title: Novel and sensitive reversed-phase high-pressure liquid chromatography method with electrochemical detection for the simultaneous and fast determination of eight biogenic amines and metabolites in human brain tissue.
abstract: A fast and simple RP-HPLC method with electrochemical detection (ECD) and ion pair chromatography was developed, optimized and validated in order to simultaneously determine eight different biogenic amines and metabolites in post-mortem human brain tissue in a single-run analytical approach. The compounds of interest are the indolamine serotonin (5-hydroxytryptamine, 5-HT), the catecholamines dopamine (DA) and (nor)epinephrine ((N)E), as well as their respective metabolites, i.e. 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), 5-hydroxy-3-indoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). A two-level fractional factorial experimental design was applied to study the effect of five experimental factors (i.e. the ion-pair counter concentration, the level of organic modifier, the pH of the mobile phase, the temperature of the column, and the voltage setting of the detector) on the chromatographic behaviour. The cross effect between the five quantitative factors and the capacity and separation factors of the analytes were then analysed using a Standard Least Squares model. The optimized method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). Our human brain tissue sample preparation procedure is straightforward and relatively short, which allows samples to be loaded onto the HPLC system within approximately 4h. Additionally, a high sample throughput was achieved after optimization due to a total runtime of maximally 40min per sample. The conditions and settings of the HPLC system were found to be accurate with high intra and inter-assay repeatability, recovery and accuracy rates. The robust analytical method results in very low detection limits and good separation for all of the eight biogenic amines and metabolites in this complex mixture of biological analytes.
authors.affiliation: Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Department of Biomedical Sciences, Universiteitsplein 1, Wilrijk B-2610, Belgium. Electronic address: debby.vandam@uantwerpen.be.
authors.forename: Debby
authors.initials: D
authors.lastname: Van Dam
authors.affiliation: Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Department of Biomedical Sciences, Universiteitsplein 1, Wilrijk B-2610, Belgium. Electronic address: yannick.vermeiren@uantwerpen.be.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Vermeiren
authors.affiliation: Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Department of Biomedical Sciences, Universiteitsplein 1, Wilrijk B-2610, Belgium. Electronic address: tony.aerts@uantwerpen.be.
authors.forename: Tony
authors.initials: T
authors.lastname: Aerts
authors.affiliation: Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Department of Biomedical Sciences, Universiteitsplein 1, Wilrijk B-2610, Belgium; Biobank, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, Antwerp 2610, Belgium; Department of Neurology/Memory Clinic, Middelheim General Hospital, ZNA, Lindendreef 1, Antwerp 2020, Belgium; University of Groningen, Department of Neurology and Alzheimer Research Center, University Medical Center Groningen (UMCG), Hanzeplein 1, Groningen 9713 GZ, The Netherlands. Electronic address: p.p.de.deyn@umcg.nl.
authors.forename: Peter Paul
authors.initials: PP
authors.lastname: De Deyn
mesh_terms: D000818:Animals; D001679:Biogenic Amines / Q000032:analysis* / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods*; D006801:Humans; D013997:Time Factors
chemical_list: D001679:Biogenic Amines
keywords: Biogenic amines; HPLC-ECD; Human brain tissue; Method optimization; Method validation