LC–MS Assists Metabolomic Study of Brain Effects of Cocaine and Ethanol Consumption in Rats

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Samples of the prefrontal cortex, striatum, and hippocampus of rats were analyzed by LC–MS to assess brain tissue response to combined consumption of ethanol and cocaine.

A group of researchers from the National University for Distance Learning, Carlos III University, and Alcalá University, all in Madrid, Spain, is reporting the first untargeted metabolomic study on the neurobiological effects of combined consumption of cocaine and ethanol, using liquid chromatography–mass spectrometry (LC–MS) to analyze tissue samples from three different sections of the brains of rats (1).

The study, accepted for publication by the Journal of Chromatography A, divided young male and female rats into six groups, not all of which intravenously self-administered both drugs, and monitored the endogenous compounds present in the prefrontal cortex, striatum, and hippocampus. Citing a scarcity of studies of the combined use of ethanol and cocaine, a frequent combination among human drug abusers, the researchers aimed to better understand how repetitive drug use causes long-term plasticity in certain brain areas, making consumption of cocaine more likely to trigger dependence on ethanol, or vice versa.

Long-term plasticity in certain brain areas can lead to maladaptive changes that result in various neurological disorders. For example, long-term potentiation (LTP), a type of plasticity that strengthens synaptic connections, can become excessive and lead to epileptic seizures. On the other hand, long-term depression (LTD), a type of plasticity that weakens synaptic connections, can lead to learning and memory deficits. Additionally, long-term plasticity can contribute to chronic pain by enhancing the strength of pain signals transmitted to the brain. Long-term plasticity also plays a role in addiction by strengthening drug-associated memories and causing craving and relapse.

Each of the three selected brain regions plays a role in drug dependency. The prefrontal cortex houses the functions of emotional and executive self-control, which become dysfunctional in those addicted to drugs. The striatum holds the key to obtaining a reward in guided behaviors. And drug use has been shown to affect learning and memory functions in the hippocampus to such a degree that addiction is allowed to be maintained or further developed.

In this context, the purpose of metabolomics is to find the largest possible amount of metabolites, typically low-molecular weight compounds (<1500 Da) in a given sample, using chemometrics and statistics to compare physiological conditions dependent on environment and genetics. Alterations in metabolic profiles may provide, among other markers, information on the toxicology mechanism that triggers drug addiction. Aside from predispositions to codependency and relapse, metabolomic findings have identified other biomarkers sensitive to toxicity from drugs of abuse: neurotransmission alterations, oxidative stress, and disruptions of energy and amino acid metabolisms.


A metabolomic approach in rats has been scarce when it comes to polyconsumption, but the researchers previously found eight differentiated metabolites in the plasma of rat subjects, mainly concerning the metabolism of amino acids like tryptophan, arginine, proline, and methionine.

Additionally, nuclear magnetic resonance (NMR) spectroscopy has been used in the past to study polydrug users of ethanol plus a psychostimulant, finding that their brain metabolite profile is different from users of just a single substance. But in this study, LC–MS with a high-resolution Orbitrap analyzer found up to 761 significant features with assigned molecular formula, 190 tentatively identified and 44 unequivocally confirmed.

Metabolic pathways, and the functions they serve, that were found to have been altered by the administration of cocaine or ethanol in the LC–MS analysis echoed previous metabolomic discoveries, including receptor systems (such as the glutamine-glutamic acid-GABA axis or catecholamine pathway), purinergic and pyrimidine pathways, fatty acids, and oxidative stress. Furthermore, the female rats were seen as more vulnerable, fulfilling the requirements of the self-administration in less time than the males.

Whether an analysis of humans would yield corresponding results has yet to be determined. The researchers admit that the practice of intravenous self-administration used in this study would be a liability in humans, but considered the use of rats an appropriate experimental approach.


(1) Marcos, A.; León, C.; Moreno-Fernández, M. et al. Untargeted metabolomic study by liquid chromatography–mass spectrometry in brain tissues on the effects of combined cocaine and ethanol self-administration in male and female young rats. J. Chromatogr. A 2023, 464047. DOI: 10.1016/j.chroma.2023.464047