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). Chemical derivatization is necessary to improve the MS response. 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 higher than those of the commercial reagent, 2-aminobenzamide, when analyzing maltopentaose, maltohexaose, and maltaheptaose, demonstrating the advantages of fast, mild labeling, simple post-treatment, better separation, and significant improvement in ionization. This method was first applied to the detection of carbohydrates in the plasma of mice with glycogen storage disease Ib (GSDIb). The free glycogen content in the plasma of diseased mice was confirmed and determined by the standard addition method to be approximately 7.1 ng mL⁻¹. Given the lack of standards and structural complexity of N-glycan complexes, a stable isotope-labeled RBH with 20 deuterons was designed and synthesized at very low cost to reduce the difficulty of N-glycan determination and enable 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 to quantify plasma N-glycans from GSDIb and normal mice. The fucosylated contents of diseased mice were abnormally increased, while the non-fucosylated contents were reduced, indicating the fucosylation level of plasma N-glycan was expected to be an indicator for the detection of GSDIb.