Development and fit for purpose validation of a quantitative LC-MS/MS method for heparan sulfate in cerebrospinal fluid as a biomarker for mucopolysaccharidosis type IIIA

Abstract Background Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome) is a fatal neurodegenerative lysosomal storage disorder caused by impaired degradation of heparan sulfate (HS). Despite rapid advances in gene and enzyme therapies, there remains a critical need for an analytically validated, quantitative biomarker that accurately reflects central nervous system (CNS) substrate burden. Such biomarker would be a valuable tool in assessing disease progression and monitoring therapeutic efficacy. Objective This study describes the method development, fit for purpose validation, and preliminary clinical application of a quantitative liquid chromatography−mass spectrometry (LC−MS/MS) assay for the HS-derived disaccharide N-sulfoglucosamine–glucuronic acid (GlcNS−GlcUA) in human cerebrospinal fluid (CSF), a critical biomarker for diagnosis, disease monitoring, and regulatory evaluation of emerging MPS IIIA therapies. Methods A structurally defined GlcNS−GlcUA reference standard and its [ 13 C 6 ]-labeled internal standard were used in a derivatization and detection workflow employing 1-phenyl-3-methyl-5-pyrazolone labeling, and LC-MS/MS. Results The method exhibited acceptable linearity across 0.005−0.500 nmol/mL (r ≥0.9976), with intra- and inter-assay imprecision ≤3.5%CV and accuracy within 95%−110% of nominal concentrations. No matrix or hemolysis interference or carryover was observed, and the analyte remained stable during freeze-thaw storage conditions. Application of the method to 12 CSF samples from patients with MPS IIIA demonstrated quantifiable GlcNS−GlcUA levels ranging from 0.0054 to 0.106 nmol/mL, confirming suitability for clinical and regulatory use. Comparison of the MPS IIIA sample results between the development laboratory and the contract research organization laboratory support robust inter-lab assay transfer. Conclusions This validated LC–MS/MS method establishes a regulatory-grade quantitative assay for measurement of CSF HS in MPS IIIA. Its high analytical sensitivity and reproducibility enable reliable assessment of CNS substrate reduction and pharmacodynamic response, supporting biomarker-driven therapeutic development and accelerated approval pathways for neuronopathic mucopolysaccharidoses.