Quantitative and Qualitative Determination of Estrogen Sulfates in Human Urine by Liquid Chromatography/Tandem Mass Spectrometry Using 96-Well Technology

Abstract

A sensitive and robust method to determine five estrogen sulfates in human urine has been developed employing high-throughput solid-phase extraction with 96-well technology, and HPLC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. The five estrogen sulfates determined include three major endogenous estrogen sulfates in the human, estrone 3-sulfate (E1-3S), estriol 3-sulfate (E3-3S), and 17 beta-estradiol 3-sulfate (E2-3S), and two biochemical synthetic estrogen sulfates, 17 beta-estradiol 17-sulfate (E2-17S) and 17 beta-estradiol 3,17-disulfate (E2-3,17S). For E2-3,17S, E3-3S, and E2-17S, external standard calibration was used for quantitation, and for the remaining two compounds, internal standard calibration using a stable isotopic labeled internal standard was employed. A total of 96 samples may be prepared with 96-well C18 extraction disk plate techniques performed by a robot within 25 min including the time for evaporation of solvent. The lower level of quantitation (LOQ) for these estrogen sulfates in human urine was determined at 0.2 ng/mL based on 100-microL aliquots of human urine using the optimum tuning parameters for each individual selected precursor ion/product ion transition. The assay was validated with a linear concentration range of 0.2-200 ng/mL, and the interassay accuracy, intraassay precision, and interassay precision do not exceed 8.6%, 12%, and 12%, respectively, by analysis of quality control samples at five concentration levels including the LOQ of 0.2 ng/mL, from four 96-well plates. The target endogenous test articles were qualitatively determined by comparing the full-scan LC/MS/MS mass spectra and retention time in test samples and reference standards. The LOQ is significantly improved compared to previous reports for the targeted compounds using LC/MS/MS. The described simple and automated sample preparation procedure recovered 91% of the target compounds. A total of 192 samples can be analyzed within 1 day (22 h). The method can measure the endogenous estrogen sulfates in urine from both gravid and nongravid subjects.

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.…”

Simultaneous determination of natural and synthetic steroid estrogens and their conjugates in aqueous matrices by liquid chromatography/mass spectrometry

“…The conjugate moiety favoured by E3 and E2 is estriol-16α-glucuronide (E3-16αG) and 17β-estradiol-3-glucuronide (E2-3G) respectively [10,11] . Estrone excretion has traditionally been as estrone-3-glucuronide (E1-3G) [11] , though subsequent studies have since identified estrone-3-sulphate (E1-3S) as the main urinary excretion product [10,12,13] . The synthetic steroid estrogen, 17α-ethinylestradiol (EE2) is used in contraceptive therapy and is predominately excreted in urine with 50-90 % favouring glucuronide conjugation on the C3, the remainder mainly as 17α-ethinylestradiol-3-sulphate (EE2-3S) [14] .…”

Simultaneous detection of monohydroxybenzo[a]pyrene positional isomers by reversed‐phase liquid chromatography coupled to electrospray ionization mass spectrometry

“…The reproducibility of this method is comparable with the LC-MS-MS method of Zearenol (2.1-3.3%) using zealenone as an internal standard (Zollner et al, 2000) and estrogen sulfate conjugates (12%) using an external standard (Zhang and Henion, 1999).…”