Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary

Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.

Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 μm particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (75∶25, v:v) and switched to a CN column (10 cm long, 5 μm particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 μm particle diameter) with acetonitrile-0.088 M acetate buffer (75∶25, v:v) as mobile phase.

Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (λ_ex= 225 nm; λ_em > 320 nm).