Capillary liquid chromatography with MS 3 for the determination of enkephalins in microdialysis samples from the striatum of anesthetized and freely–moving rats

Abstract

In vivo microdialysis sampling was coupled to capillary liquid chromatography (LC)/electrospray ionization quadrupole ion trap mass spectrometry (MS) to monitor [Met]enkephalin and [Leu]enkephalin in the striatum of anesthetized and freely–moving rats. The LC system utilized a high-pressure pump to load 2.5 µl samples and desalt the 25 µm i.d. by 2 cm long column in 12 min. Samples were eluted with a separate pump at ∼100 nl min −1 . A rapid gradient effectively separated the endogenous neuropeptides in 4 min. A comparison was made for operating the mass spectrometer in the MS 2 and MS 3 modes for detection of the peptides. In standard solutions, the detection limits were similar at 1–2 pM (2–4 amol injected); however, the reproducibility was improved with MS 3 as the relative standard deviation was <5% compared with 20% for MS 2 for 60 pM samples. For dialysate solutions, reconstructed ion chromatograms and tandem mass spectra had much higher signal-to-noise ratios in the MS 3 mode, resulting in more confident detection at in vivo concentrations. The method was successfully used to monitor the peptides under basal conditions and with stimulation of peptide secretion by infusion of elevated K + concentration. Copyright © 2005 John Wiley & Sons, Ltd.