Development of a multi-sugar LC-MS/MS assay using simple chemical derivatization with acetic anhydride

Carbohydrate analysis poses many analytical challenges, in terms of extraction, chromatographic separation, and detection. Extraction challenges are associated with poor recovery from biological matrices using conventional techniques such as protein precipitation, and solid phase and liquid-liquid extraction. Chromatographic challenges are related to the poor retention of these analytes using conventional reversed phase chromatography and resolution from structurally related endogenous compounds (mannitol and sorbitol; lactulose and sucrose). Detection challenges are associated with limited UV absorbance and poor mass spectrometric ionization efficiency. To address the aforementioned challenges, the utility of a simple chemical derivatization procedure with acetic anhydride was developed for the quantitation of four sugar probes (mannitol, lactulose, sucrose and sucralose) in cynomolgus plasma and urine. Acetylation derivatization is very well known in the literature; due to its simplicity, fast reaction time under ambient conditions, and low cost it has become a popular choice for the derivatization of polar compounds containing hydroxylic, phosphate, phenolic, or amino functional groups. This derivatization protocol was found to generate derivatives with chromatographic properties more amenable for analysis on traditional reversed stationary phases followed by mass spectrometric detection. The methodology does not require filtration of plasma or urine samples, in contrast to published protocols, and the sample volume required is as small as 10 mu L plasma or urine.