The development of a rapid magnetic-mediated solid-phase extraction and pre-concentration method of steroids in natural waters and biofluids by polymer coated Fe₃O₄ core-shell magnetite microspheres and liquid chromatography-tandem mass spectrometry

Abstract

A new rapid magnetic-mediated solid-phase extraction technique using polymer coated Fe3O4 core-shell magnetite microspheres coupled with liquid chromatography-tandem mass spectrometry detection method has been developed to analyze five endocrine disrupting chemicals and sixteen reproductive hormone metabolites in environmental and biologic matrices. Magnetic Fe3O4 cores of this micro-phase extraction (MPE) sorbent microspheres were fabricated by a solvothermal approach and thin layers of amphipolar poly(divinylbenzene-co-methacrylic acid) (pDVB-MAA) and poly(methyl methacrylate-co-divinylbenzene) (pMMA-DVB) coatings were established via suspension polymerization. The size of these two kinds of polymer coated magnetic particles ranged from 200 nm to 700 nm in diameter. Because of the high surface to volume ratio, partition equilibrium of compounds between the sample matrix and the sorbent can be achieved in two to fifteen minutes. Also, the volume of organic solvent needed to wash out compounds from the particle surface is only about 1 milliliter. The endorcrine disrupting chemicals estrone(E1), estrodiol(E2), estriol (E3), 17α-ethynylestradiol (EE2) and bisphenol-A (BPA) were extracted from nature water and waste water by pDVB-MAA coated Fe3O4 particles and detected with LC-MS/MS. The achieved MPE recoveries of the EDCs were in the ranges of 66-102% (Milli-Q water), 71.2-108.2% (tap water), 69.1-110.9% (seawater) and 55.9-110% (sewage). Method detection limits achieved were in the ranges of 1-20 pg ml-1 for tap water, 1.8-36 pg ml-1 in seawater and 2.2-19 pg ml-1 in sewage, with repeatability in the ranges of 5-18.9%, 3.5-10.5% and 3.6-16.0% respectively. pMMA-DVB coated Fe3O4 particles were used to extract these five compounds from human urine samples. The recovery of the EDCs from the urine samples were in the range of 90.3±4.0 to 102.5±1.6%, except for E3 which was relatively low. The RSD ranged from 1.6 to 4.4%, except for E3 which was 16.8%. The achieved MDL ranged from 0.007 ng ml-1 to 0.072 ng ml-1. Free E1 was detected in three real human urine samples. Conjugated E1, E2, E3 were found in two to four real human urine samples. Poly(divinylbenzene-co-methacrylic acid) (pDVB-MAA) extraction coupled with LC-MS/MS method was also developed to detect the 16 reproductive hormone metabolites of the human steroidogenesis pathways in the H295R cell line. LOQ of all hormones were in the range of 0.07-1.66 ng ml-1, except for androstanediol whose LOQ was 28.13 ng ml-1. Analysis of cell culture medium spiked with hormone standards showed that the accuracy in the determination of all the hormone metabolites after derivative was greater than 71.7% at high spike levels. RSD (n=4) values were below 20%. At the low spike levels, recoveries ranged from about 50% to 120%. The relative standard deviation of the 16 hormone metabolites was less than 18.7%. These data showed that our method is applicable to the study of the metabolite profile. This method was then applied to investigate effects of bisphenol-A (BPA) on hormone production by H295R cells. The PCA score plot showed that there was a clear separation in the profiles of the hormone metabolites between 2 ppm (C1) and 0.8 ppm (C2) BPA exposure compared with control cells. Cell samples exposed to 0.4 ppm BPA were indistinguishable from control cells, which showed that modification of human steroidogenesis metabolomic profile is very minimal if the BPA exposure concentration is less than 0.4 ppm. 17α-Hydroxypregnenolone, pregnenolone, cortisol, androstenedione, dehydroandrosterone, testosterone and androstanolone production were found to change under BPA exposure. Level of 17α-hydroxylprogesterone, on the other hand, did not change much with different dosage of BPA. We concluded that the former eight steroids can be used as biomarkers to identify BPA exposures.

Date of Award	15 Feb 2011
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hon Wah Michael LAM (Supervisor)