Quantitative Neurochemical Analysis Protocol

All chemicals, drugs and reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise noted. Water and acetonitrile are Burdick & Jackson HPLC grade purchased from VWR (Radnor, PA). 10 mM stock solution of each analyte was made in HPLC water and kept at −80 °C. Standard mixture was diluted from stock using aCSF consisting of 145 mM NaCl, 2.68 mM KCl, 1.10 mM MgSO₄, 1.22 mM CaCl₂, 0.50 mM NaH₂PO₄, and 1.55 mM Na₂HPO₄, adjusted pH to 7.4 with 0.1 M NaOH. Calibration curves were made using standards at 50 nM, 500 nM, 1000 nM, 5000 nM, 10000 nM for Gly, Ser and Tau; 5 nM, 50 nM, 100 nM, 500 nM, 1000 nM for Asp, Glu, GABA, Hist, Ado, HVA, 5-HIAA and DOPAC; 0.5 nM, 5 nM, 10 nM, 50 nM, 100 nM for ACh, 5-HT, NE, DA, NM and 3-MT. Internal standard was 1 mM Gly, Ser and Tau, 100 µM Asp, Glu, GABA, Hist, Ado, HVA, 5-HIAA, and DOPAC and 10 µM 5-HT, NE, DA, NM and 3-MT derivatized with ¹³C₆ benzoyl chloride using the same procedure as ¹²C reagent, then diluted 100 fold in DMSO containing 1% formic acid. d4-ACh (C/D/N isotopes, Pointe-Claire, Canada) was spiked into the reaction mixture to a final concentration of 100 nM.