A validated LC–MS/MS method for the determination of homocysteic acid in biological samples

Abstract

The role of homocysteic acid (HCA) in severe diseases like Alzheimer’s disease is under discussion and some recent studies correlate elevated HCA concentrations with the diagnosis of Alzheimer’s. However, non-selective and insufficiently sensitive methods have been used to quantitate HCA and results of different studies show large differences in the determined HCA concentration in samples from patients and controls, and therefore non-comparable results.An accurate and precise quantitation method for the determination of HCA in human serum, urine and CSF has been developed by using a combination of protein precipitation and solid phase extraction for sample preparation followed by an LC–MS/MS analysis using a combination of a HILIC separation and tandem mass spectrometry. The developed method has been fully validated in accordance with the guidelines provided by the US Food and Drug administration FDA and the European Medicines Agency EMA. Furthermore, the method has demonstrated its ability to determine the endogenous HCA concentration in serum and urine samples from healthy volunteers.