Quantification of estrogens by LC–MS/MS
SEPARATION
Serum E1 and E2 concentrations were analyzed by a LC–MS/MS: Agilent 1200 high-performance liquid chromatography (Agilent Technologies Inc., Santa Clara, CA, USA) coupled with an AB Sciex Triple Quad 5500 mass spectrometer controlled by Analyst Software 1.6.2 (AB Sciex, Concord, ON, Canada). Slight modifications of our previous method were included [Assay calibrators and blank solutions of 0.0–1000 pmol/L E1 (Vetranal, Sigma-Aldrich, St. Louis, MO, USA) and 0.0–1275 pmol/L E2 (Sigma-Aldrich) were prepared in water:methanol (1:1, v/v). To 300 µL of calibrator or 300 µL of serum 30 µL of internal standard (IS) containing 3 nmol/L One hundred µl of the calibrator or the sample extract was measured in one LC–MS/MS run. Chromatographic separation was performed on a tandem column where a Discovery HS F5-3 column (2.1 × 100 mm, 3 µm; Supelco, Bellefonte, PA, USA) was coupled with a SunFire C18 column (2.1 × 50 mm, 3.5 µm; Waters, Milford, MA, USA). The mobile phase was a linear gradient consisting of 40 µmol/L ammonium fluoride in water (A) and methanol (B) at a flow rate of 300 µL/min. The gradient was 0 min 50% B, 4.5–10 min 100% B, and 10.5–19 min 50% B.E1, E2, and corresponding Using Analyst Software 1.6.2 data processing tools for assay calibrators and sample quantifications we estimated the lower limits of quantification (LLOQ) to 5 pmol/L for E1 and E2 with signal to noise ratios 