Novel Approaches to High Sensitivity Capillary Liquid Chromatography-Mass Spectometry and Application to in vivo Neuropeptide Monitoring.

Abstract

Novel approaches to capillary liquid chromatography (LC) coupled to multi-stage mass spectrometry (MSn) were developed to determine endogenous neuropeptides in microdialysis samples. Important factors including sample stability, detection sensitivity, reproducibility and system robustness have been examined and improved. The method was tested for 10 different neuropeptides with limits of detection (LODs) from 0.5 pM to 60 pM for most. β-endorphin could be quantitatively determined with enhanced sensitivity by detecting a characteristic peptide produced by tryptic digestion. Quantitative determination of the peptides using external calibration was verified. In this study, the temporal resolution during detection was improved from over 20 min to 5 min. To avoid poor reproducibility caused by the reduction of the sampling time of each fraction, isotopically labeled leu-enkephalin (LE) was added as an internal standard. Relative standard deviation (RSD) for in vivo dialysates measurement was reduced from over 30% to ~10%. By lowering the temporal resolution by a factor of four, larger LE concentration changes in the striatum of rat brain were observed upon perfusion of 75 mM K+ solution. Another novel approach to the capillary LC-MS system was fraction generation using a 100 µm i.d. Tee junction. With this approach, chemically separated components were preserved as nanoliter plugs and than analyzed off-line on a mass spectrometer. At optimal ESI voltage and a flow rate range from 20 to 2000 nL/min, aqueous plugs in oil segmented flow underwent ESI while oil plugs were dripped off the emitter tip or extracted through hydrophobic Teflon tubing at higher flow rate. A sample analysis rate of 2.2 Hz was achieved and parking of selected peaks to obtain more structural sample information was achieved by changing the infusion rate of the segmented flow. An application of the system was shown to be in vivo monitoring of opioid peptide following acute injection of amphetamine. Enkephalins were measured both at basal level and after drug administration, revealing a substantial release of the peptides in rat brain. This dynamic change was dose dependent. With this high sensitivity detection system, investigations of pharmacological treatments can be carried out routinely.