Stability and analysis of 2-chloro-2'-deoxyadenosine, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine and 2-chloroadenine in human blood plasma

Cladribine (2-chloro-2'-deoxyadenosine, CdA) is a purine nucleoside analog with activity against lymphoproliferative and autoimmune disorders. 2-Chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA), a derivative of CdA with better acid stability, shows a similar in vitro spectrum of activity as CdA. 5-Chloroadenine (CAde) is the major catabolite of both CdA and CAFdA. We have developed a high performance liquid chromatography method to measure CdA, CAFdA and their metabolite CAde in plasma. This method employees an internal standard, chloroadenosine (CAdo), and a C-8 solid-phase extraction to isolate and concentrate the substances. Chromatographic separation was achieved using a C-8 reverse-phase column, with UV detection at 265 nm, which gives a limit of detection of 1 nmol/l for all substances. The method was reproducible with intra- and inter-assay coefficients of variations below 6% at 50 nmol/l and at 5 nmol/l below 23%. The average recoveries of CdA, CAde, CAFdA and the internal standard were higher than 70%. Stability studies of authentic patient samples show that samples containing CdA should be kept in a refrigerator or on ice to prevent degradation. plasma containing CAde should not be kept at -20 degrees C for longer than 10 weeks before analysis. CdA and CAFdA remain almost stable during storage at -20 degrees C for 12 weeks.