A simple and rapid HPLC-ECD method for assay of monoamine-related substrates (MA), choline (Ch), acetylcholine (ACh) and GABA in the same crude sample of mouse discrete brain areas

• Published in: Japanese Journal of Pharmacology 1989, Vol.49 (suppl), p.273-273
• Main Authors: Murai, Shigeo, Saito, Hiroko, Nagahama, Hiromichi, Miyate, Hiroki, Abe, Eiichi, Masuda, Yoshikatsu, Itoh, Tadanobu
• Format: Article
• Language: English
• ISSN: 0021-5198; 1347-3506
• DOI: 10.1016/S0021-5198(19)56656-2

A simple HPLC-ECD method was developed for assaying tyrosine, tryptophan, NE, DA, 5-HT, MHPG, DOPAC. HVA, 5-HIAA, Ch, ACh and GABA in the same sample of small brain areas. In this method, perchloric acid (PCA) extracts of tissues were directly injected onto the HPLC for MA or for Ch and ACh. The remaining part of the PCA extracts was mixed with o-phthalaldehyde/β-mercaptoethanol solution and injected onto the HPLC for GABA. The HPLC consisted of an HPLC pump (Yanagimoto), a RP-18 guard column (Eicom), an analytical column (MA-ODS, Eicom) for MA and GABA, and an analytical column (AC-GEL, Eicom) connected with an immobilized enzyme column (Eicom) for Ch and ACh. The electrochemical detector (VMD-501, Yanagimoto) was connected with a graphite electrode (WE-3 G, Eicom) for MA and GABA or a platinum electrode (WE-PT, Eicom) for Ch and ACh. The composition of the three mobile phases were as follows：0.02 M sodium acetate/0.0125 M citric acid buffer (pH 3.92) with 16% methanol, 0.033% heptane-sulfonic acid and 0.1 mM EDTA, for MA; 0.05 M phosphate buffer (pH 6.2) with 45% methanol and 5% tetrahydrofuran, for GABA; and 0.07 M phosphate buffer (pH 8.3) with 0.065% tetramethylammonium,0.3% octanesulfonic acid and 0.2 mM EDTA for Ch and ACh.