Antonina Gucciardi1, Irene Costa1, Paola Pirillo2, Mauro Naturale2, Eugenio Baraldi2, Giuseppe Giordano2.
1 Institute for Pediatric Research IRP "Città della Speranza", Padova, Italy; 2 Women’s and Children’s Health Department, University of Padova, Padova, Italy
We developed and validated a method for measurement of plasma neurotransmitters (dopamine, epinephrine, norepinephrine, serotonin) and trace amines (tyramine, octopamine, synephrine, β-phenylethylamine and tryptamine) by UPLC-MS/MS. Analytes were extracted using weak cation exchange SPE and separated by a PFP column and water-acetonitrile-methanol gradient as mobile phase, into the ACQUITY UPLC system. The metabolites were measured in MRM mode with positive electrospray ionization using a Waters Xevo TQ-S mass spectrometer. Commercially quality control (QC) materials from Chromsystems were used for validation. The method showed good precision, specificity, sensitivity and linearity, and will be useful for clinical research of neurodegenerative disease.
Introduction: Epinephrine (E), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) are classical biogenic amine neurotransmitters (NTs) and their changes are related to a variety of diseases and disorders such as insomnia, epilepsy, schizophrenia, anxiety, depression, Alzheimer's disease and Parkinson's disease. In addition to these classical amines, there exists a class of “trace amines” that are found in very low levels in mammalian tissues, and include tyramine, octopamine, synephrine, β-phenylethylamine (β-PEA) and tryptamine. There is clinical literature that supports a role for trace amines in depression as well as other psychiatric disorders and migraine. Because of their low concentrations in plasma, the limited volume of samples in clinical study, especially in pediatric research, and the potential chromatographic interference with compounds that coelute, their accurate and fast measurement in plasma is still a challenge. We developed and validated a sensitive method for measurement of these compounds, using solid phase extraction (SPE) combined with UPLC-MS/MS without derivatization.
Methods: Plasma samples (300 µL) were pretreated with 300 µL of 50 mM ammonium acetate (pH 7.0), added of labeled internal standard, and loaded onto pretreated wells of Biotage EVOLUTE WCX 25mg/1mL SPE. The wells were then washed twice with 900 µL of 90:10 H2O:MeOH, and eluted with 900+600 µL aliquots of MeOH with 5% of formic acid (FA). Samples were dried under nitrogen flow at 30°C and reconstituted in 50 µL of H2O with 0.1% FA. Chromatographic separation was achieved using an ACQUITY UPLC HSS PFP column, 1.8 µm, 2.1 mm x 100 mm. Mobile phase A consisted of 0.1% FA in H2O, and mobile phase B was 80:20 MeOH:MeCN with 0.1% FA. Compounds were detected by MRM in ESI positive ionization mode using a Waters Xevo TQ-S mass spectrometer. The chromatographic run time was within 15 min.
Certified plasma controls, purchased from Chromsystems, were used as QC at normal and pathological range for NE, E,5-HT, and DA. No certified materials were available for trace amines.
Results: All compounds eluted within 10 minutes. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated by the use of spiked plasma samples and certified CQ.
Recovery efficiency varied from 50-98.5%. Matrix effect, occurred as ionic suppression, was found in the range of 5% to 50% depending on the compound and stable isotope labeled internal standards were used to increase the accuracy of quantitation. Calibration curves were linear from 0.1 to 25-nmol/L for all analytes (5-HT ranged to 3 µmol/L) with regression coefficients higher than 0.99. Intra-and inter-day precision and accuracy were less than 15%. The lower limit of quantitation ranged from 0.05 to 0.25 nmol/L.
Conclusions: A simple and reliable UPLC-MS/MS method for the quantification of plasma biogenic and trace amines was developed and validated for the first time. The method was applied to evaluate NTs plasmatic profile of healthy adult subjects. Our method might be applied for tracking the changes of endogenous NTs in neurological disorders and neurodegenerative diseases.