Determination of phenylisothiocyanate derivatives of amphetamine and its analogues in biological fluids by HPLC-APCI-MS or DAD

Amphetamine (A), methamphetamine (MA), methylene-dioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDE), and methylenedioxymethamphetamine (MDMA), as well as eight other sympathomimetic amines (benzyl-1- phenylethylamine, ephedrine, fenfluramine, norfenfluramine, phentermine, phenylethylamine, phenylpropanolamine, and propylhexedrine), were extracted from serum or urine with ether, derivatized with phenylisothiocyanate, and subjected to high-performance liquid chromatographic (HPLC) examination in isocratic mode. Two detection arts were applied: atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) and UV-spectrometry as diode array detection (DAD) or single wavelength at 250 nm. The derivatives were well-separated and showed good chromatographic behavior. Full-scan mass spectra of drugs examined by means of APCI with collision induced dissociation (APCID) contained protonated molecular ions (M+H)+ and fragments typical for particular drugs. APCID-liquid chromatography-mass spectrometry (LC-MS) appeared very selective for differentiation of all drugs involved. The quantitation with APCID was performed using selected ion monitoring (SIM) of (M+H)+ ions and selected fragments of drugs involved and their deuterated analogues. The limits of detection ranged from 0.001 mg/L (MA, MDMA, and MDE) to 0.005 mg/L (A and MDA). In HPLC-DAD, the spectra of MDMA and MDE were practically identical with maxima of 236-240 nm. Other amphetamines showed slightly different spectra with maxima of 245-250 nm. The limits of detection in UV detection amounted to 0.01-0.03 mg/L (single wavelength detector at 250 nm) or 0.05-0.1 mg/L (DAD).