Simultaneous Determination of Lyso-Gb1 and Lyso-Gb3 in Plasma Using Salt-Assisted Liquid-Liquid Extraction Combined with LC-MS/MS.

Statistical analysis demonstrated a highly significant difference between the two groups ( p < 0.0001).

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The 5 min chromatographic run and subng/mL LOQs (Lyso-Gb1 0.380 ng/mL; Lyso-Gb3 0.756 ng/mL) met all ICH M10 criteria, with consistent accuracy/precision, stability, carry-over, dilution integrity, and reinjection performance. Clinically, the assay demonstrated a clear trend of separation between Fabry patients and healthy controls and showed excellent ROC performance (AUC = 1.000) with pragmatic cutoffs (5.80 ng/mL for Lyso-Gb1; 3.334 ng/mL for Lyso-Gb3), consistent with ranges reported in previous studies.

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