Quantitative analysis of adenosine using liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS).

Adenosine-secreting cellular brain implants constitute a promising therapeutic approach for the treatment of epilepsy. To engineer neural stem cells for therapeutic adenosine delivery, a reliable and fast analytical method is necessary to quantify cell-based adenosine release. Here we describe the development, optimization and validation of adenosine measurement using liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC-APCI-MS/MS). LC-MS/MS in positive ion mode used selected reaction monitoring at m/z of 268.2/136.1 and 302.2/170.0 for adenosine and the internal standard, respectively. The bias was within 15% of the nominal value and evaluation of precision showed a relative standard deviation lower than 15% for all measured concentrations. The lower limit of quantification of adenosine was 15.6 ng/ml. Freeze and thaw stability and processed sample stability also fulfilled the acceptance criteria. Evaluation of the matrix effect showed that the method is not affected by relative matrix effects. The major advantages of this method are the absence of an extraction phase and the combination of the high selectivity and sensitivity characteristic for the LC-MS/MS technique, with a short run time of 4.5 min. These results demonstrate that this method is a useful tool to measure adenosine concentrations in culture medium released from stem cells in vitro.

8-Cl-adenosine is an active metabolite of 8-Cl-cAMP responsible for its in vitro antiproliferative effects on CHO mutants hypersensitive to cytostatic drugs.

8-Cl-cAMP has been undergoing clinical trials as a potential chemotherapy agent, but there is much discussion in the literature as to whether the active agent is 8-Cl-cAMP itself, or its major metabolite, 8-Cl-adenosine. 8-Cl-cAMP is susceptible to the action of serum enzymes such as phosphodiesterases, and its metabolism when administered to cancer patients raises questions as to the mechanism of action of 8-Cl-cAMP. The stability of 8-Cl-cAMP when incubated with serum, and the effects of both 8-Cl-cAMP and 8-Cl-adenosine on the proliferation of variant lines of CHO cells hypersensitive to 8-Cl-cAMP were investigated. A solid-phase extraction (SPE) purification protocol and the HPLC method previously developed were used to determine 8-Cl-cAMP and 8-Cl-adenosine. Heat treatment of serum inactivated the enzymes in the culture medium responsible for activating 8-Cl-cAMP. Under these conditions 8-Cl-cAMP remained stable and there were no traces of its metabolite, 8-Cl-adenosine. Cell culture experiments showed that 8-Cl-cAMP only affected cell growth in medium that contained untreated serum. In contrast, 8-Cl-adenosine was shown to be growth inhibitory in medium containing either heat-treated or untreated serum. HPLC analysis of the culture medium from the cell culture experiments supported the hypothesis that 8-Cl-cAMP was only effective in inhibiting cell growth after metabolism to 8-Cl-adenosine. Thus further studies of this drug and its mechanism of action should focus on 8-Cl-adenosine.