Development of an HPLC/Diode Array Detector Method for the Determination of Human Plasma Adenosine Concentrations

Abstract

Assays of adenosine and its derivatives in biological fluids involve fluorimetric, radioimmunological or chromatographic analyses. Techniques currently used are tedious because they involve either an extraction using immunological methods or one or two freeze drying cycles when using high performance liquid chromatography (HPLC). In this context, we describe a "quick" HPLC method using a diode array detector for the spectral analysis and quantitation of adenosine and its derivatives in less than two hours following sampling. We compared our results to those obtained with an HPLC technique coupled to UV detection. Downloaded by [UQ Library] at 02:36 01 November 2014 ORDER REPRINTS 1830 GUIEU ET AL.Although there is a good correlation between the two techniques, the "quick" method provide values that are on average 30% higher than those obtained with the classical method. The absence of a lyophilization step in the classical method could explain this difference. The utilization of an HPLC system coupled to a diode array detector leads to a rapid and reliable assay of adenosine and its derivatives. The possibility of having a rapid assay method available could be very useful in cases of certain cardiovascular or neurological disorders.

Spare Adenosine A2a Receptors Are Associated with Positive Exercise Stress Test in Coronary Artery Disease

“…This method allows whole blood to mix quickly with the stop solution, thus preventing red blood cell uptake and adenosine degradation (20)(21)(22). After collection, samples were centrifuged and deproteinized prior to analysis using high-performance liquid chromatography.…”

Fall in oxygen tension of culture medium stimulates the adenosinergic signalling of a human T cell line

“…The technique used was previously described [10] with minor modifications. Intracellular adenosine concentration was measured using high-performance liquid chromatography equipped with a diode array detector (Chromsystems, Munich, Germany).…”

Rapid Measurement of Adenosine Concentration in Human Blood Using Fixed Potential Amperometry: Comparison with Mass Spectrometry and High- Performance Liquid Chromatography

“…The method has been previously described [9][10][11][12] Briefly, after collection, blood samples were put in ice then centrifuged (4°C, 1500 g), and deproteinized (perchloric acid, 5%, 0.25 ml/ml of plasma), again centrifuged (1500 g for 10 min) and the supernatant was pipetted off and stored at -80°C until analysis. Defreezed samples were analyzed by chromatography.…”

“…Healthy subjects and patients gave their written inform consent for blood collection. Whole blood was collected through a cubital vein using special tubes (5 mL) under vacuum containing 2 mL of cold stop solution as previously described [11,12]. The stop solution was composed of dipyridamole 0.2 mmol/l, ethylene diamine tetracetic acid disodium (4 mmol/l), erythro 9-2-hydroxy-3-nonyl adenine (5 mmol/l), α,β -methyleneadenosine 5' diphosphate (79 mmol/l); coformycine 10 µg/mL, and heparin sulfate 1 IU/mL (SIGMA Aldrich).…”

“…We have developed a method using a stop solution to inhibit adenosine degradation in body fluid. After plasma deproteinization, adenosine concentration was evaluated using high performance liquid chromatography [9][10][11][12]. Some teams have developed a tandem mass spectrometry method that require no stop solution for adenosine measurement in whole blood collected on blotting paper in the perspective of adenosine deaminase deficiency screening [13].…”