Introduction: Biogenic amines are a group of endogenous compounds which play an important role in maintaining homeostasis. These compounds are produced from amino acid precursors and exert strong physiological effects despite being at low concentrations (low pg/ml). There are a wide variety of affects and effects controlled by biogenic amines. Different conditions can be brought on by changes in the concentrations of these biogenic amines that can alter the levels or be brought about by incorrect levels. Thus biogenic amine concentrations may prove to be valuable biomarkers if the extremely low levels can be accurately and rapidly quantitated. The aim of this study was to develop and validate a method which is able to quantitate multiple biogenic amines in a single run from low sample volumes.
Methods and Materials: Quantification of the blood levels of biogenic amines was performed following extraction using derivatisation and an appropriate liquid chromatography tandem mass spectrometric method. The derivatisation was performed at mild conditions for an hour and extracted using ethyl acetate. The LC-MS/MS system used was an Agilent 1100 series HPLC and an ABSciex 4000 QTrap. The system was operated in positive ESI mode and appropriate MRM precursor-product pairs were selected. In order to validate the method a series of 8 dilutions from 0.05 2.5 ng/ml were prepared in solvent and plasma. These were then processed according to the developed method and analysed using Analyst software.
Results and discussion: The validation of the biogenic amines was performed according to the ICH guidelines. The calibration curves were fitted using linear regression (1/x weighting) over the linear range of the calibration curve. Metanephrine and melatonin were validated in plasma between 0.1 2.5 ng/ml. Serotonin, histamine and normetanephrine were validated in solvent between 0.25 2.5 ng/ml. Adrenaline, dopamine, noradrenaline and 5-hydroxy-3-indoleacetic acid were validated in solvent between 5 50 ng/ml. Metanephrine exhibited the highest sensitivity and showed linearity between 0.05 2.5 ng/ml in solvent. The catecholamines and 5-HIAA were the least sensitive of the analytes and had estimated LLOQs between 0.5 2.5 ng/ml. Stability tests show the derivatised analytes to be stable at -20°C for 5 days and recovery testing shows recoveries of over 70% for metanephrine and melatonin.
Conclusion: Sensitivity still remains an issue when working with biogenic amines. The extremely low physiological concentrations are further exacerbated by the low sample volumes used in this study. Despite the challenges the compounds were all validated according to ICH guidelines and an LC-MS/MS method was developed that was able to analyse 9 biogenic amines of different classes in a single run using only 250 ?l of human plasma, after multistep sample preparation whilst achieving high sensitivity.