High-performance liquid chromatographic assay of methylthioguanine nucleotide

This paper describes a specific and sensitive reversed-phase HPLC assay for the measurement of 6-methylthioguanine (methyl-TG) and methyl-TG nucleotides (methyl-TGNs) in red blood cells (RBCs), which is suitable for routine clinical use. Briefly, an ethyl acetate extract of RBCs is evaporated and reconstituted in 0.1 M HCl. The methyl-TG is separated from other thiopurines by reversed-phase HPLC and quantitated using UV detection. For the measurement of methyl-TGNs the free base (methyl-TG) is obtained by acid hydrolysis of the nucleotide back to the parent thiopurine. The intra-assay C.V. over the concentration range of 0.055-1.10 nmol methyl-TG per 4x10(8) (100 microl) RBCs ranged from 2.8 to 8.5%, and the mean recovery of methyl-TG over the calibration range was 61.6% (coefficient of variation, C.V., 3.8%). The lower limit of reproducibility was 0.055 nmol extracted from 100 microl RBCs. Analysis of blood samples from children with leukaemia receiving 6TG chemotherapy, revealed RBC methyl-TGNs at concentrations ranging from 323 to 1365 pmol per 8x10(8) RBCs. No methyl-TG was detected in any of the patient samples.