A Novel High-Conjugated Probe of 4-(Acridone-10-yl)-Phenylethyl Chloroformate (APE-Cl) for Rapid Detection of Amino Compounds Using HPLC with Fluorescence Detection

The fluorescence detection of amino compounds and the evaluation of their content in environmental samples are vital, not only for assessing food quality but also for studying soil organic matter. Here, we present the synthesis and application of a novel fluorescent probe, 4-(9-acridone)benzylmethyl carbonochloride (APE-Cl), for detecting amino compounds via a chloroformate reaction with fluorescence detection. The complete derivatization reaction of APE-Cl with amino compounds can be accomplished in aqueous acetonitrile within 5 min at room temperature, using 0.2 M borate buffer (pH = 9.0). APE-amine derivatives exhibited intense fluorescence with an excitation maximum at λex 254 nm and an emission maximum at λem 418 nm. All derivatives demonstrated high stability, strong fluorescence, and elevated ionization potential under atmospheric pressure chemical ionization (APCI-MS) in positive ion detection mode. The method, combined with gradient elution, provides baseline resolution of common amine derivatives on a reversed-phase C18 column. The LC separation for the derivatized amines shows good reproducibility with aqueous acetonitrile as the mobile phase. The relative standard deviations (n = 6) for each amine derivative are < 3.99%. The detection limits (at a signal-to-noise ratio of 3) per injection ranged from 1.68 to 11.2 femtomole. The established pre-column derivatization method for determining amino compounds in practical samples proved to be satisfactory.