Simultaneous separation and detection of 18 phenethylamine/tryptamine derivatives by liquid chromatography-UV absorption and -electrospray ionization mass spectrometry.

The optimal conditions for the separation and detection of a mixture of 18 phenethylamine/tryptamine derivatives were determined, using liquid chromatography/UV-absorption (LC/UV) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) methods, respectively. Complete separation could be achieved within approximately 25 min using gradient elution (A, 0.1% formic acid aqueous solution/pH 2.5; B, acetonitrile). The limit of detection (LOD at S/N = 3) obtained by LC/UV-absorption (absorption wavelength, 280 nm) was in the range from 0.3 to 3 microg/mL. In contrast, when the LC/ESI MS method was used, the LODs for primary, secondary and tertiary amines were in the ranges 0.1-3.0, 0.1-0.2, and 0.05-1.8 microg/mL, respectively. The lower LOD obtained for a tertiary amine can be attributed to the fact that its ionization efficiency (during the ESI process) is better than the others. In order to improve the LOD of a primary/secondary amine, a derivatization procedure was used in which the chemical structure was altered to a secondary/tertiary amine, via a reaction with acetic anhydride. As a result, the LODs for primary/secondary amines could be significantly improved. The characteristic mass fragmentations of the 18 phenethylamine/tryptamine derivatives, as well as the products of the reaction with acetic anhydride, were investigated, and the data were reported. A urine sample was obtained by spiking urine from a volunteer with the 18 derivatives, and after liquid-liquid extraction the sample was examined by LC/UV and LC/ESI MS, respectively. The extraction procedures used for the urine sample and the experimental conditions for the separation and detection were optimized.