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The present paper provides an overview on currently developed derivatization chemistries and techniques for determination of monoamine neurotransmitters serotonin (5-HT), norepinephrine (NE) and dopamine (DA) in microdialysis samples by microbore liquid chromatography with fluorescence detection. In mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine (BA), forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity was achieved for reaction with 1,2-diphenylethylenediamine (DPE) rather than with BA, therefore for simultaneous determination of 5-HT, NE and DA in brain microdialysates, a two-step derivatization with BA followed by DPE was developed. The detection limits for 5-HT, NE and DA were 0.2, 0.08 and 0.13 fmol, respectively, in an injection volume of 20 microL, which corresponds to concentrations of 30, 12 and 19.5 pm, respectively in standard solution prior to derivatization. The experimental data presented demonstrate the ability of the technique to simultaneously monitor neuronally releasable pools of monoamine neurotransmitters in the rat and mouse brains at basal conditions and following pharmacological treatments or physiological stimuli. These techniques play an important role in drug discovery and clinical investigation of psychiatric and neurological diseases such as depression, schizophrenia and Parkinson's disease.

The metabolism by human liver microsomes of several new illicit drugs, that is, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3- carboxamide (ADB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1- (4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC), quinolin-8-yl 1-(5-fluoropentyl)-(1H-indole)-3-carboxylate (5 F-QUPIC) and α-pyrrolidinovalerothiophenone (α-PVT), which have indole, indazole, quinolinol ester and thiophene structures, was investigated using reversed-phase chromatography and mass spectrometry. The present method is based upon the oxidation by cytochrome p450 superfamily enzymes in the microsomes. The oxidation of ADB-FUBINACA and AB-FUBINACA mainly occurred on the N-(1-amino-alkyl-1-oxobutan) moiety. However, the oxidation of AB-PINACA seemed to occur on the 1-pentyl moiety. On the other hand, QUPIC and 5 F-QUPIC, which have a quinolinol ester structure, predominantly underwent a cleavage reaction to produce indoleacetic acid type metabolites. In contrast, the metabolism reaction of α-PVT was different from that of the other tested drugs, and various oxidation products were observed on the chromatograms. The obtained metabolites are not in conflict with the results predicted by MetaboLynx software. However, the exact structures of the metabolites, except for 1-pentyl-1H-indole-3-carboxylic acid (QUPIC metabolite) and 1-(5-fluoropentyl)-1H-indole-3-carboxylic acid (5 F-QUPIC metabolite), are currently not proven, because we have no authentic compounds for comparison. The proposed approach using human liver microsome seems to provide a new technology for the prediction of possible metabolites occuring in humans.

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.

The non-targeted metabolomics analysis of biological samples is very important to understand biological functions and diseases. LC combined with electrospray ionization-based MS has been a powerful tool and widely used for metabolomic analyses. However, the ionization efficiency of electrospray ionization fluctuates for various unexpected reasons such as matrix effects and intraday variations of the instrument performances. To remove these fluctuations, normalization methods have been developed. Such techniques include increasing the sensitivity, separating coeluting components and normalizing the ionization efficiencies. Normalization techniques allow simultaneously correcting of the ionization efficiencies of the detected metabolite peaks and achieving quantitative non-targeted metabolomics. In this review paper, we focused on these normalization methods for non-targeted metabolomics by LC-MS.