B-361 An Efficient Strategy for Simultaneous Detection of Six Catecholamines in Plasma

Abstract

Catecholamines (CAs) are neuroactive substances containing catechol and amino groups. Imbalances in their levels often indicate the development of various diseases, including pheochromocytoma, paraganglioma, neuroblastoma, and hypertension. Therefore, rapid and convenient monitoring of plasma CAs levels is crucial for the diagnosis, treatment, and prognosis of related diseases. Among numerous analytical methods, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) stands as the preferred approach due to its outstanding specificity, selectivity, and accuracy. Using hybrid weakly cation-exchange reverse-adsorption magnetic beads and a semi-automated pretreatment system, six CAs (Epinephrine (E), Norepinephrine (NE), Dopamine (DA), Metanephrine (MN), Normetanephrine (NMN), and 3-Methoxytyramine (3-MT) were initially extracted from plasma samples. A series of pre-treatment reagents with simple composition and low organic solvent content were selected and optimized based on the characteristics of these CAs. After removing impurities through two elution, the analytes were released into the eluent for detection. Chromatographic separation of analytes was achieved using a reversed-phase column. Detection was performed by electrospray ionization triple quadrupole mass spectrometry in positive ion mode, with data acquired via multiple reaction monitoring. For each analyte and its isotopically labeled internal standard, two ion pairs were monitored for quantitative and qualitative analysis, respectively. The developed method was evaluated according to standard guidelines for performance metrics including linearity, detection limit, quantification limit, accuracy, specificity, matrix effects, and carryover contamination to ensure compliance with clinical application requirements. This method utilized magnetic bead technology to automate sample extraction and purification, enabling simultaneous enrichment and separation of 32 samples within 17 minutes. Combined with HPLC-MS/MS technology, it achieved dual separation of CAs, yielding detection results for six target CAs within 5 minutes. When analyzing clinical plasma samples, this method demonstrated broad linear ranges with excellent linear relationships, achieving R² values exceeding 0.999, for all six analytes (E, NE, DA, MN, NMN, and 3-MT). The limits of detection (LOD) for E, and 3-MT were 9, 20, 10, 11, and 10 pg/mL, respectively, while the limits of quantification (LOQ) were 9, 50, 24, 26, 27, and 24 pg/mL, respectively. The precision of the method was evaluated by measuring samples at different concentrations, yielding a coefficient of variation (CV) ranging from 1.25% to 5.47%. Spiked recovery was verified by adding low, medium, and high standard solutions to samples, with recovery rates ranging from 90% to 109%. The detection strategy provided in this study was straightforward, time-efficient, and highly stable, unaffected by matrix effects and carryover contamination. This study presents a simple and effective detection strategy enabling the semi-automated enrichment, separation, and detection of six catecholamines in large-scale plasma samples within a short timeframe. It provides technical support for the quantitative analysis of CAs in clinical samples and the auxiliary diagnosis of related diseases.