title: A rapid and simple method for the determination of 3,4-dihydroxyphenylacetic acid, norepinephrine, dopamine, and serotonin in mouse brain homogenate by HPLC with fluorimetric detection.
abstract: A fast and simple isocratic high-performance liquid chromatography method for the determination of 3,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in homogenate samples of mouse striatum employing the direct fluorescence of the neurotransmitters is described. The method has been optimized and validated. The analytes were separated in 15min on a reversed-phase column (C18) with acetate buffer (pH 4.0, 12mM)-methanol (86:14, v/v) as mobile phase; the flow rate was 1ml/min. The fluorescence measurements were carried out at 320nm with excitation at 279nm. The calibration curve for DA was linear up to about 2.5μg/ml, with a coefficient of determination (r(2)) of 0.9995 with a lower limit of quantification of 0.031μg/ml. Since the procedure does not involve sample pre-purification or derivatisation, the recovery ranged from 97% to 102% and relative standard deviation (RSD) was better than 2.9%, the use of the internal standard is not mandatory, further simplifying the method. Similar performance was obtained for the other analytes. As a result, thanks to its simplicity, rapidity and adequate working range, the method can be used for the determination of 3,4-dihydroxyphenylacetic acid, dopamine, norepinephrine and serotonin in animal tissues. An experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson-like disease has been used to demonstrate the method is fit-for-purpose.
authors: Laboratory of Analytical and Isotopic Mass Spectrometry, Department of Cultural Heritage, University of Salento, 73100 Lecce, Italy. Electronic address: giuseppe.debenedetto@unisalento.it.
authors: Giuseppe Egidio
authors: GE
authors: De Benedetto
authors: Laboratory of Analytical and Isotopic Mass Spectrometry, Department of Cultural Heritage, University of Salento, 73100 Lecce, Italy.
authors: Daniela
authors: D
authors: Fico
authors: Laboratory of Analytical and Isotopic Mass Spectrometry, Department of Cultural Heritage, University of Salento, 73100 Lecce, Italy.
authors: Antonio
authors: A
authors: Pennetta
authors: Laboratory of Analytical Chemistry, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
authors: Cosimino
authors: C
authors: Malitesta
authors: Section of Human Anatomy, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
authors: Giuseppe
authors: G
authors: Nicolardi
authors: Section of Human Anatomy, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
authors: Dario Domenico
authors: DD
authors: Lofrumento
authors: Section of Human Anatomy, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
authors: Francesco
authors: F
authors: De Nuccio
authors: Section of Human Anatomy, Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
authors: Velia
authors: V
authors: La Pesa
mesh_terms: D015102:3,4-Dihydroxyphenylacetic Acid / Q000737:chemistry*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004298:Dopamine / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018377:Neurotransmitter Agents / Q000737:chemistry; D009638:Norepinephrine / Q000737:chemistry*; D012701:Serotonin / Q000737:chemistry*
chemical_list: D018377:Neurotransmitter Agents; D015102:3,4-Dihydroxyphenylacetic Acid; D012701:Serotonin; D004298:Dopamine; D009638:Norepinephrine
keywords: Direct fluorescence; Dopamine; HPLC; Norepinephrine; Serotonin