Derivatization of N-Acyl Glycines by 3-Nitrophenylhydrazine for Targeted Metabolomics Analysis and Their Application to the Study of Diabetes Progression in Mice

Abstract

N-Acyl glycines (NAGlys) are an important class of metabolites in the detoxification system of the human body. They have been used in the diagnosis of several metabolic diseases. Liquid chromatography−mass spectrometry (LC−MS) is the most frequently used NAGlys detection platform. Here, we describe a simple and sensitive method of NAGlys detection by LC−MS in plasma and urine samples. This approach is based on the use of a derivatization reagent, 3-nitrophenylhydrazine. The reaction is quick in aqueous solution, and no quenching step is needed. To expand the coverage of NAGlys when standards are not available, NAGlys were first identified based on high-resolution LC−MS. Quantification was subsequently carried out on triple quadrupole LC−MS. This approach allowed a much broader measurement of NAGlys (41 NAGlys in total), especially when authentic standards are unavailable. Comprehensive analysis of NAGlys with this new method was applied in plasma and urine samples of db/db diabetic and non-diabetic db/m+ control mice. The majority of detected NAGlys were altered with high differentiation ability in plasma and urine samples from diabetic and non-diabetic mice. These identified NAGlys hold the potential to be diagnostic biomarkers for type II diabetes and diabetic complications.

Analysis of O-acetylated sialic acids by 3-nitrophenylhydrazine derivatization combined with LC-MS/MS

“…2a and b display a product ion at m / z 178.03 Da and 184.05 Da, respectively, indicating a derivatization at the carboxyl group. 33 The high abundance of ions at m / z 357.09 Da and 369.14 Da suggests that the fragment contains two 3-NPH labels, indicating the involvement of C1 and C2. As a result, this ion is assigned to the structure shown in Fig.…”

“…The 3-NPH derivatization was performed in two steps, following previous studies with minor modications. 32,33 Firstly, the dried sample was dissolved in 100 mL of 70% methanol (v/v) and mixed with 50 mL of 3-NPH (200 mM in 70% methanol) containing 5% pyridine (v/v). The solution was incubated at room temperature for 1 h. Secondly, 50 mL of EDC (120 mM in 70% methanol) was added, and the solution was incubated for another hour.…”