Highly sensitive, selective and rapid LC-MS method for simultaneous quantification of diadenosine polyphosphates in...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Diadenosine polyphosphates (ApnAs) are crucial endogenous mediators in various physiological and pathophysiological processes.
• Accurate quantification of ApnAs in biological matrices like plasma remains challenging.
• Existing chromatographic methods lack selectivity due to co-eluting plasma components.

Purpose of the Study:

• To develop and validate a highly sensitive, selective, and rapid LC-ESI-MS method for simultaneous ApnAs (n=3-6) quantification in human plasma.
• To establish a reliable method for clinical studies requiring precise ApnAs measurements.

Main Methods:

• Weak anion-exchange extraction of ApnAs from 100μl human plasma.
• Separation of ApnAs using capillary C18 columns.
• Detection and quantification using an ESI-HCT mass spectrometer in negative ion mode.

Main Results:

• A robust calibration curve (r²>0.998) was established for ApnAs in the 1.9-125nM range.
• High accuracy (91.4%-110.9%) and precision (0.1%-11.4%) were achieved for intra- and inter-day measurements.
• Mean plasma concentrations quantified: Ap3A (31.9±5.9nM), Ap4A (40.4±6.6nM), Ap5A (10.7±1.5nM), and Ap6A (10.0±18.9nM).

Conclusions:

• The developed LC-ESI-MS method is validated and effective for quantifying diadenosine polyphosphates in human plasma.
• The method's reliance on a small plasma volume (100μl) makes it highly suitable for clinical applications.
• This advancement facilitates the study of ApnAs' impact in physiological and pathophysiological contexts.