MSACL 2019 EU Abstract

Advancement of the Quantitative Measurement of Enzyme Activities in Six Lysosomal Storage Disorders via LC-MSMS

Mietha M. van der Walt; Mari van Reenen; Leréze Marais; Marli Dercksen; Barend C. Vorster; Peet J. Jansen van Rensburg
Human Metabolomics, North-West University, Potchefstroom, South Africa

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Mietha Magdalena van der Walt (Presenter) Human Metabolomics, North-West University, SA

Presenter Bio: Dr. Mietha Magdalena van der Walt is currently the Principal Investigator for the diagnostic platform for treatable Lysosomal Storage Disorders (LSDs) in South Africa, at the Centre for Human Metabolomics (CHM), North-West University. She graduated from the North-West University in 2008 with a Bachelor of Pharmacy degree (B.Pharm). She received a Doctor of Philosophy (PhD.) and Masters of Science (MSc.) degree during 2013 and 2010, respectively, both in Pharmaceutical Chemistry at the North-West University. Dr. van der Walt’s current focus is aimed at inborn errors of metabolism (IEM), specifically in the field of LSDs. She is a rated scientist at the National Research Foundation (NRF) of South Africa and over the past 6 years has contributed innovative research to the advancement in the field of Medicinal Chemistry, which is supported by her publication record & conference proceedings.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: Since treatment advances for lysosomal storage disorders (LSDs), the application of mass spectrometry (MS) techniques have expanded to screening for some of the treatable LSDs. To date, flow injection MS (FI-MS) is generally the preferred screening technique to be of diagnostic value for 6 LSDs, namely Pompe, MPS-I, Fabry, Gaucher, Niemann-Pick A/B and Krabbe disorders, from a single dried blood spot (DBS) sample. We evaluated the analytical performance and diagnostic precision of a 6-plex LSD enzyme assay utilizing the technique of liquid chromatography with tandem MS (LC-MS/MS) within the clinical laboratory setting.
Methods: The LC-MS/MS method is intended for quantitative measurement of 6 individual LSD associated enzymatic products. Quality control (QC) DBS samples were obtained from the Centers for Disease Control and Prevention (CDC) and the Perkin Elmer NeoLSD MSMS kit. The selected QC samples are associated with a range of enzyme product concentrations that span the medical decision limit (MDL) and normal range as best possible. Enzyme activities are obtained by measuring the products formed when enzymes react with synthesized substrates to create specific products. Detection of the enzyme products was performed with an Agilent 6470 Triple Quadrupole LC-MS/MS equipped with an Agilent JetStream technology ESI source (operated in positive ion mode). Chromatographic separation was achieved using a reverse-phase C18 column and a gradient flow rate of 0.40 mL/min. Dynamic multiple reaction monitoring over an 8 minute run time was performed. In addition, an FI-MS method was implemented for comparison to the LC-MS/MS method. The analytical MS methods have been evaluating by linearity, linear range and precision.
Results: Compared to the FI-MS method, the LC-MS/MS method was generally found to be more linear (e.g. LC-MS/MS R2 0.99 – 0.97 vs FI-MS R2 0.97 – 0.85) with a wider linear range for all analytes of interest. Taking CVs into account, near the MDL, the LC-MS/MS method was overall more precise (e.g. LC-MS/MS CV 2% – 16% vs FI-MS CV 2% – 44%) than the FI-MS method. The LC-MS/MS method repeatedly showed comparable results to other laboratories in the CDC proficiency scheme. In addition, two metabolites were observed with the same transitions, but different retention times, than two of the target LSD enzymatic products. This may result in an over-estimation of enzyme products, specifically when reporting enzyme activity for Gaucher and Krabbe disease without applying LC.
Conclusion: We report an LC-MS/MS method for the analysis of 6 LSD enzymatic products, in a single DBS. Emphasized are the advantages of LC-MS/MS, including high selectivity leading to precise quantification of enzyme products and subsequent enzyme activity. This improved method displays good analytical performance and meets clinical laboratory requirements. In summary, the LC-MS/MS method is proposed as an alternative to the standard FI-MS procedure.