O=C(O)CN([CH2:8][CH2:9][OH:10])CCO.N[CH2:13][C:14]([OH:16])=O.[CH3:17][C:18]#[N:19].O.[CH2:21](O)[CH3:22]>[Fe-3](C#N)(C#N)(C#N)(C#N)(C#N)C#N.[K+].[K+].[K+]>[NH2:19][CH2:18][CH2:17][C:21]1[CH:22]=[CH:8][C:9]([OH:10])=[C:14]([OH:16])[CH:13]=1To the sample solution (1.0 ml) were added 2.0 ml of 0.05M Bicine buffer solution (pH 7.0) containing 0.5M glycine, 0.1 ml of 0.1M DPE solution (ethanol solvent) and 0.1 ml of 0.08% (W/V) potassium ferricyanide solution in sequence. After allowing to stand for more than 30 minutes at 37° C., 100 μl of the reaction liquid were injected into HPLC apparatus. Conditions of HPLC were as follows: column; Ultrasphere-ODS (4.6φ×150 mm, Beckman), eluate; CH3CN--H2O (47.5:52.5 W/V), flow rate 1 ml/min. For the fluorescence detection, Shimazu FLD-I (excitation 300-400 nm, fluorescence 450-800 nm) was employed. Under these conditions, reversed-phase partition type column as mentioned above, norepinephrine, epinephrine and isoproterenol gave a single peak respectively. Although dopamine gave two peaks of an intensity ratio of 9:1, the determination was possible with either of them. The elution was completed within 12 minutes, and the lower limit of detection was 10 pg at S/N ratio of 2.
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