Validation and quantitation of six neurotransmitters in rat plasma and brain using liquid chromatography quadrupole-time-of-flight mass spectrometry

Delirium is a cognitive dysfunction observed following sedation of critically injured patients. Neurotransmitters are endogenous chemical messengers that play key roles in a variety of essential nervous system functions, and their dysregulation is believed to contribute to the development of delirium. Monitoring neurotransmitters such as acetylcholine, dopamine, serotonin, gamma-aminobutyric acid, gamma-hydroxybutyric acid, and glutamic acid for biomarkers of delirium could improve our understanding of delirium pathogenesis and provide evidence for more efficacious treatments. However, current methods have limited scopes of analysis, require large sample volumes, or use complicated extraction methods. In this study, a novel, analytical approach was developed using liquid chromatography quadrupole-time-of-flight mass spectrometry to measure neurotransmitters in limited sample. This method was used to measure neurotransmitters in plasma and brain tissue samples taken from rats treated with a benzodiazepine sedative and opioid analgesic, a drug combination commonly prescribed in intensive care units and known to precipitate delirium. The analytical method was validated for selectivity, matrix effect, accuracy and precision, dilution integrity, and stability in rat plasma and partially validated in rat brain. The calibration curve ranged from 10 to 5000 ng/mL across the analytes. Within-run and between-run bias and precision of the LLOQ and QCs in plasma and brain were within 20 % and 15 %, respectively. Analytes were stable at 2 ºC and -80 ºC. Extraction recoveries from a simple protein precipitation ranged from 85 % to 133 %. The method was successfully applied to measure analytes in rat brain homogenates from a delirium model study.

Bioanalysis; HILIC column chemistry; High resolution mass spectrometry; Neurotransmitters.