The 4 mL samples were first spiked with deuterated standard stock solution (listed in Table 1) to CA 074 final
concentration of 20 ?g L−1 deuterated standard. Solid phase extraction (SPE) was initiated by conditioning the Bond Elut Plexa cartridges (30 mg, 1 mL, Agilent Technologies, Canada) with 1 mL of 100% methyl tert-butyl ether (MTBE) followed by 1 mL 100% methanol. Samples were introduced into the cartridges by pipetting 1 mL at a
time (no vacuum), washed with ultrapure water, and dried for approximately 15 min. Elution was done with 2 × 2 mL methanol and 2 × 2 mL 90:10 methanol:MTBE. These extracts were evaporated to dryness using a Dionex SE 500 evaporator and reconstituted with 160 ?L of methanol containing lorazepam and chloramphenicol (75 ?g L−1) as instrument injection standards. SPE recoveries were determined by spiking two test tubes of 4 mL ultrapure water with 32 ?L each of the 100 ?g L−1 deuterated and regular standard stock solution. Another 4 mL sample was prepared with only ultrapure water as SPE sample blanks. Method matrix recoveries during the analysis where higher than 90%. The extracts were stored in a −20 °C freezer and were analysed within two weeks. The analysis of compounds was completed using liquid chromatography and tandem mass spectrometry (LC-MS/MS) using an Agilent 1200 HPLC coupled to an Applied Biosystems 3200 QTRAP mass spectrometer (ABSciex, Concord, ON, Canada). LC-MS/MS analysis was previously described in Wang et al. (2011). Additional parameters including the optimized parameter values and chromatographic and ionization parameters are Postmeiotic segregation
found in the supplementary material (Tables S1–S3).