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Abstract: Ion suppression effects were observed during the determination of clenbuterol in urine with solid-phase extraction/multiple- stage ion-trap mass spectrometry (SPE/MS3), despite the use of atmospheric pressure chemical ionisation. During SPE, a polymeric stationary phase (polydivinylbenzene) was applied. Post-cartridge infusion of analyte to the SPE eluate after the extraction of blank urine was performed to obtain a profile of the suppression. Single and multiple-stage NIS were performed to provide insight in the suppressing compounds. The ion suppression was mainly ascribed to two m/z values, but still no identification of the compounds was achieved from the multiple- stage MS data. No ionisable and non-ionisable complexes and/or precipitation of clenbuterol with matrix compounds were observed. A concentration dependence of the percentage of suppression was observed. Up to 70% of the signal was suppressed upon post-cartridge infusion of 0.22 mug/mL (at 5 muL/min) clenbuterol into the eluate, and this decreased to about 4% at infusion of 22 mug/mL clenbuterol. Molecularly imprinted polymers were used to enhance the selectivity of the extraction. Although matrix components were still present after extraction, no interference of these compounds with the analyte was observed. However, the bleeding of the imprint from the polymer (brombuterol) caused significant ion suppression. Copyright (C) 2002 John Wiley Sons, Ltd