Analysis of 6-mercaptopurine, 6-thioguanine nucleotides, and 6-thiouric acid in biological fluids by high-performance liquid chromatography

We present a rapid, sensitive and specific high-performance liquid chromatographic method for the analysis of 6-mercaptopurine (6MP), 6-thioguanine nucleotides (6TGN) and 6-thiouric acid (6TU), with excellent chromatographic separation of the thiopurines. Thiopurines in plasma and erythrocytes were extracted by mercuricellulose and re-eluted with beta-mercaptoethanol. For quantitative detection a reverse phase column (Lichrosorb RP-18 4 mm X 30 cm) and a UV detector were used. Detection limits were 20 nmol/l for 6MP in plasma, 250 nmol/l for 6TGN in erythrocytes, and 10 nmol/l and 15 nmol/l, respectively, for 6MP and 6TU in urine diluted 1:100 with beta-mercaptoethanol. Within-run and between-run variations were less than 10%. Recovery of 6MP added to plasma, and 6TGN monophosphate added to haemolysed erythrocytes were 91% and 81%, respectively.