Rapid preconcentration for liquid chromatography-mass spectrometry assay of trace level neuropeptides.

Abstract

Measurement of neuropeptides in the brain through in vivo microdialysis sampling provides direct correlation between neuropeptide concentration and brain function. Capillary liquid chromatography-multistage mass spectrometry (CLC-MS(n)) has proven to be effective at measuring endogenous neuropeptides in microdialysis samples. In the method, microliter samples are concentrated onto nanoliter volume packed beds before ionization and mass spectrometry analysis. The long times required for extensive preconcentration present a barrier to routine use because of the many samples that must be analyzed and instability of neuropeptides. In this study, we evaluated the capacity of 75μm inner diameter (i.d.) capillary column packed with 10μm reversed phase particles for increasing the throughput in CLC-MS(n) based neuropeptide measurement. Coupling a high injection flow rate for fast sample loading/desalting with a low elution flow rate to maintain detection sensitivity, this column has reduced analysis time from ∼30min to 3.8min for 5μL sample, with 3 pM limit of detection (LOD) for enkephalins and 10 pM LOD for dynorphin A1-8 in 5μL sample. The use of isotope-labeled internal standard lowered peptide signal variation to less than 5%. This method was validated for in vivo detection of Leu and Met enkephalin with microdialysate collected from rat globus pallidus. The improvement in speed and stability makes CLC-MS(n) measurement of neuropeptides in vivo more practical.