Determination of conjugated estrogens in human urine using carrier-mediated hollow-fiber liquid phase microextraction and LC-MS/MS

Abstract

The extraction conditions of the conjugated estrogens, including estrone-3-glucuronide (E1-3G), 17β-estradiol-3-glucuronide (E2-3G), and 17α-ethynylestradiol-3-glucuronide (EE2-3G), were optimized using carrier-mediated hollow-fiber liquid phase microextraction (HF-LPME) and then applied in the analysis of human urine samples. The optimal extraction conditions were as follows: organic phase composed of 20% (w/w) tri-octylamine (TOA) in 1-octanol; 2M NaCl as the acceptor phase; 800rpm of agitation; and 50min of extraction time. The extraction efficiency of carrier-mediated LPME was 11 times higher than that of the three-phase LPME. Estrogens and their conjugates in urine samples from pregnant and non-pregnant women were extracted and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed that carrier-mediated HF-LPME had good recoveries and linearity in the calibration curve. Estrogens in pregnant and non-pregnant women were mainly excreted in a conjugated form (94.85±4%), whereas free estrogen was detected at a ratio of 5.15%. Both free and conjugated form of E1 were the most dominant species in all forms, which accounted for 72.7% of all the measured estrogens. Consequently, the HF-LPME in carrier-mediated mode was effectively applied to extract the estrogen conjugates in only 10ml of human urine samples, which is the most significant advantage. © 2015 Balaban Desalination Publications. All rights reserved.