Determination of catecholamines and their metabolites in rat urine by ultra-performance liquid chromatography-tandem mass spectrometry for the study of identifying potential markers for Alzheimer's disease

Abstract

In order to investigate the potential links between catecholamines (CAs) and Alzheimer's disease (AD), rapid and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) methods in different ionization modes for the quantification of 14 CAs and their metabolites in rat urine without derivatization or complex sample pre-treatments were developed. After addition of the internal standard, isoproterenol, the urine samples were extracted by protein precipitation and separated on an Inertsil ODS-EP column (Shimadzu, Japan) at a flow of 1.0 ml min(-1). Tandem mass spectrometric detection was performed on a 4000Q UPLC-MS/MS in the multiple reaction monitoring mode with turbo ion spray source. Tyrosine, dopamine, noradrenaline, epinephrine, 3-methoxytyramine, normetanephrine and metanephrine were determined in positive mode, while 3,4-dihyroxy-L-phenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid, DL-3,4-dihydroxymandelic acid, DL-3,4-dihydroxyphenyl glycol, homovanillic acid, DL-4-hydroxy-3-methoxymandelic acid and 4-hydroxy-3-methoxy-phenylglycol were determined in negative mode. The methods were examined and were found to be precise and accurate within the linearity range of the assays. The intra-day and inter-day precision and accuracy of the analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard were all more than 60%. The validated methods have been successfully applied to compare CAs profiles in normal and AD rats. The results indicated the urine levels of DL-3,4-dihydroxyphenyl glycol and 4-hydroxy-3-methoxy-phenylglycol in AD rats were significantly higher than those in the normal group, and the other CAs have an opposite performance. These may attribute to the difference of some enzyme activity between rats with AD and normal. Furthermore, this may be helpful in clinical diagnostics and monitor the efficacy of AD treatment.

Keywords: Alzheimer's disease; UPLC-MS/MS; biomarkers; catecholamines; urine.