Pre-column Derivatization-High Performance Liquid Chromatography for the Determination of Aliphatic Amines with Fluorescence Detection and Mass Spectrometry Identification
Abstract A simple and highly sensitive method based on the derivatization of aliphatic amines with 2-(2-phenyl-1 H -phenanthro-[9,10- d ]imidazole-1-yl)-acetic acid (PPIA) followed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line post-column mass spectrometric identification has been developed. Optimum derivatization, giving the corresponding stable fluorescent derivatives, was obtained by the reaction of aliphatic amines with PPIA at 60 ° for 15 min in the presence of N -ethyl- N ′-(3-dimethylamino propyl)carbodiimide hydrochloride (EDC) catalyst in acetonitrile solvent. The fluorescence detection wavelength of PPIA derivatives was 380 nm (260 nm of excitation wavelength). Separation of the derivatized amines with a good baseline resolution in conjunction with a gradient elution was carried out on a reversed phase Eclipse XDB-C 8 column (4.6 mm × 150 mm, 5 μm), in this way, the contents of aliphatic amines in waste water of paper mill, telencephalon tissue of rat and acidophilus milk were determined. The identification of aliphatic amine derivatives was carried out by post-column online mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive ion mode. In virtue of the MS and MS/MS information of intermediates of derivatization reaction, the derivatization reaction mechanism was confirmed. The established method exhibits excellent reproducibility and recovery. Excellent linear responses were observed with coefficients >0.9996, and detection limits (at signal-to-noise of 3:1) were 3.1–18.2 fmol.