All three of the urine samples from a single woman were assayed together; the samples were ordered randomly and labeled such that the laboratory could not identify samples from the same woman. For each collection for each woman, 500μL of frozen urine was sent to the Laboratory of Proteomics and Analytical Chemistry, SAIC-Frederick, Inc., Frederick, MD. Given that endogenous estrogens and their metabolites are usually present in urine as glucuronide and sulfate conjugates, an initial hydrolysis step was included. Each urine sample was thawed and mixed, and 400 μL was immediately aliquoted into a clean screw-cap glass tube and 20 μL of an internal standard solution containing 1.6 ng of each of five deuterated EM (17β-estradiol-d4, estriol-d3, 2-hydroxy-17β-estradiol-d5, 2-methoxy-17β-estradiol-d5, 16-epiestriol-d3) was added, followed by 0.5 mL of 0.15 M acetate buffer, pH 4.1, containing 2 mg of ascorbic acid and β-glucuronidase/sulfatase from Helix pomatia (Type HP-2) (Sigma-Aldrich, St. Louis, MO). The deuterated EM are used to correct for loss of urinary EM during the hydrolysis, extraction, derivatization, and LC-MS2 steps of the assay procedure. Details of the assay have been published previously (35 (link), 38 (link)). In brief, quantitative data were acquired using a TSQ Quantum-AM triple quadrupole mass spectrometer coupled with a Surveyor HPLC system (Thermo, San Jose, CA). Both the HPLC and the mass spectrometer were controlled by Xcalibur software (Thermo). Quantitation of each EM in urine was carried out using Xcalibur Quan Browser (Thermo). Calibration curves for the 15 EM were constructed by plotting EM/deuterium labeled EM peak area ratios versus amounts of the EM. The amount of EM in the urine sample was then interpolated using a linear function. The overall coefficients of variation (CVs) from masked replicate quality control samples placed in each batch ranged from 1.0% (2-hydroxyestrone) to 6.5% (4-methoxyestrone).
Creatinine was measured in two batches: the first with 228 samples at the Endocrine Core Laboratory at Emory University (Atlanta, GA) using Sigma Diagnostics creatinine agents, the second with 95 samples at Dr. Nader Rifai’s laboratory at the Boston Children’s Hospital (Boston, MA). CVs were ≤4.5% in both labs.
Plasma follicular and luteal samples from each of the three collections were assayed at the same time for each woman. Estrogens and progesterone were measured at Quest Diagnostics-Nichols Institute (San Juan Capistrano, CA); details of the assay methods have been described in detail previously (39 (link)). CVs were ≤14% for plasma hormones.
Creatinine was measured in two batches: the first with 228 samples at the Endocrine Core Laboratory at Emory University (Atlanta, GA) using Sigma Diagnostics creatinine agents, the second with 95 samples at Dr. Nader Rifai’s laboratory at the Boston Children’s Hospital (Boston, MA). CVs were ≤4.5% in both labs.
Plasma follicular and luteal samples from each of the three collections were assayed at the same time for each woman. Estrogens and progesterone were measured at Quest Diagnostics-Nichols Institute (San Juan Capistrano, CA); details of the assay methods have been described in detail previously (39 (link)). CVs were ≤14% for plasma hormones.
