Simultaneous and group determination methods for designated substances by HPLC with multi-channel electrochemical detection and their application to real samples.

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.