Acetylcholine (ACh) is an important neurotransmitter that plays a crucial role in the functioning of the nervous system. The measurement of ACh in biofluid samples is often challenging due to its low concentration and polarity. In this article, we discuss how to increase the concentration of ACh in biofluid samples via SPE (solid-phase extraction).
The Problem
The issue with ACh extraction is its polarity. Traditional C18 SPE protocol, which uses methanol as an eluent, is not suitable for ACh extraction due to its positively charged nature. During the wash step, the ACh would likely be washed away. Thus, the yields of ACh are often low when using traditional SPE protocols.
Solution via HILIC
To extract ACh effectively, it is recommended to use a more amiable SPE protocol that targets charged molecules. HILIC (hydrophilic interaction chromatography) is one protocol that can aid in the identification and concentration of ACh. HILIC has a polar stationary phase and a non-polar mobile phase, which allows for better separation and concentration of polar molecules, such as ACh, in biofluid samples.
Tips for Designing a Targeted SPE Protocol
Here are some tips for designing an SPE protocol to extract ACh effectively using HILIC:
Select a hydrophilic stationary phase
Use an eluent with a strong organic solvent, such as acetonitrile, to increase ACh solubility and recovery
Optimize the sample pH to ensure maximum ACh stability and recovery
Minimize the elution volume to concentrate the ACh extract
Select a compatible downstream bioassay protocol
SPE Protocol for ACh Extraction via HILIC
To extract ACh effectively, we recommend the following SPE protocol via HILIC:
Condition the SPE cartridge with the eluent
Load the biofluid sample onto the conditioned cartridge
Wash the cartridge with the eluent to remove any interfering compounds
Elute the ACh extract in a small volume of eluent (e.g., 100-200 μL)
Concentrate the extract under a gentle stream of nitrogen gas to dryness
Reconstitute the extract in a compatible solvent for downstream bioassay
Conclusion
To increase the concentration and yield of ACh in biofluid samples, it is recommended to use an SPE protocol that targets charged molecules, such as HILIC. It is also important to optimize the protocol to ensure maximum ACh stability and recovery. By following the recommended SPE protocol and tips, it is possible to extract ACh effectively and concentrate the extract for downstream bioassay analysis.
Increasing Concentration of Acetylcholine Via Spe
Introduction
Acetylcholine (ACh) is an important neurotransmitter that plays a crucial role in the functioning of the nervous system. The measurement of ACh in biofluid samples is often challenging due to its low concentration and polarity. In this article, we discuss how to increase the concentration of ACh in biofluid samples via SPE (solid-phase extraction).
The Problem
The issue with ACh extraction is its polarity. Traditional C18 SPE protocol, which uses methanol as an eluent, is not suitable for ACh extraction due to its positively charged nature. During the wash step, the ACh would likely be washed away. Thus, the yields of ACh are often low when using traditional SPE protocols.
Solution via HILIC
To extract ACh effectively, it is recommended to use a more amiable SPE protocol that targets charged molecules. HILIC (hydrophilic interaction chromatography) is one protocol that can aid in the identification and concentration of ACh. HILIC has a polar stationary phase and a non-polar mobile phase, which allows for better separation and concentration of polar molecules, such as ACh, in biofluid samples.
Tips for Designing a Targeted SPE Protocol
Here are some tips for designing an SPE protocol to extract ACh effectively using HILIC:
SPE Protocol for ACh Extraction via HILIC
To extract ACh effectively, we recommend the following SPE protocol via HILIC:
Conclusion
To increase the concentration and yield of ACh in biofluid samples, it is recommended to use an SPE protocol that targets charged molecules, such as HILIC. It is also important to optimize the protocol to ensure maximum ACh stability and recovery. By following the recommended SPE protocol and tips, it is possible to extract ACh effectively and concentrate the extract for downstream bioassay analysis.