Influence of Organic Modifier Additives to Separate Steroids by Micellar Electrokinetic Chromatography: Determination of Solute-Micelle Association Constants at Different Acetonitrile Concentrations

Abstract

In this report, the determination of testosterone, progesterone, estrone, 17-β-estradiol, and ethynilestradiol by micellar electrokinetic chromatography (MEKC) is described. Several organic modifiers were investigated using sodium dodecyl sulfate as the surfactant agent in the background electrolyte. The effect of the acetonitrile concentration on the migration time of the steroids and on the selectivity was also studied by using different background electrolytes. Under the optimized conditions that included a sodium tetraborate (pH 9.3; 25 mM) buffer with 10 mM sodium dodecyl sulfate, 20% (v/v) of acetonitrile, 27 kV running voltage, and injection with a plug of background electrolyte (7 mbar × 1 s), the analytical performance of the method was evaluated. Good linearity (correlation coefficients, R ² ≥ 0.99) and adequate precision were achieved, with limits of detection of 1.27, 2.17, 0.6, 1.13, and 1.7 µg/mL for testosterone, progesterone, estrone, 17-β-estradiol, and ethynilestradiol, respectively. To study the effect of the acetonitrile concentration on the solute-micelle interaction, the retention factor and association constants were determined. In all cases, the association constants decreased by increasing the acetonitrile concentration from 10% to 30%, suggesting that the presence of large amounts of organic modifier decreased the steroid-micelle interactions.

Keywords:

• MEKC
• Micellar electrokinetic chromatography
• Organic modifier additive
• Solute-micelle association constant
• Steroids

Notes

– not eluted in 90 min of analysis time, electrophoretic conditions as in Figure 1.

T: Testosterone; P: Progesterone; E1: Estrone; E2: 17-β-estradiol; EE2: Ethynilestradiol.

Electrophoretic conditions as in Figure 1.

T: Testosterone; P: Progesterone; E1: Estrone; E2: 17-β-estradiol; EE2: Ethynilestradiol.

Electrophoretic conditions as in Figure 3a.

T: Testosterone; P: Progesterone; E1: Estrone; E2: 17-β-estradiol; EE2: Ethynilestradiol.

Critical micellar concentration (CMC): 0.5 mM (10% acetonitrile), 6 mM (20% acetonitrile) and 14 mM (30% acetonitrile).

Electrophoretic conditions: capillary temperature, 15°C; injection by pressure, 35 mbar × 3 s with a plug of 7 mbar × 1 s of background electrolyte and applied voltage, 27 kV.

SDS: Sodium dodecyl sulfate; T: Testosterone; P: Progesterone; E1: Estrone; E2: 17-β-estradiol; EE2: ethynilestradiol.