Determination of leukocyte DNA 6-thioguanine nucleotide levels by high-performance liquid chromatography with fluorescence detection.
A HPLC method for determination of 6-thioguanine nucleotide in DNA was developed. Leukocyte DNA was isolated from peripheral blood, derivatized with chloroacetaldehyde and the formed etheno derivatives N(2),3-etheno 6-thioguanine (epsilon6TG), 1,N(6)-etheno adenine (epsilonA) and N(2),3-etheno guanine (epsilonG) were released from the DNA backbone by hydrolysis at pH 6.0 and 80 degrees C for 60 min. After extraction of epsilon6TG by immobilized metal ion affinity chromatography (IMAC) the sample was analysed by ion-pair reversed-phase HPLC with fluorescence detection. The limit of quantification was 9.0 nM and the intra- and interday precision ranged from 2.8 to 15.5%. In a small cohort of eight children with acute lymphoblastic leukaemia (ALL), a median of one 6-thioguanine base was found for each 3000 normal bases (range 1:2000-1:11000).
Olesen
Karen-Marie
KM
Department of Pharmaceuticals and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. kmo@farma.ku.dk <kmo@farma.ku.dk>
Hansen
Steen Honoré
SH
Sidenius
Ulrik
U
Schmiegelow
Kjeld
K
D002648:Child; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000097:blood*; D006150:Guanine Nucleotides / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007962:Leukocytes / Q000737:chemistry*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000235:genetics; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013866:Thioguanine / Q000031:analogs & derivatives / Q000032:analysis; D013873:Thionucleotides / Q000032:analysis*
D006150:Guanine Nucleotides; C529216:N2,3-etheno-6-thioguanine; D013873:Thionucleotides; C003964:6-thioguanylic acid; D004247:DNA; D013866:Thioguanine