Sil 20a ht auto

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.

The LC-MS-system (Shimadzu, Kyoto, Japan) consisted of a CBM-20A controller unit, pumps (LC-20AD), a SIL-20A_HT auto sampler, a CTO-10AS column oven, a valve unit FCV-20AH₂ and an MS-8030. The mobile phase was 0.2% HCOOH. MeOH was added in the following gradient: 0–3.0 min 1% MeOH, 3.1–6.0 min 15% MeOH, 6.1–10.0 min 1% MeOH. The flow rate was set to 1 mL/min. A C18 column (Kinetex 4.6 × 100 mm 2.6 µm 100 Å) was kept at 50 °C. Samples (10 µL) were auto-injected.
For MS detection, electrospray ionization (ESI) was applied as the ion source, while the combination of a triple-quadrupole and an electron multiplier were used as analyzer and detector. Detection times were set from 3.0 min to 10.0 min.
PTCA and TTCA were measured using the positive selected ion monitoring mode in the third quadrupole (Q3-SIM), while PDCA and TDCA were measured using the negative Q3-SIM mode. PDCA and PTCA were identified both by reference substances and mass-to-charge ratio (m/z) (PDCA 154.0; PTCA 200.0). TDCA and TTCA were identified by mass-to-charge ratio (TDCA 172.0; TTCA 218.0) only. The latter were not commercially available. The area under the curve (AUC) obtained by MS analysis was normalized to the total count of all events in the range of 0.5 µm to 50 µm (see Section 4.6). The AUC per count (AUC/count) was used to compare relative amounts of specific degradation products.