Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

Abstract

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukaemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di-and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measure ment of the low concentrations of mono-, di-and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/109 cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.

Effects on transmethylation by high-dose 6-mercaptopurine and methotrexate infusions during consolidation treatment of acute lymphoblastic leukemia

“…Plasma and RBC were isolated as described [20]. RBC transfusions, an essential part of the supportive treatment during the inten sive consolidation treatment, were given between the courses, but never during a course.…”

“…RBC transfusions, an essential part of the supportive treatment during the inten sive consolidation treatment, were given between the courses, but never during a course. Dithiothreitol was added to plasma, RBC, and urine to prevent oxidation of the thiol groups [20]. Urine was collected during the 24 hr of the 6MP infusion as well as during the next 24 hr, either as a pooled 24'hr sample or as fractionated samples of approx imately 6 hr.…”

“…Detection limits for the methylated metabolites in plasma were 20-50 nM and for MetIMP 11 pmol per 100 (xL injection [20j. A previously indeterminate metabolite of 6MP [20] was identified as MeM8QHP. It was measured as >ther methylthiopurine metabolites and eluted at 30 urine c min [20].…”

HPLC Determination of the Levels of 6-Mercaptopurine Metabolites Suitable for the Clinical Risk Assessment of its Toxicity among Egyptian Children with Acute Lymphocytic Leukemia

“…Being developed earlier, the procedure of Lennard has been used exclusively in clinical studies to establish therapeutic ranges for 6-TG or toxic levels for 6-MMP, however the procedure was tedious, reagent consuming, and mercury-based waste generator. An anion exchange method for the determination of intracellular mono-, di-, and triphosphate thiopurine and methylthiopurine nucleotides in RBCs was also described by Keuzenkamp-Jansen et al [30]. Dervieux et al later worked on implementing a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS) method [19].…”

How Should Thiopurine Treatment be Monitored?— Methodological Aspects

“…A number of attempts have been made to specifically measure TGMP, TGDP, and TGTP, [10,15,21] . Most of these have been modifications of an extraction protocol developed by Rabel et al [22] .…”