Derivatization Reagent for Electrochemical Detection

The precise quantification of monoamine neurotransmitters such as dopamine (DA) and 5-hydroxytryptamine (5-HT) is an important theme in neurochemical research particularly in the investigation of the mechanism of pathogenesis of neurological diseases. Accordingly, the area has been the focus of much research effort in recent years.

Amongst the methods developed for this purpose is that introduced by the research group of University of Shizuoka which employs derivatization reagent NPCA (1) for electrochemical detection. 1 has an activated ester group which can undergo reaction with various primary amines, and a 6-hydroxychroman skeleton in which hydroxyl group is protected with a THP group. Interaction of 1 with various primary amines under mild conditions, followed by removal of the THP group affords the corresponding α-CA derivatives which have 6-hydroxychroman skeleton. These α-CA derivatives exhibit intense EC activity based on 6-hydroxychroman skeleton and can be separated efficiently by HPLC on an ODS column.

According to reports of the reactions between 1 and a variety of monoamine neurotransmitters published by this research group, the derivatization reaction can be carried out by the addition of 0.1 mM monoamine in phosphate buffer solution 10 μl and 50 mM NPCA in acetonitrile solution 10 μl to a mixture of 100 mM phosphate buffer (pH 8.0) and acetonitrile (1:1, v/v) at 40 °C. The α-CA derivatives can be obtained by subsequent acidification of this reaction mixture with H₃PO₄.

Following this, they investigated the separation of α-CA derivatives of various monoamine neurotransmitters by reverse-phase HPLC using EC detection. These studies revealed that the α-CA derivatives can be separated excellently on an ODS column by using a gradient elution with buffer solution (pH 5.0) and methanol. Furthermore, the α-CA derivatives can be detected with an applied voltage of less than +600 mV, and the basal noise level in the chromatogram can be decreased permitting high sensitivity analysis to fmol level. They have applied this method to rat urine analysis and achieved simultaneous determination of DA and 5-HT along with their monoamine oxidase (MAO) metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanilic acid (HVA). Additionally, in view of the intense EC responses exhibited by the α-CA derivatives, the possibility of application of the technique to a semi-micro HPLC system and the capillary electrophoresis-EC detection is also indicated.

The simultaneous determination of neuroactive monoamines, their MAO metabolites, and catechol-O-methyl transferase (COMT) metabolites is possible in this HPLC-EC method. Therefore, this method is useful for investigating the alterations in monoamines and their metabolites in urine from patients with several diseases such as neuroblastoma and migraine. Additionally, racemic compounds such as DL-norphenylephrine hydrochloride (DL-NPE) reacts with 1 to give diastereometric derivatives. These diastereometric derivatives can be separated on an achiral ODS column and the optical purities of DL-NPE etc. can be determined.