MSACL 2025 Abstract

Quantitative Sulfatide Measurement in Body Fluids: LC Optimization and Investigation of Non-Specific Adsorption

Kaitlyn King (1) and Xinying Hong (1, 2)
(1) Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA (2) Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Kaitlyn King, BSc - Biochemistry (Presenter) The Children's Hospital of Philadelphia

Relevant Financial Disclosures (within past 24 months, reported on Mar 18, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Background:
Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder caused by a deficiency in arylsulfatase A, leading to the accumulation of sulfatides in white matter. This accumulation results in progressive demyelination and neurodegeneration. Biochemically, MLD can be diagnosed based on the hyperexcretion of sulfatides in urine. Ex vivo gene therapy for MLD has been approved in US. Therefore, there is an urgent need to establish quantitative urinary sulfatide analysis for diagnosis and prognosis. Its measurement in CSF is also imperative for monitoring treatment efficacy.

Problem:
Previous studies have shown that the response of sulfatides is not consistent across multiple injections. Additionally, it has been reported that sulfatides are unstable after more than two freeze-thaw cycles. These findings have prompted us to investigate the underlying causes before proceeding with analytical and clinical validation.

Method Information:
We developed a UPLC-MS/MS assay to quantify 25 sulfatide species in urine and CSF.
• 20 uL urine or CSF extracted with 300 uL MeOH solution containing 3 isotope-labeled sulfatides as internal standards (IS)
• Waters ACQUITY UPLC coupled with TQ Absolute tandem mass spectrometer
• MPA: 50:50 Water:Acetonitrile, 0.1% formic acid
• MPB: 80:20 Acetonitrile:Isopropanol, 0.1% formic acid
• 7 min gradient with 0.7 mL/min flow rate
• Column: ACQUITY UPLC BEH C8 Column (1.7μm, 2.1mm x 50mm)
• Column temperature: 40 °C
• Injection volume: 10 uL
• Quantitative towards C16, C18, C24, C24:1 sulfatides. Semi-quantitative towards other
sulfatide species

Troubleshooting Steps:
We hypothesized that the unstable MS/MS response was due to the suboptimal LC condition. We conducted the LC-MS/MS analysis using two columns: the CORTECS C18 column and the ACQUITY UPLC BEH C8 column. Sulfatide samples in neat solution were repeatedly injected on both columns.
We hypothesized that the instability of sulfatide observed after multiple freeze-thaw cycles was primarily due to non-specific adsorption, rather than degradation of the sulfatide itself. This is
attributed to the high stability of sulfatide, particularly under neutral conditions, as well as the inactivity of the only sulfatide-degrading enzyme, ARSA, at neutral pH. Standard polypropylene and polystyrene containers used for the collection of urine and CSF create conditions for non- specific adsorption of analytes due to their electrostatic and hydrophobic properties. Furthermore, these matrices contain lower concentrations of proteins and lipids, which increases the possibility of non-specific adsorption during sample collection and processing. The number of freeze-thaw cycles the sample undergoes is another factor that could impact the degree of adsorption, which is an important consideration for sample storage conditions in a clinical setting.

We assessed potential non-specific adsorption in different matrices by performing sequential transfer and freeze-thaw experiments. Samples were tested in triplicate at three levels: baseline (no spike), low-level spike, and high-level spike. Standard mixes were spiked into pooled non- affected specimens, and each level was transferred across five separate primary sample collection containers for each matrix, followed by ten freeze-thaw cycles. Extractions were performed at transfers 1, 3, 5 and freeze-thaw cycles 1, 3, 5, and 10. Data were analyzed and compared across extractions for each matrix.

Results:
The sulfatide response, after normalization to the internal standard (IS), was found to be unstable when analyzed on the CORTECS C18 column. A 15–40% increase in response was observed after 50 injections. Surprisingly, the use of isotope-labeled IS did not mitigate this issue. On the contrary, the response was stable on the BEH C8 column across 200 injections.
We observed a significant decrease in sulfatide levels with each subsequent transfer in both urine and CSF collection containers. There was no evidence of analyte loss from multiple freeze-thaw cycles in either matrix.

Outcome:
Switching to the C8 column has stabilized the response, even for sulfatide species without an isotope-labeled internal standard. The non-specific adsorption observed in all tested collection containers for urine and CSF suggests that the addition of a blocking agent is critical for clinical diagnostics. Further studies are required to determine whether similar losses occur in plasma. If minimal loss is observed in plasma, bovine serum albumin (BSA) may be considered as a potential blocking agent due to its ability to compete for analyte binding. Alternatively, hexadecylpyridinium chloride monohydrate (HDP) should be tested as a blocking agent if plasma sulfatide loss is observed.