ODP179 Development of Derivatization-free ID-LC/MS/MS Method for Simultaneous Measurement of Human Serum Monosaccharides

Abstract

Abstract According to the CDC's National Diabetes Statistics Report for 2020, it is estimated that 34.2 million, 10.5% of the U. S. population, have diabetes. High fructose intake has also been associated with increased de novo lipogenesis in the liver leading to non-alcoholic fatty liver disease (NAFLD). Emerging evidence suggests that measuring fructose, galactose, and other saccharides in addition to glucose may be helpful for diagnosis of metabolic disorders and other diseases. Therefore, there is a need for an accurate and precise laboratory method for measuring serum monosaccharide panels. Traditional monosaccharide assays used in patient care often can only quantitate one monosaccharide at a time. We aim to develop a high-throughput analytical method for simultaneous measurement of serum monosaccharides that is suitable for large scale epidemiologic studies. Our assay is based on a derivatization-free ID-LC/MS/MS procedure. Certified primary reference materials of monosaccharides from NIST (Gaithersburg, MD) were used for assay calibration. The serum samples were spiked with 13 C labeled internal standards. The monosaccharides were isolated from serum matrix by acetonitrile protein precipitation and filtration in 96-well filter plate (0.2um, porous PTFE). Aliquot of the acetonitrile filtrate was applied on an AB SCIEX Triple Quad™ 6500 LC/MS/MS system for analysis. The monosaccharides including glucose, galactose, fructose, mannose, and potential interferences were resolved from each other on Unison UK Amino column (3×250mm) eluted with acetonitrile and water gradient. Selective reaction monitoring in negative electrospray ionization mode was used for quantitation of monosaccharides in the samples. The measurement ranges covered 10–402 mg/dL for glucose, which is sufficient to monitor the serum glucose levels in normal, hypo-, and hyperglycemia patient samples. Using 4 levels of NIST SRM 965b (values assigned using GC-MS based reference measurement procedure), the method demonstrated excellent measurement accuracy of over 98% for serum glucose. The imprecision of serum glucose measurement was within 3%. Imprecision and accuracy of lower abundant serum monosaccharides were lower than 6% and above 96%, respectively. The application of 96-well format filter plate and automated liquid handler system significantly improves the throughput of sample preparation. The current analytical method can be used to simultaneously measure the concentrations levels of a panel of serum monosaccharides with appropriate accuracy and precision. Free of derivatization, simple sample procedure and application of automation system significantly improves throughput of the assay which are suitable for routine serum monosaccharides assay in clinical laboratories and for large biomonitoring studies. Presentation: No date and time listed