A column-switching HPLC-MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disorders.
Lysosomal storage disorders comprise a group of rare genetic diseases in which a deficit of specific hydrolases leads to the storage of undegraded substrates in lysosomes. Impaired enzyme activities can be assessed by MS/MS quantification of the reaction products obtained after incubation with specific substrates. In this study, a column-switching HPLC-MS/MS method for multiplex screening in dried blood spot of the lysosomal enzymes activities was developed. Mucopolysaccharidosis type I, Fabry, Gaucher, Krabbe, Niemann-Pick A/B and Pompe diseases were simultaneously assayed. Dried blood spots were incubated with substrates and internal standards; thereafter, supernatants were collected with minor manipulations. Samples were injected, trapped into an online perfusion column and, by a six-port valve, switched online through the C18 analytical column to perform separation of metabolites followed by MS/MS analysis. A total of 1136 de-identified newborn screening samples were analyzed to determine references for enzymes activity values. As positive controls, we analyzed dried blood spots from three patients with Pompe, one with Fabry, one with Krabbe disease and two with MPS I, and in all cases the enzyme activities were below the cutoff values measured for newborns, except for an MPS I patient after successful hematopoietic stem cell transplantation.
Mass Spectrometry Laboratory, Department of Women's and Children's Health, University of Padova, Italy.
Antonina
A
Gucciardi
Elisa
E
Legnini
Iole Maria
IM
Di Gangi
Carlo
C
Corbetta
Rosella
R
Tomanin
Maurizio
M
Scarpa
Giuseppe
G
Giordano
D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D059788:Dried Blood Spot Testing / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods; D006801:Humans; D007068:Iduronidase / Q000097:blood / Q000378:metabolism; D007231:Infant, Newborn; D016014:Linear Models; D016464:Lysosomal Storage Diseases / Q000175:diagnosis* / Q000201:enzymology; D008059:Mucopolysaccharidosis I / Q000175:diagnosis* / Q000201:enzymology; D015997:Neonatal Screening / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods
D007068:Iduronidase
MPS I; column-switching HPLC/MS; dried blood spot; lysosomal storage disorders; newborn screening