Determination of substance P and calcitonin gene-related peptide by various analytical method

In this study, Substance P and Calcitonin Gene-Related Peptide analysis in rat brains and brain stem tissues were determined by using Liquid Chromotography-Tandem Mass Spectrometry and Enzyme Linked-Immunosorbent Analysis methods. Firstly mass spectrometry parameters for Substance P and CGRP were optimized after acheving separation with C8 column (150x2.1 mm, 3.5 µm) and mobile phase containing 0.2% formic acid in water-acetonitrile then the method have been validated. Mass spectrum was obtained in positive polarity for Substance P and CGRP, and ion transitions were used at m/z 674.3→[600+254.4] and 952.2→1214.0, respectively. Linearity range was 5.85-438.52 ng/mL for Substance P and 0.21-155.28 ng/mL for CGRP. The limit of quantification was 5.85 ng/mL for Substance P and 0.21 ng/mL for CGRP. Accuracy was shown with recoveries higher than 98% for Substance P, higher than 87% for CGRP. The method precision was shown by intra-day and inter-day precision lower than 15% RSD for Substance P and 11 % RSD for CGRP. This method was applied to rat brain and brain stem tissues. Accuracy in brain tissue was shown with recoveries higher than 97% and precision was shown lower than 7%RSD for Substance P. Accuracy in brain stem tissue was shown with recoveries higher than 98% for Substance P, higher than 95% for CGRP and precision was shown lower than 7% RSD for Substance P and 13%RSD for CGRP. According to tissue samples analysis (n=6), Substance P and CGRP basal endogene concentrations in brain stem tissue were calculated as 415.10±124.80 ng/g and 132.07±196.77 ng/g respectively, while Substance P concentration in brain was calculated as 57.95±38.01 ng/g. CGRP concentration in brain tissue could not be calculated because of lower levels than limit of quantification of proposed method. Substance P and CGRP concentration were determined in brain and brain stem tissues by using Enzyme Linked-Immunosorbent Analysis kit procedure. Substance P concentration wasn't calculated in brain and brain stem tissues. CGRP concentration was calculated as 2351.24±292.49 ng/g in brain stem, 1567.96±55.47 ng/g in brain.