The application of multiplexed, multi‐dimensional ultra‐high‐performance liquid chromatography/tandem mass spectrometry to the high‐throughput screening of lysosomal storage disorders in newborn dried bloodspots

Abstract

Lysozomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the analytical challenges associated with published assays including complex sample preparation and potential interference from excess residual substrate. Solutions to these challenges are presented in the form of on-line two-dimensional chromatography to eliminate off-line liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the use of ultra-high-performance liquid chromatography (UHPLC) to separate excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method.

Newborn Screening for Lysosomal Storage Disorders in Hungary

“…An experienced operator can perform the cleanup of 4,500 samples, the daily workload of an average size screening laboratory, within 4-6 h. Recent introduction of online HPLC sample cleanup technology would save the time-consuming cleanup procedure as well as consumables but requires longer analysis time (LaMarca et al 2009) or more sophisticated equipment (Kasper et al 2010). However, using online HPLC cleanup on MS/MS, the instrument very likely can be used only for LSD screening and is blocked for other analysis.…”

Simplified Newborn Screening Protocol for Lysosomal Storage Disorders

“…This expansion has practical value because new substrates for the screening for MPS II, IVA, and VI are on the horizon (4 -7 ). To illustrate this point, we integrated the screening of GALC activity with our published pentaplex assay (18 ) and evaluated the resulting GALC activities in collaboration with the New York screening laboratory by performing parallel measurements. Although our GALC enzyme activities were lower than those measured by the New York laboratory, we could clearly differentiate Simplified Screening for Lysosomal Storage Disorders the affected Krabbe patient and healthy newborns [patientЈs GALC activity was below the cutoff for retesting of Ͻ12% according to Orsini et al (9 )].…”

“…We recently introduced a method for screening for LSDs that uses Turboflow chromatography technology (18 ). In the current study, we modified and optimized the system for high-throughput screening of 6 different LSDs in a single assay.…”

Tandem Mass Spectrometry of Sphingolipids: Application in Metabolic Studies and Diagnosis of Inherited Disorders of Sphingolipid Metabolism

“…Enzymology, in combination with tandem mass spectrometry, is useful in lysosomal storage disorders screening and in evaluations of enzyme activity (Kasper, et al, 2010;Li, et al, 2004;Spacil, et al, 2011;Turecek, et al, 2007). One practical application of this technique is the analysis of lysosomal -glucocerebrosidase activity using glucosylceramide with C12:0 fatty acid as the enzyme substrate.…”

“…Instead, HPLC is combined with mass spectrometry. The HPLC step purifies the sample, removing any potential interfering compounds (Kasper, et al, 2010;Spacil, et al, 2011).…”