Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

Area of Science:

• Analytical Chemistry
• Biochemistry
• Chromatography

Background:

• Previous methods for quantifying dinucleoside polyphosphates using ion-pair reversed-phase perfusion chromatography had detection limits in the micromolar range.
• There was a need for more efficient chromatographic assays to improve separation, reduce analytical run times, and enable mass spectrometric analysis.

Purpose of the Study:

• To develop a more efficient chromatographic assay for dinucleoside separation, reducing analytical run times.
• To establish conditions for chromatographic assays compatible with MALDI-mass spectrometric analysis of fractions.
• To compare the performance of different chromatography columns for dinucleoside polyphosphate analysis.

Main Methods:

• Comparison of conventional silica reversed-phase, perfusion, and monolithic reversed-phase C18 chromatography columns.
• Analysis of the effects of different ion-pair reagents (tetrabutylammonium hydrogensulfate - TBA, triethylammonium acetate - TEAA), flow rates, and gradients.
• Separation and analysis of synthetic and human platelet-derived diadenosine polyphosphates.

Main Results:

• Monolithic reversed-phase C18 columns demonstrated superior sensitivity and resolution compared to perfusion and conventional reversed-phase columns.
• Diadenosine polyphosphates were baseline separable using monolithic C18 columns with either TBA or TEAA as the ion-pair reagent.
• TEAA was found to be compatible with MALDI mass spectrometry, facilitating the identification of unknown nucleoside polyphosphates.
• Increased flow rates with monolithic columns reduced analysis time by approximately 50%, particularly with TBA.

Conclusions:

• Monolithic reversed-phase C18 columns offer a significant advancement in dinucleoside polyphosphate analysis, enhancing sensitivity, resolution, and time efficiency.
• These columns are recommended over perfusion or conventional silica reversed-phase columns for future analytical assays.
• The use of monolithic silica C18 columns is expected to facilitate the discovery and quantification of novel dinucleoside polyphosphates, advancing research into their biological functions.