| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1221137 | Journal of Pharmaceutical and Biomedical Analysis | 2014 | 5 Pages |
•Development and validation of an HPLC-FD method for selected neurotransmitters.•Easy, one step sample preparation not requiring the use of an internal standard.•Isocratic elution and absence of an ion pairing agent make the method more robust.•Application to mouse striatum homogenates demonstrates the method is fit-for-purpose.
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 15 min on a reversed-phase column (C18) with acetate buffer (pH 4.0, 12 mM)–methanol (86:14, v/v) as mobile phase; the flow rate was 1 ml/min. The fluorescence measurements were carried out at 320 nm with excitation at 279 nm. The calibration curve for DA was linear up to about 2.5 μg/ml, with a coefficient of determination (r2) of 0.9995 with a lower limit of quantification of 0.031 μg/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.
Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide
