In the central nervous system, CGRP coexists with SP and is co-released from primary sensory neurons, that are involved in blood flow, pain and inflammation. It has been reported that there was no change in SP levels but CGRP concentration increased during migraine attacks. Several studies indicate that brain-skin connections and SP and CGRP could be important as the key of stress mediators. Stress increases the number of both SP and CGRP positive sensory nerve fibers in mice model.
Since peptides are few in quantity in biological samples, the separation and quantification of them is the most challenging issue in peptidomics. Generally, RIA and ELISA methods are widely used for quantification of neuropeptides. Although these methods are sensitive, most of the antiserum preparations are not unique to a single peptide and can cross-react with other peptides. In addition to these methods, only a certain number of neuropeptides can be analyzed at the same time. This limits the recognition of peptides. Effective peptide quantification from biological samples requires highly selective, sensitive, robust and accurate liquid chromatography-mass spectrometric (LC-MS) methods. Neuropeptidomic studies based on mass spectrometry include the identification of endogenous neuropeptides in the nervous system in biological samples and have an increasing prevalence in neuroscience research. New peptides can be discovered using this method. It is possible to simultaneously identify and quantify multiple neuropeptides in a biological sample and when applied to disease models and important roles in pathophysiological mechanisms can be identified and used as drug targets or biomarkers.
In this study, researchers aimed to perform SP and CGRP in brainstem tissues using LC-MS/MS, because of the presence of neuropeptides in biological samples at very low concentrations. Separation parameters were optimized successfully and validated according to the bioanalytical validation guideline published by the EMA . Acceptable validation results were achieved. Basal endogen concentrations of Substance P and CGRP in rat brainstem tissue were determined.
A new, highly sensitive, reliable, selective, rapid, fully validated LC-MS/MS method has been developed for to identify and quantify SP and CGRP in rat brainstem tissue. It will contribute to diagnostic studies of diseases in the fields of medicine and physiology researchers working in the field of neuroscience. It could be used in illuminating various scientific hypotheses which would be based on measurements in biological tissues.