Abstract:

This study was conducted to develop a robust and efficient high-performance liquid chromatography (HPLC) method for the simultaneous quantification of tyrosine (Tyr), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), epinephrine (E), and dopamine (DA) in human blood samples. The analytical system combined ultraviolet (UV) and electrochemical detection, with vanillin serving as the internal standard. The mobile phase comprised acetonitrile and a buffer solution (25 mmol/L sodium acetate, 25 mmol/L citric acid, 0.01 mmol/L EDTA-2Na, 2.0 mmol/L sodium 1-octanesulfonate, pH 3.76) at a volumetric ratio of 6:94. Chromatographic separation was achieved at room temperature, with a flow rate of 1.0 mL/min and an injection volume of 20 μL. The electrochemical detector was operated at 800 mV with a 10 nA range. The method exhibited excellent linearity for all five analytes (R² > 0.996), with limits of detection (LOD) ranging from 50 to 150 ng/mL and limits of quantification (LOQ) below 0.5 μg/mL, satisfying analytical requirements. Intra-day precision (RSD) was consistently below 5.4%, while inter-day precision remained under 15%. Sample stability was maintained at 4 °C, and extraction recovery rates were consistently high. This HPLC approach allowed for accurate and reliable measurement of catecholamine levels in human blood, offering high precision, satisfactory accuracy, and robust stability, underscoring its practical applicability in clinical and research settings.

Key words: HPLC method, Plasma sample, Internal standard method, Catecholamine, UV-ECD