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

• 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.

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