Development and validation of a high-throughput LC-MS/MS method for simultaneous quantification of Lyso-GL1 and Lyso-GL3 in dried blood spots for rare disease screening.

To develop and validate a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of glucosylsphingosine (Lyso-GL1) and Lyso-GL3 (Lyso-GL3) in dried blood spots (DBS). Target analytes were extracted from DBS using methanol-water solution with isotopic internal standards for quantification. Chromatographic separation was performed on a C18 column (2.1 × 50 mm, 1.7 μm) at 50 °C, with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) under gradient elution (0.4 mL/min). Extraction conditions (45 °C, 1300 rpm, 1.5 h) were optimized to enhance recovery, which is a simplified one-step extraction protocol replacing laborious conventional pretreatment methods (e.g., solid-phase extraction). Method validation included linearity, precision, sensitivity, and clinical verification using 20 DBS samples. The limits of quantification (LOQ) for Lyso-GL1 and Lyso-GL3 were 0.5 ng/mL and 0.8 ng/mL (S/N ≥ 10), respectively. Linear ranges were 2.716-51.088 ng/mL (R2 = 0.9997) for Lyso-GL1 and 2.416-49.846 ng/mL (R2 = 0.9991) for Lyso-GL3, with recoveries of 85%-115%, intra-/inter-batch precision CV <15%, and sensitivity down to 0.5 ng/mL. Clinical validation demonstrated robust method stability. This method is the first to apply DBS technology for simultaneous detection of Lyso-GL1 (Gaucher disease biomarker) and Lyso-GL3 (Fabry disease biomarker), featuring simplified sample pre-treatment, enables high-throughput screening for Gaucher and Fabry diseases, leveraging the stability and convenience of dried blood spots for rare disease screening.