Analysis of Choline, Acetylcholine, and Monoamine Neurotransmitters by Polymer-Based Ion Chromatography and Mass Spectrometry

Monoamine neurotransmitters (adrenaline, dopamine, noradrenaline, and acetylcholine) are released from the brain and peripheral nervous system in minute amounts. These neurotransmitters play a role in arousal, emotion, and cognition. The detection of an increase or decrease of these neurotransmitters through physiological changes or the use of drugs can be of importance for treating psychiatric disorders, mood, and thought processes.

Shodex IC YS-50, a polymer based ion chromatography column for cation analysis with non-suppressor methods, has demonstrated the separation of these compounds at low concentration levels by MS with SIM (selected ion monitoring) mode.

Experimental Conditions

Separation was carried out by Shodex IC YS-50 (4.6 mm i.d. × 125 mm L). Column temperature was set at 30 °C and flow rate used was 1.0 mL/min. The following isocratic eluent conditions were applied: 4 mM HNO₃ (aq.)/ CH₃CN = 70/30. Sample contained 0.01–1.0 µM of each neurotransmitter. Injection volume of 20 µL was used for the experiments. HPLC system was coupled with an ESI-MS and SIM mode was used for detection.

Figure 1: Chromatogram of neurotransmitters and choline. Column: Shodex IC YS-50; column temperature: 30 °C; injection volume: 20 µL; eluent: 4 mM HNO3 (aq)/CH3CN=70/30; flow rate: 1.0 mL/min; detector: ESI-MS (SIM).

Results

Choline, acetylcholine, and monoamine neurotransmitters were analyzed successfully by the HPLC–MS method (Figure 1). Of note, the method achieved baseline separations of choline and acetylcholine which previously required a post-column immobilized enzyme reactor with addition of an ion-pair reagent to the eluent. The calibration curves for each of the neurotransmitters and choline were shown to be highly linear for all species (Figure 2).

Figure 2: Calibration curves for neurotransmitters and choline. Column: Shodex IC YS-50; column temperature: 30 °C; injection volume: 20 µL; eluent: 4 mM HNO3 (aq)/CH3CN=70/30; flow rate: 1.0 mL/min; detector: ESI-MS (SIM).

Conclusions

Shodex IC YS-50, a polymer-based ion chromatography column coupled with ESI-MS is feasible for the analysis of choline, acetylcholine, and monoamine neurotransmitters at low levels of concentration. The presented method using the YS-50 column provides a simple and rapid analysis of neurotransmitters, and the eluent conditions used for ion chromatography are desirable for ESI-MS measurements.

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