Sensitive determination of 8-chloroadenosine 3',5'-monophosphate and 8-chloroadenosine in plasma by high-performance liquid chromatography

Abstract

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is progressing through clinical evaluation as an anticancer drug. There is debate as to whether 8-Cl-cAMP is the active principal or its cytotoxic metabolite 8-Cl-adenosine. Separate high-performance liquid chromatographic methods are described for (i) 8-Cl-cAMP and its nucleotide metabolites (with 8-Br-cAMP as internal standard), and (ii) 8-Cl-adenosine. Both methods use a reversed-phase (Spherisorb ODS-2) stationary phase and a mobile phase consisting of sodium phosphate buffer (10 mM, pH 3.5) and methanol but with gradient elution for the nucleotides and isocratic elution for 8-Cl-adenosine. 8-Cl-cAMP and related nucleotides are extracted from plasma using strong anion-exchange solid-phase extraction (SPE) and 8-Cl-adenosine is extracted using reversed-phase (C8) SPE. Both techniques enabled analyses to be performed at high detector sensitivity with minimal interference. Limit of detection in plasma was 10 ng/ml for both 8-Cl-cAMP and 8-Cl-adenosine. When applied to the analysis of plasma samples from a patient treated with a low dose continuous infusion of 25 micrograms/kg/h, steady-state concentrations centred around 60 ng/ml 8-Cl-cAMP were determined. In the same patient 8-Cl-adenosine was not detected. Application of this methodology will aid in the further development of 8-Cl-cAMP as a potential new form of anticancer treatment.