Ion suppression effects in liquid chromatography–electrospray-ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectra libraries

Abstract

Ion suppression effects during electrospray-ionsation mass spectrometry (ESI-MS) caused by different sample preparation procedures for serum were investigated. This topic is of importance for systematic toxicological analysis for which LC–ESI-MS has been developed with transport-region collision-induced dissociation (ECI-CID) and mass spectra library searching. With continuous postcolumn infusion of two test compounds—codeine and glafenine—the ion suppression effects of extracted biological matrix obtained after a standard liquid–liquid extraction, a mixed-mode solid-phase extraction (SPE) method, a protein precipitation method and a combination of precipitation with polymer-based mixed-mode SPE have been investigated. Extracted ion chromatograms of codeine ([M+H] +, m/ z 300) and glafenine ([M−H] −, m/ z 371) were used for monitoring ion suppression. Severe ion suppression effects for codeine and glafenine were detected in positive and in negative ionisation modes, respectively, in the LC-front peak after serum clean-up with SPE (acid/neutral fraction) and protein precipitation as well as with protein precipitation combined with SPE. Less ion suppression of codeine in positive mode was found with liquid–liquid extraction of serum samples. No ion suppression was detected with the second fraction of the mixed-mode SPE (using RP-C 8 and cation-exchange phase) in both ionisation modes. All suppression effects were caused by polar and unretained matrix components, which were present after extraction and/or protein precipitation. However, no specific ion suppression was seen after elution of the polar LC-front throughout the whole gradient. It could be demonstrated, that ion suppression is not generally present at any retention time when using reversed-phase HPLC with rather long gradient programs, but may play an important role in case of high-throughput LC–MS analysis, when the analyte is not separated from the LC-front, or in flow injection analysis without chromatographic separation.