Ethinyl estradiol

A racemic mix of BF isomers (98% purity) was purchased from Chem Service, Inc. Ethinylestradiol (EE2 ≥ 98% purity) was purchased from Sigma Aldrich. Ethanol (Decon Laboratories Inc.) was 200 proof. A stock solution of BF in 100% ethanol (EtOH) was prepared, and diluted solutions in embryo media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄, in pH 7) were made at every exposure day. Methanol (MeOH) and acetonitrile (MeCN), both OPTIMA™ grade, were from Fisher Scientific. Deionized water of 18.2 Ω was prepared with an Milli-Q water purification system (Millipore). Formic acid (FA - OPTIMA™ grade) was obtained from Fisher Scientific. Dopamine hydro-chloride (DA) (1 mg/mL in MeOH), and neat solid standards of 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were from Sigma Aldrich. Standard of isotopically labeled compound used as internal standard (IS), dopamine-1,1,2,2-d4 hydrochloride (DA-d4), was from Sigma Aldrich. All standards were >98% purity. Stock solutions of DOPAC, HVA, and DA-d4 were prepared at 1 mg/mL in methanol, and stored at −80 °C. The concentrations were not corrected for purity or salt content. Solutions of working standards and internal standard (IS) were prepared in MeOH. Calibration standards were prepared in 0.1% formic acid in DI water. All the solutions were kept in −80 °C.