Development of a liquid chromatography–tandem mass spectrometry method for 4-aminobenzoic acid ethyl ester–derivatized glycosaminoglycans and its application to synovial fluid

• Simultaneous analysis of 19 ABEE-derivatized GAG disaccharides by HILIC-mode LC-MS/MS. • Optimized pretreatment enabled accurate GAG profiling in synovial fluid. • Applicability to human synovial fluid highlights its utility for comprehensive GAG analysis. A novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) in HILIC-mode method for quantitative analysis of glycosaminoglycan (GAG) disaccharides derivatized with 4-aminobenzoic acid ethyl ester was developed. The method enables highly sensitive and selective analysis of 19 GAG-derived disaccharides and was designed for application to trace biological samples. Through the optimization of analytical conditions, separation was established within 40 min, which was 20 and 30 min shorter than our previous HILIC‑mode LC‑MS/MS and fluorescence HPLC methods, respectively. A pretreatment procedure combining actinase E digestion with ethanol precipitation was shown to be optimal for efficient release of GAGs from proteoglycans. The method was validated in terms of linearity, accuracy, repeatability, and limits of quantification (LOQ) and detection (LOD). Furthermore, recovery and matrix factors were also verified. The application of the developed method to human synovial fluid samples enabled preliminary qualitative and quantitative profiling of GAG disaccharides, suggesting its potential utility for comprehensive GAG profiling, including KS‑derived species, in joint‑related studies.