Methodological aspects of an off-line combination of preparative isotachophoresis and high-performance liquid chromatography with mass spectrometry in the analysis of biological matrices.

This work reports on some methodological aspects of an off-line combination of preparative ITP and HPLC with mass spectrometric detection (pITP-HPLC-MS) and its potential applications to the analysis of high molecular mass compounds present in complex biological matrices from the analytical chemistry perspective. Lysozyme served as the model analyte and human saliva as the complex biological matrix in this study. A mixture of five low-molecular mass compounds was found and successfully used in the pITP experiments as discrete spacers to isolate the analyte from the interferents present in the complex biological matrix and to minimize their disturbance effect on the final MS analysis. The experiments at the pITP stage were performed in the cationic mode. On-column conductivity detectors were used for the detection of ITP zones. Lysozyme was found in the human saliva samples using just deconvolution of the MS data after background correction. The MS data obtained from HPLC-MS analysis of pITP fractions exhibited the great analytical potential of the combination of pITP-HPLC-MS resulting from the ITP clean-up effect as well as the ITP preconcentration of the analyte present at low concentration levels in complex biological matrices.