Simultaneous Quantification of Biogenic Amines and their Metabolites in Mice Tissue by Combining Ultraviolet and Integrated Pulsed Amperometric Detectors

We developed a reversed-phased high-performance liquid chromatographic method combining ultraviolet detection and integrated pulsed amperometric detection for the simultaneous quantification of dopamine, 5-hydroxyindolacetic acid, homovanillic acid, serotonin, 3,4-dihydroxyphenylacetic acid, norepinephrine and epinephrine. All target components were completely separated in a C18 column with isocratic elution of 5% acetonitrile solution containing 8 mM HClO₄ and 0.20 mM 1-octanesulfonic acid as an ion pairing reagent. This method showed limits of detection of 0.03-0.1 ng and limits of quantification of 0.10-0.3 ng with linear regression coefficients of 0.9998-1.0000. All inter-day and intra-day precision values were below 9.58%, and the average recoveries were 93.71-109.82% for mouse striatum samples. The mean levels of the seven components in striatal brain tissue in a mouse model of Parkinson's disease decreased by 2-12 times compared to those of a control group. In particular, the decrease in dopamine, 5-hydroxyindolacetic acid and 3,4-dihydroxyphenylacetic acid was statistically significant (P < 0.05). Orthogonal partial least squares discriminant analysis confirmed that the seven components are useful biomarkers. The significance of the developed method lies in its ability to simultaneously analyze seven biogenic amines related to Parkinson's disease by combining two detectors, offering a simple and cost-effective approach for clinical and biological labs.