title: Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard.
abstract: Thiopurines are S-substituted antimetabolites that are widely used in the treatment of hematological malignancies and as immunosuppressants. Because of extensive inter-individual variation in drug disposition and the significant toxicity associated with thiopurine therapy, there is a need for improved individualized treatment. We here present a fast and sensitive method for quantifying the pharmacological end-point of thiopurines, 6-thioguanine (TG) in chromosomal DNA. Purine nucleobases are released from DNA, etheno-derivatized with chloroacetaldehyde, separated by HILIC and quantified by tandem mass spectrometry using endogenous chromosomal guanine as internal standard. The method is linear up to at least 10 pmol TG/μg DNA and the limit of detection and quantification are 4.2 and 14.1 fmol TG/μg DNA, respectively. The matrix (DNA) had no effect upon quantification of TG. SPE recovery was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45-1190 fmol/μg DNA) with intra- and inter-day RSDs of less than 11%. The method uses 2 μg DNA/sample, which can easily be obtained from these patients.
authors.affiliation: Department of Pediatrics, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Jack H
authors.initials: JH
authors.lastname: Jacobsen
authors.forename: Kjeld
authors.initials: K
authors.lastname: Schmiegelow
authors.forename: Jacob
authors.initials: J
authors.lastname: Nersting
mesh_terms: D002138:Calibration; D002648:Child; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000097:blood / Q000737:chemistry* / Q000187:drug effects; D004273:DNA, Neoplasm / Q000097:blood / Q000737:chemistry; D006147:Guanine / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D007962:Leukocytes / Q000737:chemistry; D057230:Limit of Detection; D016014:Linear Models; D015122:Mercaptopurine / Q000378:metabolism / Q000627:therapeutic use; D008727:Methotrexate / Q000378:metabolism / Q000627:therapeutic use; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000188:drug therapy / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013866:Thioguanine / Q000032:analysis*
chemical_list: D004273:DNA, Neoplasm; D006147:Guanine; D004247:DNA; D015122:Mercaptopurine; D013866:Thioguanine; D008727:Methotrexate