title: LC-MS/MS analysis of plasma lyso-Gb3 in Fabry disease.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  A robust LC-MS/MS methodology is presented for plasma lyso-Gb(3) quantification.
authors.affiliation: Service of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Boutin
authors.forename: René
authors.initials: R
authors.lastname: Gagnon
authors.forename: Pamela
authors.initials: P
authors.lastname: Lavoie
authors.forename: Christiane
authors.initials: C
authors.lastname: Auray-Blais
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002853:Chromatography, Liquid; D000795:Fabry Disease / Q000097:blood*; D005260:Female; D006017:Glycolipids / Q000097:blood* / Q000737:chemistry; D006801:Humans; D008297:Male; D008875:Middle Aged; D015394:Molecular Structure; D013107:Sphingolipids / Q000097:blood* / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D055815:Young Adult
chemical_list: D006017:Glycolipids; D013107:Sphingolipids; C063288:globotriaosyl lysosphingolipid