Development of Glycosaminoglycans Analysis Method for Dried blood spot as a Second-tier Test in Mucopolysaccharidoses Screening

Significance of the Topic

The accumulation of undegraded glycosaminoglycans (GAGs) underlies the pathology of mucopolysaccharidoses (MPS), leading to progressive multisystem damage. Conventional newborn screening based on lysosomal enzyme activity often yields false positives due to pseudodeficiency. Implementing a robust second-tier GAG assay using dried blood spots (DBS) can improve diagnostic specificity, reduce unnecessary follow-up testing, and expedite early interventions.

Objectives and Study Overview

This study aimed to develop and validate an LC-MS/MS method for quantifying signature GAG disaccharides from DBS. Key goals were to optimize enzymatic digestion conditions, establish chromatographic and mass spectrometric parameters, and assess method performance in healthy newborns and confirmed MPS II patients.

Methodology

Sample Preparation

• Two 3 mm DBS punches were extracted with water and subjected to incubation with a mixture of chondroitinase B, heparitinase, and keratanase II in Tris-HCl buffer containing BSA and internal standards.
• After enzymatic digestion, samples were precipitated with methanol, centrifuged, and supernatants collected for analysis.

Used Instrumentation

UHPLC system: Shimadzu Nexera X3 with amide-based HILIC column at 40 °C, flow rate 0.3 mL/min and gradient elution using ammonium formate buffers.
Mass spectrometer: Shimadzu LCMS-8060 triple quadrupole in negative ESI MRM mode, interface temp 300 °C, DL temp 250 °C, heat block 400 °C, nebulizing gas 3 L/min, drying gas 10 L/min, heating gas 10 L/min.

Main Results and Discussion

The method quantified five target disaccharides: Di-4S (dermatan sulfate), DiHS-0S and DiHS-NS (heparan sulfate), and mono- and di-sulfated keratan sulfate. Calibration curves showed excellent linearity (R>0.994), with LLOQs of 25 ng/mL for most analytes and repeatability %RSD below 5 % for retention times and peak areas. In analysis of 100 healthy newborns versus three MPS II patients, heparan sulfate markers were significantly elevated in patient samples, distinguishing affected individuals from controls.

Practical Benefits and Applications

The proposed second-tier GAG assay offers:

• High specificity to reduce false positives in MPS newborn screening.
• Minimal DBS sample requirement and streamlined workflow.
• Quantitative data supporting clinical decision making for confirmatory testing.

Future Trends and Potential Uses

Integration of this assay into multiplexed screening platforms could expand coverage to other lysosomal storage disorders. Advances in high-throughput robotics and data analytics may further shorten turnaround times and improve cost-effectiveness. Emerging mass spectrometry technologies could enable panel expansion to additional biomarkers, enhancing newborn screening programs worldwide.

Conclusion

A novel LC-MS/MS method for GAG disaccharide quantification from DBS has been established with robust performance. The assay reliably differentiates MPS II patients from healthy newborns and serves as an effective second-tier test to enhance screening accuracy and patient care.