Two-dimensional analysis of phenolic acids in seedling roots by high performance liquid chromatography-electrospray ionization-ion mobility spectrometry

Abstract

An online two-dimensional high-performance liquid chromatography-electrospray ionization (silica capillary tube with polyamide coating)-ion mobility spectrometry (HPLC-ESI-IMS) method was developed and applied to identify nine phenolic acids in seedling roots. The separation was performed by gradient elution consisting of methanol and 0.1% acetic acid (v/v) on a narrow diameter reverse C₁₈ column with ammonia as the sheath fluid. Ion mobility spectrometry (IMS) was employed to assist in the determination of the co-eluted compounds that were not separated by HPLC to achieve a highly selective and versatile two-dimensional analysis. Upon optimization of the mobile phase, sheath fluid and column flow rates, the resulting IMS spray voltage was 4.5 kV, and the drift tube temperature was 95 °C. Consequently, the HPLC-ESI-IMS technique enabled effective separation of coumaric acid, protocatechuic acid, chlorogenic acid, vanillic acid, caffeic acid, ferulic acid, sinapic acid, benzoic acid and salicylic acid according to their acquired mobility drift time differences. The limit of detection (LOD) ranged from 0.05 to 5 ng and the recovery of the spiked samples ranged from 85.10% to 100.51%. Collectively, this sensitive and efficient two-dimensional HPLC-ESI-IMS method developed in this study may be applicable for the isolation and identification of the phenolic acids secreted by the plant roots.