Clinical application of liver diseases diagnosis using ultrahigh-sensitive liquid chromatography-mass spectrometry for sialic acids detection

Abstract

Sialic acids are a group of nine-carbon N-acetylated derivatives of neuraminic acid containing a keto group at position C2 (ketononose), which play important roles in many biological processes. The simultaneous detection of free sialic acid (FSA) and total sialic acid (TSA) is always challenging due to three orders of magnitude difference. An accurate and robust chemical derivatization-based liquid chromatography-mass spectrometry (LC-MS) method was proposed here to quantify both TSA and FSA in human serum with only 1 μL human serum consumption. The derivatization method with Girard's P (GP) reagent provided an ultrasensitive analysis of sialic acids as only [GP+SA-H2O]+ ions derivatized from SA could be detected by LC-MS. The limit of quantification (LOQ) of SA was less than 5 fg (S/N = 47), which was the most sensitive measurement published for SAs in biomatrices. In addition, no matrix effect existed after 10000-fold dilution of serum extracts. The recovery rates were in the range of 98.1–114.0% and the coefficient of variations (CV) was within 5%. The method has been successfully applied for the quantification of TSA and FSA in serums of patients with different liver diseases. The specificities of TSA and FSA for the early diagnosis of severe hepatopathies were higher than most of the lab blood test indicators with area under the curve (AUC) of 0.900 and 0.882.