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 P, 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 × 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 µL/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 P, 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 P, 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 © 2009 John Wiley & Sons, Ltd.