A quantitative HPLC–MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos

Abstract

An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.

Influence of the perivitelline space on the quantification of internal concentrations of chemicals in eggs of zebrafish embryos (Danio rerio)

“…Peak integration was checked visually and an external calibration was used (1/x weighted) for quantification. Further details are described by Brox et al (2014). …”

“…The analyte concentration stayed constant during the whole experiment and embryos did not show lethal effects at any sampling time. Experiments were performed according to Brox et al (2014). Briefly, embryos were collected using a light microscope and the experiment was started approximately 4 h post-fertilization (hpf).…”

“…Collection of the ZFE, washing procedure and extraction was performed as described elsewhere (Brox et al, 2014). Briefly, at their time of sampling all ZFE (9 ZFE per replicate) were transferred onto a metal mesh and were washed using 50 mL of stirred bidestilled water.…”

“…Additionally commonly used solvents may change the permeability of the chorion (Kais et al, 2013). Until now only a limited number of studies investigate the uptake of chemicals in early life-stages of the ZFE in combination with (Brox et al, 2014;Stanley et al, 2009) or without (Kühnert et al, 2013;Petersen and Kristensen, 1998) dechorionation. The dechorionation of early life-stages of the ZFE is proposed for exposure experiments starting at 24 hpf (Henn and Braunbeck, 2011).…”

“…The observed effects are often compared to external concentrations but for a better understanding of toxic effects the internal concentrations should be known (Escher and Hermens, 2004). We have recently published an analytical method to determine internal concentrations in ZFE (Brox et al, 2014).…”