Estriol

Stable isotope dilution HPLC-MS/MS was used to quantify 15 estrogens and estrogen metabolites (Figure 1), as previously detailed (22 (link)). Six labeled internal standards were used: deuterated 2-hydroxyestradiol, 2-methoxyestradiol and estriol (C/D/N Isotopes Inc, Pointe-Claire, QC, Canada); deuterated 16-epiestriol (Medical Isotopes Inc, Pelham, NH, USA); and ¹³C-labeled estrone and estradiol (Cambridge Isotope Laboratories, Andover, MA, USA).
The serum method detects 15 estrogens and estrogen metabolites which circulate primarily as sulfated and/or glucuronidated conjugates. Five estrogen metabolites (estrone, estradiol, estriol, 2-methoxyestrone and 2-methoxyestradiol) were measured in unconjugated forms. The serum sample was split into two aliquots to measure the combined concentration of each of the 15 metabolites (sum of conjugated plus unconjugated forms) and the unconjugated forms. To measure the combined parent estrogen or estrogen metabolite level, an enzyme with sulfatase and glucuronidase activity was added to the samples to cleave any sulfate and glucoronide groups (22 (link)). To measure the unconjugated forms, the enzyme was not included in sample preparation. For metabolites with both combined and unconjugated measurements, the concentration of the conjugated form was calculated as the difference between the combined and unconjugated estrogen measurements. The limit of detection for each estrogen and estrogen metabolite measured was 10 fg on column (approximately 0.33–0.37 pmol/L) (22 (link), 23 (link)). No samples had undetectable hormone levels. Laboratory coefficients of variation (CV) of masked technical replicates across batches were <6.0% for all hormones measured. Intraclass correlation coefficients (ICCs) ranged from 0.93–0.996 (median value 0.98).

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.

Stable isotope dilution LC-MS/MS was used to quantify 15 estrogens and estrogen metabolites including: estrone, estradiol, 2-pathway metabolites (2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestrone-3-methyl ether); 4-pathway metabolites (4-hydroxyestrone, 4-methoxyestrone, and 4-methoxyestradiol); and 16α -pathway metabolites (16α-hydroxyestrone, estriol, 16-ketoestradiol, 16-epiestriol, and 17-epiestriol,). Details of the method have been published previously (22 (link)). For this study, six labeled internal standards were used: deuterated 2-hydroxyestradiol, 2-methoxyestradiol and estriol (C/D/N Isotopes Inc, Pointe-Claire, QC, Canada); deuterated 16-epiestriol (Medical Isotopes Inc, Pelham, NH, USA); and ¹³C-labeled estrone and estradiol (Cambridge Isotope Laboratories, Andover, MA, USA).
In serum, this method detects 15 estrogens and estrogen metabolites which circulate, at least in part, as sulfated and/or glucuronidated conjugates to facilitate storage, transport, and excretion. Five of the estrogens (estrone, estradiol, estriol, 2-methoxyestrone and 2-methoxyestradiol) were also measured in unconjugated forms in circulation. The serum sample was split into two aliquots, one to measure the combined concentration of each of the 15 metabolites (that is, the sum of conjugated plus unconjugated forms); the other, to measure the unconjugated forms. To measure the combined level of the conjugated plus unconjugated parent estrogen or estrogen metabolite level, an enzyme with sulfatase and glucuronidase activity was added to the samples to cleave any sulfate and glucoronide groups (22 (link)). To measure the unconjugated forms only, addition of the enzyme is not included in the sample preparation steps. For those metabolites with both combined and unconjugated measurements, the concentration of the conjugated form was calculated as the difference between the combined estrogen measurement and the unconjugated estrogen measurement, for estradiol that calculation was (conjugated E2 = combined E2 – unconjugated E2). The limit of detection for each estrogen and estrogen metabolite measured using this LC-MS/MS assay was 10 fg on column (approximately 0.33–0.37 pmol/L) (22 (link), 23 (link)). There were no samples in the current study with undetectable levels for any of the hormones measured. Laboratory coefficients of variation (CV) of masked technical replicates across batches were <6.0% for all hormones measured. Intraclass correlation coefficients (ICCs) ranged from 0.93–0.996 with median value of 0.98.