title: Hydrophilic interaction chromatography combined with dispersive liquid-liquid microextraction as a preconcentration tool for the simultaneous determination of the panel of underivatized neurotransmitters in human urine samples.
abstract: A simple and sensitive method using dispersive liquid-liquid microextraction (DLLME) followed by liquid chromatography coupled to mass spectrometry (LC-MS) with a hydrophilic interaction chromatography (HILIC) column was developed for the simultaneous determination of 13 compounds of different polarities, comprising monoamine neurotransmitters (dopamine, norepinephrine, epinephrine and serotonin) along with their respective precursors and metabolites, in human urine samples. The microextraction procedure was based on the fast injection of a mixture of ethanol (disperser solvent) and dichloromethane (extraction solvent) into a human urine sample, forming a cloudy solution in the Eppendorf tube. After centrifugation, the sedimented phase was collected and subsequently analyzed by LC-HILIC-MS in about 12min without a derivatization step. The separation was performed on an XBridge Amide™ BEH column 3.0×100mm, 3.5mm and the mobile phase consisted of phase A: 10mM ammonium formate buffer in water pH 3.0 and phase B: 10 mM ammonium formate buffer in acetonitrile, under gradient program elution. Tyrosine, tryptophan, 5-hydroxytryptophan, dopamine, epinephrine, norepinephrine, serotonin, 3-methoxytyramine, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxy-l-phenylalanine and norvaline (internal standard) were detected in the positive ionization mode. While vanillylmandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxybenzylamine (internal standard) were detected in the negative ionization mode. Parameters influencing DLLME and LC-HILIC-MS were investigated. Under the optimum conditions, the proposed method exhibited a low detection limit (5-10ngmL(-1)), and good linearity with R between 0.9991 and 0.9998. The recoveries in human urine samples were 99.0%±3.6%. for the 13 studied biogenic amines with intra- and inter-day RSDs of 0.24-9.55% and 0.31-10.0%, respectively. The developed DLLME-LC-MS method could be successfully applied for the determination of trace amounts of polar endogenous compounds, such as neurotransmitters, in human urine samples, including samples with a reduced volume obtained from pediatric patients.
authors.affiliation: Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gen. J. Hallera 107, 80-416 Gdańsk, Poland.
authors.forename: Lucyna
authors.initials: L
authors.lastname: Konieczna
authors.affiliation: Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gen. J. Hallera 107, 80-416 Gdańsk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Roszkowska
authors.affiliation: Department of Pediatrics, Hematology and Oncology, Medical University of Gdańsk, Dębinki 7, 80-211 Gdańsk, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Niedźwiecki
authors.affiliation: Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gen. J. Hallera 107, 80-416 Gdańsk, Poland. Electronic address: tbaczek@gumed.edu.pl.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Bączek
mesh_terms: D015102:3,4-Dihydroxyphenylacetic Acid / Q000032:analysis; D006916:5-Hydroxytryptophan / Q000032:analysis; D000097:Acetonitriles / Q000737:chemistry; D002853:Chromatography, Liquid; D004298:Dopamine / Q000031:analogs & derivatives / Q000032:analysis; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D059627:Liquid Phase Microextraction; D013058:Mass Spectrometry; D018377:Neurotransmitter Agents / Q000652:urine*; D010649:Phenylalanine / Q000032:analysis; D012997:Solvents / Q000737:chemistry; D016482:Urinalysis / Q000379:methods*
chemical_list: D000097:Acetonitriles; D018377:Neurotransmitter Agents; D012997:Solvents; D015102:3,4-Dihydroxyphenylacetic Acid; C022133:3,4-dihydroxybenzylamine; D010649:Phenylalanine; D006916:5-Hydroxytryptophan; C001746:3-methoxytyramine; D004298:Dopamine; C032159:acetonitrile
keywords: Dispersive liquid–liquid microextraction (DLLME); Human urine sample; Hydrophilic interaction chromatography (HILIC); Liquid chromatography coupled to mass spectrometry (LC–MS); Neurotransmitters