A novel method for detecting amino acids derivatized with phenyl isothiocyanate by high-performance liquid chromatography–electrospray ionization mass spectrometry

Abstract High-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) is an important technique for amino acid analysis. As a common derivatization reagent, phenyl isothiocyanate (PITC) is widely used in this field. However, researchers have long faced the critical problem of coupling efficient separation with simultaneous, sensitive MS detection. In this work, a novel HPLC–ESI-MS method based on PITC derivatization was established for detecting amino acids. Complete separation of 15 amino acid derivatives was achieved with an Agilent Eclipse plus C18 column maintained at 30 °C in 29 min. The mobile phase was 0.05% formic acid aqueous solution (A) and 70:30 ( v / v ) acetonitrile–water (B). The spectral data of PITC-amino acids are indicative of losses of 17 Da (NH 3 ), 18 Da (H 2 O) and/or 46 Da (CO + H 2 O) from the parent protonated molecules. Assessment of this method revealed that response curves were linear within the range of 630–20,156 μmol L −1 . LOD and LOQ ranged within 111–816 and 311–2721 pmol μL −1 , respectively. The method was validated with precision values of 0.924–6.578% (intraday, n  = 6) and 3.327–8.962% (interday, n  = 6 in 3 d). Therefore, the proposed method is a simple, robust alternative to current methods of detecting amino acids.