B-185 High Throughput ID-LCMSMS Method for Simultaneous Quantitation of Monosaccharides in Human Serum

Abstract

Abstract Background Blood glucose is one of the most common clinical tests used for diagnosis and treatment of diabetes, with clinical analyzers being the typical instrumentation used in the clinical laboratory. While glucose is an important monosaccharide for management of diabetes, dietary fructose has shown associations with non-alcoholic fatty liver disease (NAFLD). However, only limited data on serum fructose in the general population are available. Clinical analyzers that can measure glucose and other monosaccharides simultaneously from human serum are unavailable. A high throughput ID-LC/MS/MS method is needed to allow simultaneous quantitation of monosaccharides from human serum to help better understanding the associations of monosaccharides with different diseases. Methods Calibrators, serum samples, and internal standards were transferred and subsequently processed on an automated liquid handler platform. Acetonitrile was added to precipitate monosaccharides from serum matrix and the precipitate was removed through filtration with 96-well protein removal plate. Clean extracts were injected on a LC-MS/MS system using a LC gradient consisting of acetonitrile and water. Quantitation was performed using stable-isotope labeled standards. Certified reference material NIST glucose SRM 917c and commercially available fructose, mannose and galactose were used for calibration. The analytical performance was assessed using NIST glucose SRM 965b serum materials and commercial serum material with monosaccharides spiked at different levels. Results The method demonstrated high level of specificity for glucose, fructose, mannose, and galactose with all monosaccharides being chromatographically baseline-resolved within 9 minutes. No interferences were detected when monitoring the QI/CI ratio in commercial serum materials. The method is highly linear (all R2 = 0.999) between 10–380 mg/dL and 0.04–12 mg/dL for glucose and other monosaccharides, respectively. The limit of detection was 0.19 mg/dL for glucose and 0.02–0.03 mg/dL for galactose, mannose, and fructose. For glucose, the imprecision and bias, determined with NIST SRM 965b measured over 5 days in duplicate, <4%CV and −1.44%, respectively. Inter-day imprecision, determined with samples at concentrations between 0.05 mg/dL to 9 mg/dL, was between 4 and 12%CV for all monosaccharides. The method demonstrated good agreement when compared with the CDC glucose GC-MS RMP with an intercept and slope of −1.7 (CI −4.05 to 0.38) and 1.03 (CI 1.01–1.06), respectively. The Pearson’s coefficient is 0.999. This method allows for measurement of 85 samples in one batch in a semi-automated manner. Conclusion The described high throughput LC/MS/MS method for simultaneous quantitation of four monosaccharides is sufficiently sensitive, selective, accurate and precise for measuring monosaccharides in epidemiological studies. Its suitability as comparator method for method comparison studies with regular clinical analyzers and point-of-care devices are currently ongoing.