Method development for the separation of steroids by capillary electrochromatography

A rapid capillary electrochromatography (CEC) method was developed to separate five structurally related steroid compounds from the production line of steroid hormones. The separation was performed on a Hypersil C8 MOS and Unimicro C18 stationary phases using acetonitrile (ACN), methanol (MeOH), and tetrahydrofuran (THF) as organic modifiers and tris(hydroxymethyl)aminomethane (Tris) as buffer additive. The Hypersil C8 MOS stationary phase performed best together with ACN as organic modifier and Tris buffer. The method was extensively tested for ruggedness with respect to sensitivity to temperature, ACN composition, pH change, concentration of Tris buffer, injected plug length, and run-to-run and day-to-day repeatability. The minimal detectable concentration and amount were investigated for quantification purposes. The developed CEC method was shown to be fast, rugged, and well suited for quantification of the steroids under study.