Stacking enhanced determination of steroids by CE

This study outlines a simple method for pH‐mediated stacking of natural and synthetic steroids facilitated with carboxymethyl‐β‐CD. Sample stacking (10 kV, 60 s) is accomplished with 23 mM carboxymethyl‐β‐CD in 50 mM 3‐[cyclohexylamino]‐1‐propanesulfonic acid buffered at pH 10. Following stacking, steroidal compounds are separated in less than 5 min with a running buffer of 13 mM hydroxypropyl‐β‐CD, 30 mM SDS in 200 mM phosphate buffered at pH 2.5. Using a 60 s electrokinetic injection, the limits of detection of estradiol, ethynyl estradiol, estrone, hydroxyprogesterone, progesterone, and 11‐ketotestosterone range from 2 to 14 nM. For all steroids, the within‐day and day‐to‐day reproducibility in migration time is ≤1 and ≤2% RSD, respectively. The within‐day and day‐to‐day reproducibility in peak area is ≤9 and ≤22% RSD, respectively. The method is applied to fish plasma and holds potential to profile multiple steroids in a single biological sample.