Highly Sensitive Analysis for Ophthalmic Acid by PEG-Based Derivatization Method with HILIC-MS

Abstract

We have developed a highly sensitive method for the determination of ophthalmic acid (OPH) in human plasma using a derivatization technique with a polyethylene glycol (PEG) reagent combined with hydrophilic interaction liquid chromatography and electrospray ionization-mass spectrometry (HILIC-ESI-MS). In this study, the amino group of OPH was derivatized with a PEG reagent containing an N-hydroxysuccinimide ester to increase the molecular volume of the analyte. The obtained derivative was directly injected into the LC-MS system. Under optimized analytical conditions, PEG-derivatized OPH was successfully retained on the HILIC column, and highly sensitive detection was achieved by negative ESI-MS. The limit of detection was determined to be 0.67 nM (1.67 fmol on column), and the relative standard deviations of the peak areas obtained during intra- and inter-day determinations were within 8.5%. Furthermore, this method can be used to quantify endogenous OPH in pooled human plasma samples.