LC-MS/MS analysis of plasma lyso-Gb3 in Fabry disease

Clin Chim Acta. 2012 Dec 24;414:273-80. doi: 10.1016/j.cca.2012.09.026. Epub 2012 Oct 2.


Background: Fabry disease is a complex, multisystemic and clinically heterogeneous disease, with elevated excretion of globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)) accumulating in biological fluids caused by deficiency of the enzyme, lysosomal α-galactosidase A. Our aims were to propose a tandem mass spectrometry fragmentation mechanism for lyso-Gb(3), to develop and validate a simple, and robust methodology for the measurement of plasma lyso-Gb(3) using LC-MS/MS in large Fabry cohorts and in controls. Response to treatment was also evaluated.

Method: A solid-phase extraction procedure was used to process plasma samples. The 1-β-D-glucosylsphingosine (GSG) internal standard was chosen for its commercial availability. A liquid chromatography method was devised to allow the co-elution of the GSG internal standard with lyso-Gb(3), thus compensating for system variability and reducing the matrix effect. A multiple reaction monitoring method was developed, working in positive electrospray ionization.

Results: The validation of the method provided good accuracy and precision: intraday and interday biases of less than 8% and 5%, respectively, and intraday and interday CVs of <12% and 7%, respectively. Limit of detection was 0.7 nmol/l and limit of quantification was 2.5 nmol/l. Plasma samples were stable for up to 6h at room temperature, 48 h at 4 °C, and 20 weeks at -20 °C. Regarding untreated Fabry patients, the mean lyso-Gb(3) concentrations were 170 nmol/l for males and 9.7 nmol/l for females, and for treated patients, 40.2 nmol/l for males and 7.5 nmol/l for females.

Conclusion: A robust LC-MS/MS methodology is presented for plasma lyso-Gb(3) quantification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Child
  • Chromatography, Liquid
  • Fabry Disease / blood*
  • Female
  • Glycolipids / blood*
  • Glycolipids / chemistry
  • Humans
  • Male
  • Middle Aged
  • Molecular Structure
  • Sphingolipids / blood*
  • Sphingolipids / chemistry
  • Tandem Mass Spectrometry
  • Young Adult


  • Glycolipids
  • Sphingolipids
  • globotriaosyl lysosphingolipid