Sensitive Quantification of GlcSph and GalSph in Human CSF Plasma and Brain

Title: Highly-sensitive simultaneous quantitation of glucosylsphingosine and galactosylsphingosine in human cerebrospinal fluid by liquid chromatography/tandem mass spectrometry

Journal: Journal of Pharmaceutical and Biomedical Analysis

Published: 2022

Background

Mutations in the GBA gene, which encodes glucocerebrosidase (GCase), are associated with Gaucher disease (GD) and are the most common genetic risk factors for Parkinson's disease (PD). Glucosylsphingosine (GlcSph) serves as a pharmacodynamic biomarker for GCase-targeted therapies, while galactosylsphingosine (GalSph, also known as psychosine) is a biomarker for Krabbe disease (KD). Although plasma and brain levels of GlcSph have been measured previously, quantifying GlcSph in cerebrospinal fluid (CSF) from healthy individuals or GBA-PD patients had not been successful due to extremely low concentrations. Moreover, due to their isobaric nature, GalSph can interfere with GlcSph quantification without proper separation. This study developed a highly sensitive LC-MS/MS method capable of quantifying both GlcSph and GalSph in healthy human CSF, plasma, and brain samples, allowing better understanding and monitoring for therapies.

Materials & Methods

Samples: Human CSF, plasma, and brain tissue from healthy donors.

Chemicals: Standards of GlcSph, GalSph, d5-GalSph, and 13C6-GlcSph.

Sample Preparation:

CSF samples were mixed with internal standards and extracted using a cationic exchange solid-phase extraction method.
Plasma and brain homogenates were processed with organic solvent mixtures, centrifuged, evaporated, and reconstituted.

LC-MS/MS Analysis:

A Nexera X2 UHPLC system and tandem mass spectrometers (Qtrap 6500 for CSF; API 5000 for plasma and brain) were used.
Two Halo HILIC columns connected in tandem were employed for better separation in CSF analysis.

Method Validation:

Evaluated linearity, precision, accuracy, matrix effects, recovery, and stability according to standard bioanalytical guidelines.
Lower limit of quantification (LLOQ) was set at 0.1 pg/mL.

Results

Strategy for Quantitation of Endogenous GlcSph and GalSph

The study successfully developed a robust LC-MS/MS method to simultaneously quantify GlcSph and GalSph in human CSF, plasma, and brain samples with high sensitivity and specificity. A major achievement was the effective chromatographic separation of the two isobaric compounds using two HILIC columns in tandem and optimizing the mobile phase (0.1% formic acid), which improved signal-to-noise ratio by more than 10-fold.

The method achieved extremely low limits of quantification (LLOQs): 0.1 pg/mL for CSF, 0.03 ng/mL for plasma, and 0.3 ng/mL for brain homogenates. Calibration curves were linear over a wide range, and precision and accuracy for QC samples met regulatory criteria. GlcSph and GalSph showed excellent stability under various conditions, confirming the method's robustness for clinical and research use.

GlcSph and GalSph Concentrations in CSF, Plasma, and Brain

In CSF and brain, GalSph levels were higher than GlcSph, whereas in plasma, GlcSph was more abundant.
GlcSph concentration in CSF was dramatically lower than in plasma (325-fold) and brain (4364-fold).
Some CSF samples showed artificially high GlcSph concentrations likely due to blood contamination; therefore, only clear, colorless CSF samples were considered valid.
GalSph concentrations in CSF were less affected by blood contamination compared to GlcSph.

Comparison of GlcSph and GalSph Levels in Healthy and Diseased States

Healthy individuals showed very low GlcSph concentrations in CSF (1.07 ± 0.71 pg/mL), in contrast to GD patients, whose levels are approximately 10-fold higher.
These findings support the use of GlcSph as a highly specific biomarker for Gaucher disease.
Mild to moderate accumulation of GlcSph might also occur in GBA-associated Parkinson's disease (GBA-PD), though large cohort studies are needed for confirmation.
GalSph concentration in healthy adult plasma matched previously reported values from newborn DBS studies, suggesting consistency across different biological matrices.
Given the role of GalSph accumulation in Krabbe disease (KD), CSF GalSph measurement could serve as a surrogate for brain GalSph levels in evaluating brain-targeted therapies.

Chromatograms of GlcSph and GalSph in human (A) CSF, (B) plasma, and (C) brain

GlcSph and GalSph concentrations in human (A) CSF, (B) plasma, and (C) brain. The circles represent individual concentrations, while the bars show mean concentrations.

* For Research Use Only. Not for use in diagnostic procedures.

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