Reducing Adsorption To Improve Recovery and in Vivo
Detection of Neuropeptides by Microdialysis with LC-MS
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Abstract
Neuropeptides
are an important class of neurochemicals; however,
measuring their concentration in vivo by using microdialysis sampling
is challenging due to their low concentration and the small samples
generated. Capillary liquid chromatography with mass spectrometry
(cLC-MS) can yield attomole limits of detection (LOD); however, low
recovery and loss of sample to adsorptive surfaces can still hinder
detection of neuropeptides. We have evaluated recovery during sampling
and transfer to the cLC column for a selection of 10 neuropeptides.
Adding acetonitrile to sample eliminated carryover and improved LOD
by 1.4- to 60-fold. The amount of acetonitrile required was found
to have an optimal value that correlated with peptide molecular weight
and retention time on a reversed phase LC column. Treating AN69 dialysis
membrane, which bears negative charge due to incorporated sulfonate
groups, with polyethylenimine (PEI) improved recovery by 1.2- to 80-fold.
The effect appeared to be due to reducing electrostatic interaction
between peptides and the microdialysis probe because modification
increased recovery only for peptides that carried net positive charge.
The combined effects improved LOD of the entire method by 1.3- to
800-fold for the different peptides. We conclude that peptides with
both charged and hydrophobic regions require combined strategies to
prevent adsorption and yield the best possible detection. The method
was demonstrated by determining orexin A, orexin B, and a novel isoform
of rat β-endorphin in the arcuate nucleus. Dialysate concentrations
were below 10 pM for these peptides. A standard addition study on
dialysates revealed that while some peptides can be accurately quantified,
some are affected by the matrix