Abstract A critical assessment of perchloric acid (PCA) brain tissue extracts for precise identification and quantitation of brain metabolites using in vitro proton nuclear magnetic resonance ( 1H NMR) spectroscopy was studied. One pulse with a presaturation NMR experiment was used. The chemical shifts and coupling networks of the major brain metabolites as a function of pH were characterized by using individual model compounds and a model mixture solution. We found that the conditions of the PCA solution are essential for accurate interpretation of NMR spectra of brain metabolites. The maximum spectral resolution was obtained at pH 4.92. Caution is necessary when using high resolution 1H NMR spectroscopy to identify and quantify brain metabolites.