Steroid determination in fish plasma using capillary electrophoresis

Abstract

A capillary separation method that incorporates pH-mediated stacking is employed for the simultaneous determination of circulating steroid hormones in plasma from Perca flavescens (yellow perch) collected from natural aquatic environments. The method can be applied to separate eight steroid standards: progesterone, 17alpha,20beta-dihydroxypregn-4-en-3-one, 17alpha-hydroxyprogesterone, testosterone, estrone, 11-ketotestosterone, ethynyl estradiol, and 17beta-estradiol. Based on screening of plasma, the performance of the analytical method was determined for 17alpha,20beta-dihydroxypregn-4-en-3-one, testosterone, 11-ketotestosterone, and 17beta-estradiol. The within-day reproducibility in migration time for these four steroids in aqueous samples was < or =2%. Steroid quantification was accomplished using a calibration curve obtained with external standards. Plasma samples from fish collected from the Choptank and Severn Rivers, Maryland, USA, stored for up to one year were extracted with ethyl acetate and then further processed with anion exchange and hydrophobic solid phase extraction cartridges. The recovery of testosterone and 17beta-estradiol from yellow perch plasma was 84 and 85%, respectively. Endogenous levels of testosterone ranged from 0.9 to 44 ng/ml, and when detected 17alpha,20beta-dihydroxypregn-4-en-3-one ranged from 5 to 34 ng/ml. The reported values for testosterone correlated well with the immunoassay technique. Endogenous concentrations of 17beta-estradiol were < or =1.7 ng/ml. 11-Ketotestosterone was not quantified because of a suspected interferant. Higher levels of 17alpha,20beta-dihydroxypregn-4-en-3-one were found in male and female fish in which 17beta-estradiol was not detected. Monitoring multiple steroids can provide insight into hormonal fluctuations in fish.

Assessment of aptamer‐steroid binding using stacking‐enhanced capillary electrophoresis

“…Sweeping and stacking have been integrated in CE to improve the detection limits achievable with absorbance detection []. In pH‐mediated sample stacking [], the steroidal compounds form a guest‐host complex with carboxymethyl‐β‐cyclodextrin. The complex is maintained at pH 10 in the sample vial and electrokinetically injected.…”

“…The micelles migrate toward the detection window under reversed polarity and the hydroxypropyl‐β‐cyclodextrins, which are neutral, are effectively immobile in the acidic background electrolyte. The method has been reported for the separation of up to eight different steroids in 4 min []. Sample introduction with pH‐mediated stacking provides nanomolar detection limits with conventional UV‐visible absorbance detection [].…”

“…The final solution removed from the beads contained both estradiol and DNA. These samples were injected into the separation capillary using the method of sample stacking previously reported []. Following stacking, the estradiol and DNA were separated using reversed polarity in background electrolyte comprised of 30 mM SDS, 13 mM hydroxypropyl‐β‐cyclodextrin, and 200 mM sodium phosphate buffered at pH 2.5.…”

Data collection in support of the Endocrine Disruption (ED) assessment for non‐target vertebrates

“…Plasma samples from P. flavescence were analysed for circulating steroid hormones after ethyl acetate extraction, followed by further processing with anion exchange and hydrophobic solid phase extraction cartridges (Bykova et al 2010, reference 18954 in Annex B -). This allowed removal of fatty acids which were also extracted with ethyl acetate.…”

Stacking and simultaneous determination of estrogens in water samples byCEwith electrochemical detection

“…In order to enhance the detection limit of CE, online preconcentration is a useful technique to improve the concentration sensitivity of the detector by taking advantage of small sample volume requirement . ITP , field‐enhanced sample stacking , dynamic pH junction , sweeping , and ACN stacking are commonly used online sample preconcentration techniques. Among them, ITP is based on the use of discontinuous buffers in respect to ions with different mobility at the same velocity, which is often considered as both, a concentration and purification method.…”