Development and Validation of a Multiplex LC-MS/MS Assay for Lysosphingolipid Quantification in the Diagnosis of Gaucher and Fabry Diseases

İngilizce (OpenAlex)

Objectives: Lysosphingolipids (LysoSLs), the N-deacylated forms of sphingolipids, are increasingly recognized as toxic metabolites that accumulate in various lysosomal storage disorders (LSDs), collectively known as sphingolipidoses.These compounds, due to their amphiphilic nature and bioactive properties, can contribute to cellular dysfunction and disease pathogenesis.Recent studies have demonstrated that specific LysoSLs, such as hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), and lysosphingomyelin (LysoSM), are elevated in plasma, serum, or dried blood spots from patients with Gaucher, Fabry, Krabbe, or Niemann-Pick diseases.These findings have paved the way for the use of LysoSLs as sensitive and specific biomarkers in the diagnosis, prognosis, and therapeutic monitoring of sphingolipidoses.This study aimed to develop and validate a method for the simultaneous quantification of three LysoSLs-HexSph, LysoGb3, and LysoSM-using liquid chromatography-tandem mass spectrometry (LC-MS/MS), for use in the diagnosis and follow-up of sphingolipidoses.Materials and Methods: A multiplex analytical method was optimized for quantifying HexSph, LysoGb3, and LysoSM by ultra-fast LC coupled to tandem MS (UFLC-MS/MS) in positive-ion mode with electrospray ionization (LC-20 AD UFLC XR; Shimadzu 8040, Japan), employing multiple reaction monitoring.The method was validated for linearity, accuracy, precision, limit of detection, limit of quantification, and recovery rates.Following method validation, plasma samples were collected from 14 patients diagnosed with Gaucher disease or Fabry disease at Akdeniz University Hospital and from 20 healthy adult volunteers.Sample preparation involved methanol/acetone/water extraction prior to analysis of LysoSL concentrations.Results: The total analysis time for all three analytes was determined to be 10 minutes.Calibration curves demonstrated strong linearity, with coefficients of determination (r²) of 0.995 for LysoSM, 0.982 for HexSph, and 0.961 for LysoGb3.Plasma concentrations of LysoSLs were measured in both the patient and control groups.Significantly elevated levels of LysoSM and HexSph were observed in Gaucher patients compared to healthy controls.While LysoGb3 levels were higher in Fabry patients than in controls, the difference did not reach statistical significance.Conclusion: This validated multiplex LC-MS/MS assay enables rapid and reliable simultaneous quantification of key LysoSLs in plasma.The assay holds promise as a diagnostic and monitoring tool for sphingolipidoses and may be further expanded to include additional biomarkers relevant to other LSDs.Quantification of LysoSLs can support clinical decision-making, including therapeutic selection and monitoring of patients undergoing enzyme replacement therapy.