A liquid chromatography-mass spectrometry method for the simultaneous measurement of 15 urinary estrogens and estrogen metabolites: assay reproducibility and interindividual variability.
BACKGROUND
Accurate, reproducible, and sensitive measurements of endogenous estrogen exposure and individual patterns of estrogen metabolism are needed for etiologic studies of breast cancer. We have developed a high-performance liquid chromatography-tandem mass spectrometry method to quantitate simultaneously 15 urinary estrogens and estrogen metabolites (EM): estrone; estradiol; 3 catechol estrogens; 5 estrogens in the 16alpha pathway, including estriol; and 5 methoxy estrogens.


METHODS
Overnight urines were obtained from 45 participants. For the reproducibility study, two blinded, randomized aliquots from 5 follicular and 5 luteal premenopausal women, 5 naturally postmenopausal women, and 5 men were assayed in each of four batches. Assay coefficients of variation and intraclass correlation coefficients were calculated with ANOVA models. Data from the additional 25 participants were added to compare EM levels by menstrual/sex group and assess interindividual variability.


RESULTS
For each EM, overall coefficients of variation were < or = 10%. Intraclass correlation coefficients for each menstrual/sex group were generally > or = 98%. Although geometric mean EM concentrations differed among the four groups, rankings were similar, with estriol, 2-hydroxyestrone, estrone, estradiol, and 16-ketoestradiol accounting for 60% to 75% of total urinary EM. Within each group, interindividual differences in absolute concentrations were consistently high; the range was 10- to 100-fold for nearly all EM.


CONCLUSIONS
Our high-performance liquid chromatography-tandem mass spectrometry method for measuring 15 urinary EM is highly reproducible, and the range of EM concentrations in each menstrual/sex group is quite large relative to assay variability. Whether these patterns persist in blood and target tissues awaits further development and application of this method.
Falk
Roni T
RT
National Cancer Institute, 6120 Executive Boulevard, EPS-5137, Rockville, MD 20892-7234, USA. falkr@mail.nih.gov
Xu
Xia
X
Keefer
Larry
L
Veenstra
Timothy D
TD
Ziegler
Regina G
RG
D000328:Adult; D000368:Aged; D000704:Analysis of Variance; D002853:Chromatography, Liquid / Q000379:methods*; D004967:Estrogens / Q000652:urine*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004967:Estrogens