High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the ne w anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple pro tein precipitation protocol, using methanol. Chromatographic separations we re performed using a HiChrom HIRPB (25 cmX4.6 mm I.D.) column, with mobile phase of acetonitrile-ammonium formate buffer (0.05 M) (22:78, v/v), with f inal pH adjusted to 3.6 with formic acid. The how-rate was maintained at 1. 2 mi min(-1). Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visib le range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The Validated quantification range of the method was 0.05-10.0 mu g ml(-1) in mouse plasma. The inter-day relative standard dev iations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 mu g ml(-1) to 2.9 % and 3.3% at 10.0 mu g ml(-1) for AQ4N and AQ4, respectively. The intra-da y RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0. 05 mu g ml(-1) to 7.6% and 11.5% at 10.0 mu g ml(-1) for AQ4N and AQ4, resp ectively. The overall recovery of the procedure for AQ4N was 89.4+/-1.77% a nd 76.1+/-7.26% for AQ4. The limit of detection was 50 ng ml(-1) viith a 10 0 mu l sample volume. The method described provides a suitable technique fo r the future analysis of low levels of AQ4N and AQ4 in clinical samples. (C ) 2000 Elsevier Science B.V. All rights reserved.