Identification, characterization and quantification of specific neuropeptides in rat spinal cord by liquid chromatography electrospray quadrupole ion trap mass spectrometry

Substance P and CGRP play a central role in neuropathic pain development and maintenance. Additionally, dynorphin A is an endogenous ligand of opioid receptors implicated in the modulation of neurotransmitters including neuropeptides, such as substance P and CGRP. This manuscript proposes a method to characterize, identify and quantify substance R CGRP and dynorphin A in rat spinal cord by HPLC-ESI/MS/MS. Rat spinal cords were collected and homogenized into a TFA solution. Samples were chromatographed using a microbore C(8) 100 x 1 mm column and a 19 min linear gradient (0:100 -> 40:60, ACN:0.2% formic acid in water) at a flow rate of 75 mu L/min for a total run time of 32 min. The peptides were identified in rat spinal cord based on full-scan MS/MS spectra. Substance R CGRP and dynorphin A were predominantly identified by the presence of specific b CID fragments. Extracted ion chromatogram (XIC) suggested selected mass transitions of 674 -> [600 + 254], 952 -> [1215 + 963] and 717 -> [944 + 630] for substance P, CGRP and dynorphin A can be used for isolation and quantitative analysis. A linear regression (weighted 1/x) was used and coefficients of correlations (r) ranging from 0.990 to 0.999 were observed. The precision (%CV) and accuracy (%NOM) observed were 10.9-14.4% and 8.9-14.2%, 8.8-13.0% and 91.0-110.2% and 97.2-107.3% and 91.8-97.3% for substance P, CGRP and dynorphin A respectively. Following the analysis of rat spinal cords, the mean endogenous concentrations were 110.7, 2541 and 779.4 pmol/g for substance R CGRP and dynorphin A respectively. The results obtained show that the method provides adequate figures of merit to support targeted peptidomic studies aimed to determine neuropeptide regulation in animal neuropathic and chronic pain models. Copyright (C) 2009 John Wiley & Sons, Ltd.