Urinary Analysis of 16 Estrogens and Estrogen Mmetabolites (EMs) by LC-MS/MS (P13-013-19)

To develop and validate a better extraction method for the measurement of 16 estrogens and estrogen metabolites (EMs) in human urine using a previously validated LC-MS/MS method (Anal. Chem.2005, 77, 20, 6646–6654). The current liquid-liquid extraction method has a lot of matrix interference that makes accurate quantification more difficult. 250 µL of urine is combined with 500 µL of acetate buffer (pH 4.6), 50 µL of 50 mg/mL ascorbic acid, and glucuronidase/sulfatase (β-Glucuronidase from Helix pomatia, Sigma). Incubate at 37 °C for 4 h. Practically all of the EMs in urine is in conjugated form. 12 stable isotopically labeled EMs are added as internal standards. Samples are mixed and equilibrated for 1 hour at room temperature. Sample is then diluted with 1 mL of 1 M ammonium hydroxide solution, vortexed, then loaded onto a 2 mL 12cc StatraDE (Phenomenex©) tube. Then sample is eluted with 2 X 5 mL dichloromethane. Eluant is then dried and samples are dansylated prior to LC-MS/MS analysis. EMs were separated using a Synergi 2.5 µm Hydro-RP 100Ȧ 100 x2 mm column (Phenomenex) and a 50 minute gradient of water/methanol/acetone, all containing 0.1% formic acid. Mass spectrometry conditions for each analyte were optimized. A proof-of-performance study was conducted to assess the reproducibility of the method, including both within- and across-batch variability, for each of the 16 EMs. Relative to the previously published liquid/liquid extraction method, using Strata DE columns for urine samples gives cleaner extracts with better recovery. Substantially less interference by adjacent peaks is noted for many of the EMs. Reproducibility is acceptable. The new extraction method is also less time consuming than the liquid/liquid extractions previously used for EMs. This new solid phase extraction method for measuring EMs in urine is efficient and does not require additional sample volume. Results are reproducible for measuring 16 EMs in urine. National Cancer Institute.