Determination of substance P in rat spinal cord by high-performance liquid chromatography electrospray quadrupole ion trap mass spectrometry

Abstract

Substance P is a neuropeptide that belongs to the tachykinin neuropeptide family. It is an 11-amino acid polypeptide with the amino acid sequence: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met. It is synthesized as a larger protein and then enzymatically converted into the active undecapeptide. Substance P is widely distributed in the central and peripheral nervous systems. In the central nervous system, substance P participates in various behavioral responses and in regulating neuronal survival and degeneration. In the spinal cord, substance P participates in neurotransmission of pain and modulates autonomic reflexes. A rapid and selective method was developed for the determination of substance P concentration in rat spinal cord. The method consisted of a tissue homogenization, dilution, centrifugation and analysis by full-scan liquid chromatography electrospray quadrupole ion trap mass spectrometry (LC-ESI-QIT). The separation was achieved using a 50 x 2.1 mm C(18) analytical column combined with a gradient mobile phase composed of methanol: 0.1% formic acid in water set at a flow rate of 0.2 mL/min. An analytical range of 10-500 pmol/g was tested to analyze rat spinal cord. The LOD observed was 10 fmol injected on column. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. In conclusion, a rapid and sensitive LC-ESI/MS/MS method was developed to identify and quantify substance P in rat spinal cord.