Determination of Intracellular Levels of 6-Mercaptopurine Metabolites in Erythrocytes Utilizing Capillary Electrophoresis with Laser-Induced Fluorescence Detection

• Published in: Analytical biochemistry 1995, Vol.224 (1), p.315-322
• Main Authors: Rabel, S.R., Stobaugh, J.F., Trueworthy, R.
• Format: Article
• Language: English
• Subjects: Electrophoresis; Erythrocytes - chemistry; Fluorescence; Guanine Nucleotides - blood; Guanosine Diphosphate - analogs & derivatives; Guanosine Diphosphate - blood; Guanosine Triphosphate - analogs & derivatives; Guanosine Triphosphate - blood; Humans; Mercaptopurine - metabolism; Thionucleotides - blood
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1995.1046

Capillary electrophoresis proved to be a useful technique for the analysis of intracellular levels of 6-thioguanosine mono-, di-, and triphosphate with analysis times of 20 min. Conditions required for baseline separation of the thioguanine nucleotides consisted of a 25 mM KH 2PO 4 (pH 8.0) buffer and a separation voltage of +28 kV. Laser-induced fluorescence detection (λ ex = 325 nm, λ em = 410 nm) of the thioguanine nucleotide metabolites of 6-mercaptopurine (6-MP) was possible following oxidation of the thiol functionality. Tedious extraction procedures involving mercury cellulose resins or phenyl mercury adduct formation, which had been required previously for the selective extraction of thiopurines from erythrocytes, were unnecessary due to the overall specificity of the approach. However, the inclusion of 50 mM EDTA in the sample preparation was required to inhibit the anabolic/catabolic enzymatic activity, which was responsible for the degradation of the analytes. The method demonstrated linearity from 5 to 1700 pmol/100 μl red blood cells for the three analytes (RSDs ≤ 8%). The feasibility of the method was demonstrated for the quantitation of 6-thioguanine nucleotides in patients receiving either oral or intravenous 6-MP therapy.