MSACL 2023 Abstract

Simultaneous Quantification of Monosaccharides in Human Serum: Development of a High-Throughput Derivatization-free ID-LC-MS/MS Method

Chui Y. Tse, Li Zhang, Komal Dahya, Fidelia Pokuah, Otoe Sugahara, Uliana Danilenko, and Hubert W. Vesper
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA, 30341

Chui Tse, MS (Presenter) Centers for Disease Control and Prevention

Relevant Financial Disclosures (within past 24 months, reported on Feb 17, 2023) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Monosaccharides play an important role in human metabolism. While glucose is the major biomarker for diabetes diagnosis and treatment, emerging evidence has suggested relationships between monosaccharides and other diseases. Elevated mannose levels in blood have shown association with insulin resistance in diabetic patients. High fructose consumption has been associated with increased de novo lipogenesis in the liver leading to non-alcoholic fatty liver disease (NAFLD). Simultaneous measurement of glucose, fructose, mannose, and other monosaccharides may be helpful to understand their roles in different diseases.

OBJECTIVES: Limited data is available about levels of different monosaccharides in the population. Although some enzymatic assay kits can measure a few monosaccharides simultaneously, the analytical variance in quantification of these monosaccharides with different concentrations can be high. GC/MS methods often require lengthy sample preparation which can significantly impact the throughput. To address the limitations, there is a need for an accurate and precise laboratory method for measuring serum monosaccharide panels. We aim to develop a high-throughput analytical LC/MS/MS method for serum monosaccharides measurement that is suitable for large scale epidemiologic studies.

METHODS: The assay was calibrated using certified primary reference material NIST SRM 917c for glucose. The 13C labeled monosaccharides were spiked into serum samples and calibrators as internal standards. Using an automated liquid handler system, acetonitrile was added to precipitate monosaccharides from serum matrix and extracts were cleaned up in a 96-well protein removal plate. Monosaccharides from the samples were quantitated using a LC/MS/MS system with acetonitrile and water LC gradient.

RESULTS: Monosaccharides, including glucose, fructose, mannose, and galactose, were fully resolved under 9 minutes demonstrating a high level of specificity. The analytical measurement range for glucose covered 10 – 380 mg/dL (R2 = 0.999). Other monosaccharides were linear between measurement range of 0.3 – 12.2 mg/dL (R2 = 0.999). The assay sensitivity allowed detection of 0.27 mg/dL for glucose and 0.025 – 0.046 mg/dL for other monosaccharides. Average measurement accuracy of 97% and imprecision of 3% for serum glucose were achieved using serum-based reference material NIST SRM 965b across 8 days measurement. Spiked recovery and inter-day imprecision of other monosaccharides were above 95% and between 3-8%, respectively. Comparison with the CDC glucose GC-MS RMP using 19 single donor serum samples (glucose concentration range from 23.11 to 376.53 mg/dL) showed good agreement from Deming regression analysis. The CV% of the IS-norm MF was between 3-12% across 6 serum samples demonstrating minimal influence of the matrix on all monosaccharides analysis. Relative ME% calculated by comparison of the CV% values of standard line slopes across four different serum lot samples is less than 2% for all analytes which served as a measure of minimum relative ME between different subjects. A preliminary study of 83 human serum samples were analyzed with detection of glucose, fructose, and mannose from all samples. A statistically significant positive correlation (r=0.58; P < 0.001) was observed between glucose and mannose levels with both monosaccharides normally distributed across the population while no correlation was observed neither between glucose and fructose, nor mannose and fructose.

CONCLUSION: The current analytical method is sufficiently accurate and precise to measure concentration levels of a panel of serum monosaccharides from human serum. Using a simple and derivatization free sample procedure with automation system has significantly improved the throughput of the assay for routine serum monosaccharides measurements in clinical laboratories and for large biomonitoring studies.