IMPROVED METHOD FOR DETERMINATION OF β-PHENYLETHYLAMINE IN HUMAN PLASMA BY SOLID-PHASE EXTRACTION AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

A simple, selective, and reproducible method was developed for the quantitative determination of β-phenylethylamine (PEA) in human plasma. The method involved sample clean-up procedure by a solid-phase extraction using a Sep-Pak C18 cartridge in the presence of phenylpropylamine (PPA) as an internal standard followed by pre-column fluorescence derivatization with o-phthalaldehyde, 2-mercaptoethanol. PEA and PPA were separated by reversed-phase high performance liquid chromatography (HPLC) on a C18 reversed-phase column with a mobile phase consisting of a 0.0375 M acetate (pH 5.5)/acetonitrile (50:50, v/v) buffer and detected fluorometrically. A linear relationship was achieved between the peak area ratios of PEA/PPA and PEA concentrations over the range of 250 to 2000 pg/injection (one injection=40 μL). The limit of detection for PEA at a signal-to-noise ratio of 3 was 4 pg/injection (100 pg/mL) in a standard solution. Total analysis was achieved in less than 25 minutes with the average PEA recovery of 94.1%. The reproducibility and the repeatability of the method, assessed by calculating the mean C.V. of peak area ratio (PEA/PPA) and that of the retention times of the analytes, were in the range of 1.1–3.3% and 0.14–0.42%. The average plasma PEA level in healthy volunteers was 1129.8 ± 268.1 pg/mL (n=40, age 39.3 ± 10.3 years (mean ± S.D.)).