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Additional resources for Amines & Their Metabolites (NEUROMETHODS) (Neuromethods 2 Series I Neurochemistry)

Example text

Von Redlich and Glick (1965) have published a review of previous methods and propose a modified method for small amounts of Fluoresence Analysrs ofAmrnes and Their Metabolltes 15 trssue or body flurds that mcorporates the findings of several authors. In accord with Shore et al. (1959), these authors report negligible interference by 5-HT. Kremzner and Pfeiffer (1966) first reported the identification of SPD as a substance interfering with the fluorescence assay of HA following OPT condensation Graham et al.

1981) have found that a slight modification of the OPT assay gave better agreement with the HPLC-ED method. Westermk (1982) has compared values for DA obtained by measurement of fluorescence after ethylene diamme condensation, DOPAC and HVA measured by the fluorescence procedure of Westerink and Korf (1977), and 5-HIAA assayed as its OPT condensate with values for the same four compounds obtained using HPLC-ED. He found a good correlation between the two methods for DOPAC and HVA, a poorer correlation for DA, and an unacceptable agreement for 5-HIAA, with the fluorescence methods being suspect m the latter two cases.

3. ESTIMATION 1,2-diaminoethane Two samples (1 mL each) of the extract are employed for analysis. 1 kg) IS added to one of the samples to act as an mternal standard. After heating both tubes in the dark in a water bath (60°C for 20 mm), they are cooled m ice. 3 mL) is added to each. 3 mL). Fluorescence IS measured at A,,, 385 nm; h,,, 450 nm. The overall procedure is summarized in the flow diagram m Fig. 7. W HCI of perchlorlc acrd and solid KC1 15,OOOg, Extraction Addltron 5 mm wrth n-butyl acetate I I of Tris solution Add&on I of 1,2-diammo- ethane reagent I FerncyanIde oxldatlon I Read HVA fluorescence A,, 315 nm A,, 430 nm I Heat m the dark (6O”C, 20 min) I Cool on ice and a;d HCl I I Neutralize with 1,2-dlaammoethane !

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