HILIC-MS/MS Analysis of Adenosine in Patient Blood

Christina Virgiliou,Helen Gika,Nikolaos Fragakis,Vassilios Vassilikos,Melani Sotiriadou,Georgios Theodoridis,Spiros Gerou

doi:10.3390/separations8110222

Christina Virgiliou, Helen Gika + Show 5 more

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https://doi.org/10.3390/separations8110222

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Abstract

Adenosine is a purine ribonucleoside with important roles in various physiological processes. A number of studies have indicated the importance of adenosine in cardiovascular diseases including syncope; however, the accurate determination of adenosine in human blood is challenging due to the molecule’s instability. In the present study, we report a simple method for the pre-treatment of blood samples and the development of a fast and efficient hydrophilic interaction chromatographic tandem mass spectrometry method for the analysis of adenosine in patient blood. During collection, samples were mixed directly with a solvent mixture containing 95% acetonitrile and 10 mM ammonium formate in a Vacutainer tube, resulting in successful prevention of adenosine metabolic processes and direct blood sample deproteinization. The method was validated according to bioanalytical industry guidelines and found to be accurate, repeatable, specific and sensitive with LLOQ 0.005 μg/mL, thus allowing its application in the analysis of real clinical samples.

Highlights

Adenosine is a purine ribonucleoside that plays important roles in many physiological processes, acting as an extracellular regulatory molecule [1]
Adenosine is a polar molecule (Supplementary Materials Figure S1) with low molecular weight, 267.2413 g/mol. Such molecules typically show better retention on HILIC columns in comparison to reversed-phase materials. This increased retention is expected to minimise the impact of interferences from the analysis of real-life samples: the analyte does not elute in the solvent front together with various other sample components
mass spectrometry (MS)-MS data were obtained for adenosine after direct infusion of the standard solution (1 μg/mL), and the predominant single charged precursor ion [M+H]+ of the analyte at m/z 268 was selected

Summary

Introduction

Adenosine is a purine ribonucleoside that plays important roles in many physiological processes, acting as an extracellular regulatory molecule [1]. Little is known about the concentration ranges of adenosine in human blood or factors that influence adenosine levels, due mainly to the fact of technical limitations during blood collection. The short half-life of circulating adenosine, which is counted in seconds, makes sample collection and detection of adenosine a challenging task [8]. Since the half-life of adenosine in blood is short, various collection protocols have been developed for the prevention of adenosine metabolism. Vacutainer tubes enable the direct mixing of venous blood with a “STOP” solution that prevents adenosine uptake by red cells. Instead of using STOP solution, simple protein precipitation with perchloric acid has been proposed in the literature for the effective quantitation of adenosine in blood [11]. Validation experiments revealed that adenosine concentrations in deproteinised blood samples were unstable

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