Analysis of Choline and Acetylcholine in Rat Cerebrospinal Fluid Samples Using the Nexera UC-MS/MS System

Importance of the Topic

Choline and acetylcholine play critical roles in cellular membrane structure and neurotransmission, respectively. Monitoring their levels in cerebrospinal fluid (CSF) provides insights into neurological function and metabolic activity, making sensitive and reliable analytical methods essential in bioanalysis.

Objectives and Study Overview

This study evaluates the performance of supercritical fluid chromatography coupled to tandem mass spectrometry (SFC-MS/MS) for quantifying choline and acetylcholine in rat CSF. Two approaches are compared:

• Direct injection of microdialysate into the SFC-MS/MS system.
• Online supercritical fluid extraction (SFE) combined with SFC-MS/MS of CSF dried on filter paper.

Methodology

Chromatographic separation used an Inertsil CN stationary phase with CO₂ as the primary mobile phase and methanol–ammonium formate (95:5) as modifier. A gradient program varied modifier concentration from 10 % to 50 % over 15 minutes at 2.5 mL/min and 40 °C. Detection employed electrospray ionization in positive MRM mode, targeting m/z 104.1→60.1 for choline and 146.1→87.1 for acetylcholine. Calibration covered 10–1000 μg/L, with six replicate injections at each concentration to assess repeatability and linearity.

Used Instrumentation

• Shimadzu Nexera UC SFC system
• Triple quadrupole mass spectrometer LCMS-8050 (ESI, MRM mode)
• Inertsil CN-3 column (250 mm × 4.6 mm, 5 μm)
• Advantec GA-200 filter paper for sample impregnation

Main Results and Discussion

The SFC-MS/MS method achieved baseline separation of both analytes with excellent linearity (r² = 0.9993 for choline and 0.9982 for acetylcholine). Limits of quantitation were 30 μg/L for choline and 10 μg/L for acetylcholine. Peak area repeatability remained below 8 % RSD across the calibration range. Direct analysis of rat CSF via microdialysis yielded a choline concentration of approximately 230 μg/L, while acetylcholine remained at trace levels below LOQ. The online SFE-SFC-MS/MS workflow improved signal-to-noise and allowed quantitative detection of acetylcholine at low microgram-per-liter levels, with measured concentrations of 297 μg/L for choline and 1.7 μg/L for acetylcholine in dried-paper samples.

Benefits and Practical Applications

The described methods offer:

• Rapid analysis with minimal sample preparation.
• Robust quantitation in aqueous matrices using supercritical CO₂ mobile phase.
• Enhanced sample stability through dried matrix storage on filter paper.
• Online coupling of extraction and analysis for improved throughput and consistency.

Future Trends and Potential Use

Advances may include miniaturized online microdialysis–SFE-SFC platforms, broader application to other neurotransmitters and metabolites, and integration into in vivo monitoring or clinical diagnostics. Automation of dried-matrix sampling and high-throughput screening are expected to expand the method’s utility.

Conclusion

The combination of SFC-MS/MS and online SFE provides a fast, reproducible, and sensitive approach for measuring choline and acetylcholine in rat CSF. The dried-paper sample technique enhances convenience for storage and transport without compromising analytical performance.