BACKGROUND: Reports of the use ofmultiplex enzyme as-

disease,Niemann-Pick disease typesA andB, andKrabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories. METHODS: We optimized enzyme reaction conditions and procedures for the assay, including the concentra-tions of substrate (S) and internal standard (IS), assay cocktail compositions, sample clean-up procedures, and mass spectrometer operation. The S and IS for each en-zyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay accord-ing to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60patientswith a lyso