Synthesis of Rhodamine B Hydrazine Derivatives and Their Application in Glycan Analysis

Abstract

Due to the high molecular weight and polarity, glycans suffer low ionization efficiency in electrospray ionization mass spectrometry (ESI-MS). It is necessary to chemically derivatize them for MS response improvement. In this work, rhodamine B hydrazine(RBH) derivatives with different end groups were synthesized, among which RBH with tetraethyl end groups performed best. After optimization, the signals were 8-30 times better than those of commercial reagents 2-aminobenzamide when analyzing maltopentaose, maltohexaose and maltaheptaose, showing advantages of fast and mild labeling, simple post-treatment, better separation and significant improvement of ionization. This method was first applied to the detection of carbohydrates in plasma of mice with glycogen storage disease Ib (GSDIb). The content of free glycogen in the plasma of diseased mice was confirmed and determined by standard addition method to be about 7.1 ng/mL. For N-glycans complex in structures and inaccessible of standards, stable isotope labeled RBH with 20 deuterons was designed and synthesized at a very low cost to reduce the difficulty in N-glycan determination and realize the relative quantification. With Python for spectral analysis, 19 N-glycans were successfully identified with only 1 μL of mouse plasma. Subsequently, the method was used for relative quantification of plasma N-glycans from GSDIb mice and normal mice. The fucosylated contents of diseased mice were abnormally increased, while the non-fucosylated reduced, indicating the fucosylation level of plasma N-glycan was expected to be an indicator for the detection of GSDIb.