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 composition, 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 thenew S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with lysosoma