mercaptopurine and 6-methylthioguanine

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.

Topics: Antimetabolites, Antineoplastic; Child; Chromatography, High Pressure Liquid; Erythrocytes; Humans; Hydrogen-Ion Concentration; Hydrolysis; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Thioguanine

A reversed-phase high-performance liquid chromatographic (HPLC) procedure was developed to quantify intracellular lymphocyte 6-thioguanine, methylmercaptopurine and methylthioguanine. The free base of each metabolite was obtained by acid hydrolysis, which allowed for a total determination of thiopurine metabolites. 6-Thioguanine was analyzed on an octadecylsilane column using acetonitrile-10 mM sodium phosphate (11:89), pH 7, containing 0.06% tetrabutylammonium chloride. 6-Thioguanine was oxidized with potassium permanganate, and fluorescence was measured at 330 nm excitation and 410 nm emission. Methylmercaptopurine and methylthioguanine were separated on a cyanopropylsilane column using methanol-40 mM sodium phosphate (22:78), pH 2.7, and detected by ultraviolet absorbance at 314 and 290 nm, respectively.

Topics: Chromatography, High Pressure Liquid; Fluorescence; Humans; Lymphocytes; Mercaptopurine; Thioguanine

A reversed phase high performance liquid chromatographic procedure was developed to quantify 6-thioguanine, 6-mercaptopurine, methylthioguanine, and methylmercaptopurine in red blood cells. The free base of each thiopurine was liberated from the respective nucleoside and nucleotide moiety by acid hydrolysis, which allowed for a determination of the total thiopurine present. 6-Thioguanine and 6-mercaptopurine were analyzed on an octadecylsilane column using methanol + 20 mM sodium phosphate (15:85), pH 7.5, containing 0.07% tetrabutylammonium chloride. Detection was by potassium permanganate oxidation and fluorescence detection at 290 nm excitation and 400 nm emission. Methylmercaptopurine and methylthioguanine were analyzed on a cyanopropylsilane column using methanol + 40 mM sodium phosphate (18:82), pH 2.7, and then ultraviolet absorption at 314 nm and 290 nm, respectively. The method was used to quantify the four primary thiopurines present in red blood cells of an acute lymphoblastic leukemia patient. The procedure may be a therapeutic monitoring technique that quantifies the cytotoxic drug burden in patients receiving azathioprine or 6-mercaptopurine therapy.

Topics: Child; Chromatography, High Pressure Liquid; Erythrocytes; Humans; Male; Mercaptopurine; Microchemistry; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Quality Control; Thioguanine