220 ph meter

A Shimadzu HPLC system [Shimadzu CMB-20A controller, LC-20AB pump, DGU-20A₃ degasser, SPD-M20A diode array detector, RF-20A XS fluorimeter detector, CTO-20A column oven, and SIL-20A HT auto sampler] was used for monitoring the oxidative degradation of EE2. Chromatographic separation of EE2 and its degradation intermediates was achieved using an Agilent Microsorb-MV 100-5 C18 (250 mm x 4.6 mm, 5 μm) column. HPLC analysis conditions were: 25-uL injection volume, 40 °C column temperature, and isocratic elution using 40% acetonitrile and 60% water at 1-mL min^-1 flow rate. The HPLC diode array detector was set to a 200–450 nm range and the fluorimeter detector was set to λ_ex = 220 and λ_em = 305 nm. The retention times of EE2 and its estrogenic degradation intermediates under these conditions were, respectively, 5.2, 3.0, and 3.4 minutes. ESI-MS analyses were performed using a Finnigan LCQ MS ion trap with ESI detection. A Bruker 500 MHz NMR instrument was used for ¹H and ¹³C NMR studies (1D and 2D) at 300 K. The pH measurements were acquired with a Corning 220 pH meter calibrated with standard buffer solutions at pHs 4, 7, and 10.