A fully automated and validated 2D online solid-phase extraction (SPE) high-performance liquid chromatographic method was described for the direct simultaneous determination of clenbuterol (CLEN) and doxazosin (DOX) in spiked human plasma samples. Chromatographic separation of the analytes was achieved by column-switching. SPE column packed with restricted access material for depleting matrix and a reversed phase HPLC column for eluting analytes were used with UV detection on 250 nm. A 10 mM acetate buffer (pH: 4) and acetonitrile mixture was used in gradient elution with 1 mL/min flow rate, as mass spectrometry friendly mobile phase. Calibration graphs were prepared for spiked plasma, and good linearity was achieved over a dynamic range of 75.0 ng/mL-200.0 µg/mL for CLEN and 37.5 ng/mL-100.0 µg/mL for DOX. Detection and quantification limits were 8.47 and 11.94 ng/mL, 25.66 and 36.18 ng/mL for CLEN and DOX, respectively. Online column-switching liquid chromatography technique combines the sample preparation by online SPE and separation steps in one allowing direct determination of drugs in biological fluids. This article could be assessed as a good example of combination of online sample preparation and separation, thus simultaneous determination of drugs in plasma samples.
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