Analysis of Globotriaosylceramide (Gb 3 ) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb 3 ). Accumulation of Gb 3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb 3 and creatinine. Samples are diluted with an internal standard solution containing Gb 3 (C17:0) and creatinine-D 3 , centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb 3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb 3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb 3 . Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb 3 /creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb 3 ) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb 3 calibration curve Support Protocol 3: Preparation of the Gb 3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.