Many psychotropic substances are illegally available on the streets and/or via the Internet. This wide distribution has become a serious social problem. To control this problem, many substances have been controlled as 'designated substances' (Shitei-Yakubutsu) in Japan since April 2007 by the Pharmaceutical Affairs Law, including tryptamines, phenethylamines and piperazines. In the present study, simultaneous determination methods using HPLC with multi-channel electrochemical detection (MECD) were developed for the designated substances. The proposed methods utilizing online electrochemical oxidation are the first report on the simultaneous determination of various designated substances. The methods involve direct determination and require no complicated pretreatments such as fluorescence labeling. The designated substances were separated by reversed-phase chromatography using a TSK-gel ODS-100V (4.6 × 250 mm, i.d., 3 µm) and gradient elution by a mixture of potassium phosphate buffer, methanol and acetonitrile. The total separation of 31 designated substances was successfully performed but required long chromatographic run times. Thus, the designated substances were divided into three groups: (1) tryptamines, (2) phenethylamines and (3) piperazines and others. They were then analyzed by HPLC-MECD as another separation method. The suitable applied voltages for each designated substance were determined based upon the hydrodynamic voltammogram. The limits of detection (signal-to-noise ratio of 3) of the designated substances for the most suitable voltages were in the range of 17.1 pg (5-MeO-MIPT) to 117 ng (indan-2-amine). The calibration curves based on the peak heights were linearly related to the amounts of the designated substances (R(2)> 0.999). Good accuracy and precision by intra-day assay and inter-day assay were also obtained using the present procedures. The proposed methods were applied to the analyses of the designated substance in several real samples.

A solution of optically pure kynurenine (KYN), i.e., D-KYN or L-KYN, was administered intravenously to male Sprague-Dawley rats (10 mg kg(-1) ml(-1)). The time-course of changes in the concentrations of urinary monoamines and their metabolites such as 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), dopamine, and 3-methoxytyramine were investigated by reversed-phase high-performance liquid chromatography with electrochemical detection after precolumn derivatization with (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA). We observed a stereoselective difference in the effects of the KYN enantiomers. Only D-KYN, not L-KYN, caused a significant increase in urinary 5-HT levels within 30 min after its administration. With regard to the metabolites, urinary 3-MT level was increased by D-KYN administration. On the other hand, no significant change in the DA level was observed after administration of either D-KYN or L-KYN. These results suggest that D-KYN could affect the activity of neuroactive amines, especially 5-HT, in vivo.

Enantiomeric separation of D- and L-serine on an octadecylsilica column was investigated using (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (R-NPCA), which was developed for a pre-column derivatization reagent for electrochemical detection. In addition, (2S)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (S-NPCA) was newly synthesized from (S)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (S-alpha-CA), and the enantiomeric separation of D- and L-serine using S-NPCA was also examined. The enantiomeric separation of D,L-serine was achieved using the R- or S-NPCA as a chiral derivatization reagent, and the elution orders of the enantiomers were reversed between R- and S-NPCA. The elution orders of D- and L-serine unexpectedly reversed between the phosphate buffer at pH 4.0 and pH 2.2, both of which were used in the mobile phase. Separation factors obtained using R- and S-NPCA were similar-1.09 and 1.07, respectively. The detection limit was approximately 940 fmol on the column (signal-to-noise ratio 3) when the applied voltage was +650 mV.

Summary

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