Measuring Fifteen Endogenous Estrogens Simultaneously in Human Urine by High-Performance Liquid Chromatography-Mass Spectrometry

Abstract

A sensitive, specific, accurate, and precise high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for measuring the absolute quantities of 15 endogenous estrogens and their metabolites in human urine has been developed and validated. The method requires a single hydrolysis/extraction/derivatization step and only 0.5 mL of urine, yet is capable of simultaneously quantifying estrone and its 2-, 4-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, and 2-hydroxyestrone-3-methyl ether; estradiol and its 2-, 4-methoxy and 2-, 16alpha-hydroxy derivatives, 16-epiestriol, 17-epiestriol, and 16-ketoestradiol in pre- and postmenopausal women as well as men. Standard curves are linear over a 10(3)-fold concentration range with the standard error of the estimate (SEE) and the relative standard error of the estimate (RSEE) for the linear regression line ranging from 0.0131 to 0.1760 and 1.2 to 7.3%, respectively. The lower limit of quantitation for each estrogen is 0.02 ng/0.5 mL urine sample (2 pg on column), with the percent recovery of a known added amount of compound (accuracy) of 96-107% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-5% relative standard deviation (RSD) for samples prepared concurrently and 1-12% RSD for samples prepared in separate batches. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate, precise, and specific measurement of endogenous estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry

“…For the analysis of steroidal estrogens by LC-ESI-MS/MS, pre-column derivatization by reaction with dansyl chloride at the C-3 hydroxyl groups of these compounds is by far the most popular technique, and is now widely used in both biomedical [14,[23][24][25][26][27][28][29] and environmental [30][31][32] applications. There are, however, aspects of the analysis of estrogens as dansyl derivatives that are not desirable.…”

“…There are, however, aspects of the analysis of estrogens as dansyl derivatives that are not desirable. With dansyl derivatization and LC-ESI-MS/MS of steroidal estrogens, the vast majority of the ion current after collision-induced dissociation (CID) of the [M+H] + ion is carried by a single ion derived from the protonated 5-(dimethylamino)-naphthyl moiety [14,[23][24][25][26], and very little of it by estrogen-derived ions, thereby making MS/MS and third stage MS (MS 3 ) analyses structurally uninformative. The identification of unknown estrogens and their metabolites is therefore not significantly aided by the formation and analysis of dansyl derivatives.…”

An alternative method for the determination of estrogens in surface water and wastewater treatment plant effluent using pre-column trimethylsilyl derivatization and gas chromatography/mass spectrometry

“…So it is very important to determine the concentration of estrogens in environmental water. Considering low volatility of steroid estrogens, LC/MS (Ferguson et al 2001;López and Barceló 2001;Hu et al 2005;Cui et al 2006) and LC/MS/MS (Zhang and Henion 1999;Baronti et al 2000;Johnson et al 2000;Xu et al 2004Xu et al , 2005Xu et al , 2006Maurício et al 2006;Matejicek et al 2007) methods have recently been developed for the determination of these estrogenic compounds. However, at present, the application of LC/MS and LC/MS/MS in environmental analyses of estrogenic steroids appears to be limited for their complex operation and capital costs.…”

Multiplexed quantitation of endogenous estrogens and estrogen metabolites in human peritoneal fluid

“…To the dried sample 40 μL of 0.1 M sodium bicarbonate buffer (pH 9.0) and 40 μL of dansyl chloride solution (1 mg/mL in acetone) were added. After vortexing, the sample was heated at 60°C (Reacti-Therm III™ Heating Module, Pierce) for 5 min to form the EM and d-EM dansyl derivatives (EMDansyl and d-EM-Dansyl, respectively) [11,12,[14][15][16]. The purpose of dansylation is to improve MSionization efficiency of EMand improve the overall sensitivity of the method.…”

“…Although CE provides high resolution and rapid separation which is ideal for analyzing isomers of endogenous estrogens and EM, the two previously reported MEKC-UV methods lack the required sensitivity for the quantitation of endogenous estrogens and their metabolites in biological samples [9,10]. During the past several years our laboratory has been actively developing MS/MS-based methods for quantitatively measuring biologically active endogenous estrogens and EM in human urine and serum [11,12]. The current manuscript details the use of a capillary LC-MS/MS method to accurately and precisely measure the absolute quantities of endogenous estrogens and their metabolites in as little as 50 μL of human peritoneal fluid.…”