A reversed phase high performance liquid chromatography approach in determining total red blood cell concentrations of 6‐thioguanine, 6‐mercaptopurine, methylthioguanine, and methylmercaptopurine in a patient receiving thiopurine therapy

Abstract

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.

Ecto-5′-Nucleotidase and Thiopurine Cellular Circulation: Association with Cytotoxicity

“…As a first step, we measured levels of intracellular 6-TGNs after treating HEK293 cells with TGrib or TGMP. The assay involved determination of the concentration of free 6-TG after the hydrolysis of 6-TGNs with increased temperature and acidic pH, a commonly used approach (Lennard, 1987;Erdmann et al, 1990Erdmann et al, , 1991Lennard and Singleton, 1992;Dervieux and Boulieu, 1998b;Mawatari et al, 1998). After exposure of the cells to 5 M TGrib, the intracellular 6-TGN concentration was 250 pmol/10 6 cells ( Fig.…”

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Erythrocyte Thiopurine Nucleotides and Effect of Thiopurine Methyltransferase Gene Variants on These Metabolites in Patients Receiving Azathioprine/6-Mercaptopurine Therapy

“…Chromatographic methods for the determination of intracellular thiopurine nucleotide concentrations in erythrocytes are available (21)(22)(23)(24) and are based on acid hydrolysis of the thiopurine nucleotide moieties to thiopurine bases (22)(23)(24)(25). We report a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the quantification of erythrocyte 6-TGNs and 6-MMPNs and describe the contribution of the TPMT gene locus to thiopurine nucleotide concentrations in a large population of patients with inflammatory bowel disease receiving thiopurine therapy.…”

How Should Thiopurine Treatment be Monitored?— Methodological Aspects

“…A number of different methods have been described, the majority of which are based on hydrolysis of the TGN back to thioguanine using acid, possibly followed by some sort of sample clean up [4][5][6] . Typically, the thioguanine is detected using UV-absorbance at either around 320 nm [4] , or 340 nm [6] .…”