A Start: A Novel Approach Using LC-MS/MS for the Diagnosis of MCADD

Abstract

The purpose of this report is to inform its readers on the progress made on developing a more reliable diagnosis method for medium-chain acetyl-CoA dehydrogenase deficiency (MCADD), a commnly inhereted fatty acid oxidation disorder, with the goal of adapting the current high pressure liquid chromatography (HPLC) method to a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) model. Firstly, the enzyme reaction was established for LC-MS/MS analysis with the metabolites phenylpropanoyl-CoA and (PP-CoA) and cinnamoyl-CoA (C-CoA) in MilliQ. Based on the molecular weight, fragmentation and charge both metabolites were tuned, and a multiple reaction monitoring (MRM) quantitation method was formed and optimized. The solution of pure metabolites in MilliQ was used repeatedly to optimize the peak formation and retention time until stable quality and reproduction of peaks was reached using the Luna ® Omega column. The existing HPLC methodology for MCAD enzyme activity was used as a starting point for the enzyme function assay. From there on out, using literature and experimentation, the rest of the road was paved. Exchanging non-MS friendly elements of the HPLC method, namely hydrochloric acid, triethylamine, and di-potassium hydrogen phosphate for their more volatile MS friendly counterparts – room temperature acetonitrile and ammonium bicarbonate. Additionally, to increase the formation of C-CoA, the incubation time of the enzyme assay was increased from 10 to 20 minutes. However, when introducing the biological sample made from isolated leukocytes to the LC-MS/MS we ran into unusable results in the form of splitting peaks due to the matrix effect and possibly interference from ferrocenium hexafluorophosphate and the higher acetonitrile concentration in the analyte solution. This paired with the exchanging and conditioning of a new column, lead to us being unable to finish the new MCADD analysis method.