Comparison of Liquid Chromatography-Tandem Mass Spectrometry, RIA, and ELISA Methods for Measurement of Urinary Estrogens

Abstract

Absolute and relative concentrations of estrogens and estrogen metabolites are important for clinical decisions as well as for epidemiologic, experimental, and clinical research on hormonal carcinogenesis. RIA and ELISA are routinely used for measuring estrogen metabolites in blood and urine due to efficiency and low cost. Here, we compare absolute and ranked concentrations of estrone, estradiol, and estriol measured by indirect RIA and of 2-hydroxyestrone and 16α-hydroxyestrone measured by ELISA to the concentrations obtained using a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which measures 15 estrogen metabolites concurrently. We used overnight urine samples collected from control women (362 premenopausal and 168 postmenopausal) participating in a population-based case-control study of breast cancer among Asian American women ages 20 to 55 years. When comparing RIA or ELISA levels to LC-MS/MS, absolute concentrations for the five estrogen metabolites ranged from 1.6 to 2.9 and 1.4 to 11.8 times higher in premenopausal and postmenopausal women, respectively (all P < 0.0001). However, LC-MS/MS measurements were highly correlated [Spearman r (r s ) = 0.8-0.9] with RIA and ELISA measurements in premenopausal women and moderately correlated (r s = 0.4-0.8) in postmenopausal women. Measurements of the 2-hydroxyestrone:16α-hydroxyestrone ratio, a putative biomarker of breast cancer risk, were moderately correlated in premenopausal women (r s = 0.6-0.7) but only weakly correlated in postmenopausal women (r s = 0.2). LC-MS/MS had higher intraclass correlation coefficients (≥99.6%) and lower coefficients of variation (≤9.4%) than ELISA (≥97.2% and ≤14.2%) and RIA (≥95.2% and ≤17.8%). Comparison with the LC-MS/MS method suggests that the widely used RIA and ELISA estrogen metabolite measures may be problematic, especially at low estrogen metabolite levels characteristic of postmenopausal women. Cancer Epidemiol Biomarkers Prev; 19(1); 292-300. ©2010 AACR.

Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat

“…In non-reproductive organs, such as the brain, the synthesis of steroid hormones has been shown through the detection of cytochrome P450scc and the mRNAs encoding the enzymes that synthesize steroid hormones. Because of the broad specificity of antibodies against small molecules, the accurate determination of steroid levels in organs is difficult using RIA and ELISA (25,26). We have developed a method for the determination of steroids using an efficient preparation of organs with two columns, with the determination being performed using LC-TOF MS and LC-MS/MS analysis; highly accurate results were obtained (Table II).…”

A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis

“…These have been developed to measure the endogenous estrogen metabolites in biological specimens using RIA or enzyme immunoassay (EIA) ( 13,14 ), and GC-MS ( 4,(15)(16)(17)(18) or LC-MS (19)(20)(21). Although immunoassays have limited applicability due to an overestimation by cross reactions (22)(23)(24)(25), LC-MS-based profi ling is a proven technique with high sensitivity and selectivity in estrogen analysis combined with chemical derivatization for the phenolic hydroxyl groups of estrogens as follows: a ) pentafl uorobenzyl (PFB) derivatization with ECAPCI-MS ( 21 ), b ) dansyl ( 20 ) and picolinoyl ( 19 ) derivatizations with LC-ESI/MS, and c ) N-methyl-2-pyridyl ( 26 ), 1-(2,4-dinitro-5-fl uorphenyl)-4,4,-dimethylpiperazine ( 27 ), or N-methyl-nicotinyl derivatizations with LC-ESI/MS ( 28 ). These methods are capable of quantifying estrogens in the low pg/ml ranges, but they are time consuming because they require derivatization ( 19 ) and a long analytical run ( 29 ).…”

“…Recently, a LC-MS/MS method provided more information on 15 estrogen metabolites present in urine samples obtained from healthy premenopausal and postmenopausal women. Five (E1, 17 ␤ -E2, E3, 2-OH-E1, and 16-keto-E2) of these 15 estrogens were determined to be abundant estrogens in both groups ( 20,25 ). Few studies have evaluated the excretion of urinary estrogen metabolites in postmenopausal osteoporosis, which is known to be due primarily to an estrogen defi ciency ( 6 ).…”

Improved Confidence Intervals for the Ratio of Coefficients of Variation of Two Lognormal Distributions

“…The radioimmuno assay (RIA) method has been routinely used for its efficiency and low cost in measuring estrogen metabolites in blood and urine. Faupel-Badger et al [2] have compared concentrations of estrogen metabolites by RIA with the concentrations obtained using a novel and high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the purpose of illustration, Nam and Kwon have used the quality control data of 16 measurements by RIA method from each of 3 postmenopausal women, and 28 measurements from a postmenopausal woman using LC-MS/MS, and 25 measurements obtained from another postmenopausal woman using LC-MS/MS.…”