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Abstract INTRODUCTION
Nucleotide-activated sugars play pivotal roles in various biochemical processes, including glycosylation of proteins and lipids. They are also involved in the interaction and communication of cells and act as extracellular mediators of inflammation. Particularly, uridine diphosphate glucose (UDP-Glc) serve as precursor for other nucleotide sugars and as sugar donor in the synthesis of glycogen and glycolipids as well as its stereoisomer, uridine diphosphate galactose (UDP-Gal). Despite their importance in physiological and pathological processes and their potential as biomarkers, nucleotide-activated sugar composition in biological samples remains poorly studied.
OBJECTIVES
To improve our understanding of metabolic pathways involving nucleotide-activated sugars and diagnosis of genetic glycosylation disorders, clinically validated methods to measure these compounds in biological samples are urgently needed but currently still lacking. In this study, we developed and validated an analytical method to separate and quantify UDP-Glc and UDP-Gal in human plasma samples using ultrahigh-performance liquid chromatography coupled with tadem mass spectrometry (UHPLC-MS/MS). It provides a solid method for clinical analysis of UDP-sugars and can be further expanded easily to cover novel nucleotide-activated sugars in various biological samples.
METHODS
A calibration curve was prepared in human plasma containing UDP-Glc and UDP-Gal at the concentrations of 0.0, 0.5, 1.0, 5.0, 10.0, 50.0, 100 and 200.0 ng/mL. QCs were prepared in plasma at four concentrations: 2.0, 20.0, 80.0, and 160 ng/mL. Calibration standards and QC samples were freshly prepared on each day of the assay. The optimized extraction procedure for plasma samples was developed. Briefly 100 uL of each calibration standard, QC, and human plasma sample were placed in a glass culture tube and 20 µL internal standard (IS) solution (250 ng/mL 13C6-UDP-Glc in H2O) were added. Each sample was then mixed with 80 uL 2% formic acid in H2O (v/v). Samples were loaded on a solid phase extraction (SPE) column (Supelco ENVI-Carb™ SPE column, 100 mg/1 mL), pre-conditioned with 1 mL of elution solution (60% acetonitrile in water containing 0.30% formic acid adjusted to pH 9.0 with ammonia), followed by 1 mL of H2O. The column was washed with 1 mL of H2O. UDP-Glc and UDP-Gal were then eluted with 660 uL of elution solution and collected in glass tubes. The extracts were evaporated to dryness under nitrogen. The residue was reconstituted with 200 uL of H2O, and transferred to autosampler vial for UHPLC-MS/MS analysis. UDP-sugars were separated on a Hypercarb porous graphitic carbon (PGC) LC column (3 um, 50 x 2.1 mm; ThermoFisher Scientific) maintained at 35 °C. Mobile phase A (MPA) consisted of 0.1% FA in water with 20 mM ammonium acetate (pH 9.5), and mobile phase B (MPB) was acetonitrile. The gradient LC method with a the total run time was 20 min with a flow rate of 0.2 mL/min was used. UPHLC-MS/MS analysis was performed on an Acquity UPLC H-class with a quaternary solvent manager (QSM) pump interfaced with a triple quadrupole 7500 mass spectrometer (Sciex, Ontario, Canada) operated in multiple reaction ion monitoring (MRM) mode. Optimized source parameters and MRM settings in negative ionization mode for each analyte and IS were established. Sciex OS software was used for LC-MS/MS system control, data acquisition, and processing.
RESULTS
We optimized the SPE procedure by adapting previous methods reported and improved the recovery by modifying sample pre-processing, loading buffer and washing solutions. Various SPE cartridges with different stationary phases and polarities were tested to improve extraction efficiency. Several HPLC columns and mobile phases were evaluated to enable the detection of nucleotide sugars with high sensitivity while minimizing interference. A porous graphitic carbon (PGC) column was chosen for its performance in separation of structurally related analytes, i.e., UDP-Glc and UDP-Gal. Compared to existing methods, we enhanced UDP-sugar recovery (> 95%) with improved SPE method during sample preparation and achieved a higher chromatographic specificity and consistency. We validated the developed method in accordance with guidelines for bioanalytical method validation by the Food and Drug Administration (FDA) and followed CAP/CLIA guidelines. Importantly, linearity, lower limit of quantification (LLOQ), accuracy and precision, recovery and matrix effects, selectivity, carryover, as well as UDP-Glc and UDP-Gal stability in human plasma at various conditions were examined. Linearity was evaluated by analyzing samples containing UDP-Glc and UDP-Gal at seven different concentrations in triplicate. Calibration curves were constructed by plotting the peak area ratios of analytes to their reference IS against the analyte concentrations. Calibration curve linearity was assessed by the correlation coefficient (R^2) value. Accuracy and precision of the assay were established by analyzing four different levels of QC samples in replicates on five different days. Both inter- and intra- day precision and accuracy results were assessed. Acceptable precision and accuracy for the LLOQ was ≤ 20%, and ≤ 15% for all other calibrators and QCs. Carryover effects were evaluated using a blank sample following the highest calibrator and the peak areas were ≤ 20% than that obtained at the LLOQ. As a proof-of-concept experiment, we applied the developed method to a small group of deidentified pediatric patient plasma samples and estimated the clinical normal range for these compounds.
CONCLUSIONS
A sensitive, validated and reliable MS-based method was developed to profile and quantify UDP-sugars, applicable to biological and clinical matrices and with versatility to be expanded to include other nucleotide-activated sugars. These results confirm the method's robustness, reliability, and suitability for routine bioanalysis in a clinical assay.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
