title: High-throughput assay of 9 lysosomal enzymes for newborn screening.
abstract: BACKGROUND
  There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS).
  
  
  METHODS
  The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode.
  
  
  RESULTS
  Analysis of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories.
  
  
  CONCLUSIONS
  HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.
authors: Department of Chemistry, University of Washington, Seattle, WA, USA.
authors: Zdenek
authors: Z
authors: Spacil
authors: Haribabu
authors: H
authors: Tatipaka
authors: Mariana
authors: M
authors: Barcenas
authors: C Ronald
authors: CR
authors: Scott
authors: Frantisek
authors: F
authors: Turecek
authors: Michael H
authors: MH
authors: Gelb
mesh_terms: D002851:Chromatography, High Pressure Liquid; D057166:High-Throughput Screening Assays; D006801:Humans; D007231:Infant, Newborn; D016464:Lysosomal Storage Diseases / Q000175:diagnosis*; D008247:Lysosomes / Q000201:enzymology*; D015997:Neonatal Screening; D053719:Tandem Mass Spectrometry