title: Quantification of free sialic acid in urine by HPLC-electrospray tandem mass spectrometry: a tool for the diagnosis of sialic acid storage disease.
abstract: BACKGROUND
  Sialic acid storage diseases (SSDs) are severe autosomal recessive neurodegenerative disorders caused by a transport defect across the lysosomal membrane, which leads to accumulation of sialic acid in tissues, fibroblasts, and urine. Defective free sialic acid transport can be established by quantification of free sialic acid in urine.
  
  
  METHODS
  Urine sample size was adjusted to the equivalent of 100 nmol of creatinine. After addition of 2-keto-3-deoxy-d-glycero-d-galactonononic acid as internal standard, samples were diluted with water to an end volume of 250 microL. We used 10 microL for HPLC-tandem mass spectrometric analysis in the negative electrospray ionization mode, monitoring transitions m/z 308.3-->m/z 86.9 (sialic acid) and m/z 267.2-->m/z 86.9 (internal standard). The overall method was validated and studied for ion suppression, interfering compounds, and pH effects. Samples from controls (n = 72) and SSD patients (n = 3) were analyzed.
  
  
  RESULTS
  The limit of detection was 3 micromol/L. Intraassay imprecision (CV; n = 10) was 6%, 3%, and 2% at 30, 130, and 1000 mmol/mol creatinine, respectively; corresponding interassay CV (n = 10) were 5%, 5%, and 2%. Recovery was 109% (100-1000 mmol/mol creatinine). The mean (SD) [range] excretion rates (mmol/mol creatinine) were 31.3 (16.6) [0.7-56.9] at 0-1 year (n = 20), 21.2 (9.8) [6.3-38.3] at 1-3 years (n = 15), 14.4 (8.2) [1.7-32.9] at 3-10 years (n = 25), and 4.6 (2.6) [0-9.8] above age 10 years (n = 12). SSD patients 1.2, 3.9, and 12 years of age had concentrations of 111.5, 54.2, and 36.1 mmol/mol creatinine, respectively.
  
  
  CONCLUSIONS
  The HPLC-tandem MS method for free sialic acid in urine is more rapid, accurate, sensitive, selective, and robust than earlier methods and may serve as a candidate reference method for free sialic acid in diagnosis of SSD.
authors.affiliation: Academic Medical Center, University of Amsterdam, Emma Children's Hospital and Department of Clinical Chemistry, Amsterdam, The Netherlands.
authors.forename: Fredoen
authors.initials: F
authors.lastname: Valianpour
authors.forename: Nicolaas G G M
authors.initials: NG
authors.lastname: Abeling
authors.forename: Marinus
authors.initials: M
authors.lastname: Duran
authors.forename: Jan G M
authors.initials: JG
authors.lastname: Huijmans
authors.forename: Willem
authors.initials: W
authors.lastname: Kulik
mesh_terms: D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D019158:N-Acetylneuraminic Acid / Q000652:urine*; D015203:Reproducibility of Results; D029461:Sialic Acid Storage Disease / Q000175:diagnosis*; D021241:Spectrometry, Mass, Electrospray Ionization; D013400:Sugar Acids / Q000652:urine
chemical_list: D013400:Sugar Acids; C050008:3-deoxyglycero-galacto-nonulosonic acid; D019158:N-Acetylneuraminic Acid