|Joseph Herman, ThermoFisher Scientific, Franklin, MA |
Victor DeJesus, Center for Disease Control, Atlanta, GA
Bori Shushan, Clinical Mass Spec Consultants, Toronto, ON
|Lysosomal storage disorders (LSDs) are a group of over 40 genetic disorders caused by inborn errors of metabolism. Individuals with LSDs are characterized by low activities of particular enzymes found in the lysosome, resulting in the accumulation of molecules that are meant to be processed by these enzymes. While LSDs are each rare diseases, as a group they are estimated to affect 1 in 7,700 live births worldwide. Most LSDs are progressive and life threatening; if left untreated, they can cause physical debilitation, mental retardation and early death. Examples of LSDs include Tay-Sachs, Gaucher, Fabry and Pompe disease. Newborn screening assays for the five most common LSDs have been reported using tandem mass spectrometry (MS/MS) to measure the formation of product from incubations of dried blood spots (DBS) (1,2). Both on-line and off-line solid phase extraction (SPE) have been used to reduce matrix effects from the extracts from incubated DBS samples. The off-line SPE is time consuming but allows one minute per sample run times. The on-line SPE reduces sample preparation time but the MS/MS run is four minutes per sample, given that it requires a liquid chromatography step to separate all products and internal standards present in each sample. There is also interference from the substrate with the on-line SPE method that may not be completely removed by the methodology described in the paper (2).|
We describe an on-line clean-up method for LSD newborn screening enzymatic assays using Turbulent Flow Chromatography (TFC) and Ultra High Pressure Liquid Chromatography (UPLC). The proposed sample preparation method allows direct injection of the incubated DBS samples without further purification. The TFC method provides a 1.5 minute per sample analysis time after DBS incubations, and efficiently separates the substrates from the products chromatographically ensuring no cross-talk between substrate and product signals. The assay was validated against pure standards and DBS quality control materials supplied by the Centers for Disease Control and Prevention (CDC).
(1) Li Y., Scott R., Chamoles N.A., Ghavami A., Pinto B.M., Turecek F. and Gelb M.H.; “Direct Multiplex Assay of Lysosomal Enzymes in Dried Blood Spots for Newborn Screening”, Clin Chem 50(10), 1785-1796 (2004)
(2) La Marca G., Casetta B., Malvagia S., Gurrini R., and Zammarchi E.; “New Strategy for the Screening of Lysosomal Storage Disorders: The Use of the Online Trapping-and-Cleanup Liquid Chromatography/Mass Spectrometry”, Anal Chem 81(15), 6113-6121 (2009)