Modelling and optimization of the separation of steroids eluted with a micellar mobile phase of sodium dodecyl sulphate containing acetonitrile

The equation 1k′ = c0 + c1μ + c2ϑ + c3μϑ + c4ϑ2 (μ is the surfactant concentration and ϑ is the amount of modifier) proved to be satisfactory in the description of the retention of several steroids (boldenone, dydrogesterone, medroxyprogesterone, methandrostenolone, methyltestosterone, medroxyprogesterone acetate and testosterone) and two diuretics (amiloride and chlorthalidone) when eluted with micellar mobile phases containing sodium dodecyl sulphate (SDS) and acetonitrile. Modelling of the retention and an adequate selection of the experimental design allowed the rapid and reliable optimization of the separation of a mixture of steroids. The retention of steroids was moderately affected by an increase in the concentration of the surfactant. The elution strength of acetonitrile was relatively low and thus, a large amount of the modifier was required to obtain adequate retention times. However, the small variation in the resolution achieved in mobile phases of increasing eluent strength permitted the separation of the low (boldenone, medroxyprogesterone, methyltestosterone, medroxyprogesterone acetate) from the high (progesterone, clostebol acetate and testosterone propionate) retained steroids in the same chromatogram in less than 40 min.