple bond) and its fully reduced product 4-phenyl-2-butanone, as well as the intermediate 4-phenyl-2-butene-2-one (containing a C=C double bond), the oven was initially held at 200 °C for 1 min, then ramped at 5 °C min⁻¹ to 230 °C and held for 1 min. Octanol was used as an internal standard. In vitro ketoisophorone reduction to levodione was analyzed using the same method. Elution times are as follows: 4-phenyl-3-butyne-2-one (5.53 min), 4-phenyl-2-butene-2-one (6.76 min), 4-phenyl-2-butanone (4.55 min), ketoisophorone (3.65 min), levodione (4.08 min) and octanol (2.80 min).

For analysis of in vivo ketoisophorone biostransformation, the oven was held at 200 °C for 15 min. Elution times are as follows: octanol (2.80 min), ketoisophorone (3.76 min), levodione (7.25 min), phorenol (8.35 min) and 4-hydroxyisophorone (12.90 min).