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 an alog with activity against lymphoproliferative and autoimmune disorder s. 2-Chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA), a derivative of CdA with better acid stability, shows a similar in vitro spectrum o f activity as CdA. 5-Chloroadenine (CAde) is the major catabolite of b oth CdA and CAFdA. We have developed a high performance liquid chromat ography method to measure CdA, CAFdA and their metabolite CAde in plas ma. This method employees an internal standard, chloroadenosine (CAdo) , and a C-8 solid-phase extraction to isolate and concentrate the subs tances. Chromatographic separation was achieved using a C-8 reverse-ph ase column, with UV detection at 265 nm, which gives a limit of detect ion of 1 nmol/l for all substances. The method was reproducible with i ntra- 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 k ept in a refrigerator or on ice to prevent degradation. plasma contain ing 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.