Determination of trace constituents in biological and environmental samples usually requires a pre-concentration step. While solid-phase extraction (SPE) has been widely used, it is slow, labor intensive and adversely affected by analytical errors from handling. On-line SPE eliminates some of the flaws but often suffers from solvent compatibility problems with the subsequent chromatography separation. In this study, we are presenting a technical solution for overcoming some of these compatibility issues, by utilizing a fully automated, focused SPE sample transfer technique utilizing narrow-band solvent plugs, for seamless hyphenation with high-performance liquid chromatography (HPLC) or flow injection mass spectrometry (MS). A wide range of pharmaceutical compounds was studied in different sample matrices. Short plugs of high elution strength solvent were generated by means of an electrically actuated sample loop and enrichment and transfer steps monitored using on-line SPE-MS. The impact of the solvent plugs on chromatographic separation was studied using hyphenated SPE-LC-MS. By carefully examining elution profiles of solvent plugs of different compositions, optimum conditions for quantitative elution within well-defined volumes were found for all substances. In addition, the highly focused elution bands resulted in excellent retention time and peak area reproducibilities when injected on-line onto HPLC columns. Finally, to demonstrate proof-of-principle, the fully integrated on-line SPE-LC-MS system was applied to the analysis of spiked urine and river water samples.