Stability of 15 estrogens and estrogen metabolites in urine samples under processing and storage conditions typically used in epidemiologic studies

Background: In preparation for large-scale epidemiologic studies of the role of estrogen metabolism in the etiology of breast and other cancers, we examined the stability of estrogens and estrogen metabolites (EM) in urine during processing and storage protocols. Methods: Fifteen EM were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in first morning urines from 3 premenopausal women. Linear regression was used to model log EM concentrations for each woman, with and without adding ascorbic acid (0.1% w/v), during storage at 4°C (7-8 time points, up to 48 hours), during long-term storage at -80°C (10 time points, up to 1 year), and by freeze-thaw cycles (up to 3). Results: Without ascorbic acid, concentrations (pmol/mL) of nearly all EM changed <1% per 24 hours of storage at 4°C, and <1% during storage at -80°C for 1 year; similarly, thawing and refreezing samples 3 times was not consistently associated with losses for any EM. Ascorbic acid had no clear beneficial effect on EM stability in these experiments. Conclusions: Given the large inter-individual variability in urinary EM concentrations, changes of the magnitude observed here are unlikely to cause substantial misclassification. Furthermore, processing and storage conditions studied here are adequate for use in epidemiologic studies.