TY - JOUR
T1 - Determination of leukocyte DNA 6-thioguanine nucleotide levels by high-performance liquid chromatography with fluorescence detection
AU - Olesen, Karen-Marie
AU - Hansen, Steen Honoré
AU - Sidenius, Ulrik
AU - Schmiegelow, Kjeld
PY - 2008/3/15
Y1 - 2008/3/15
N2 - 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).
AB - 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).
KW - Child
KW - Chromatography, Affinity
KW - Chromatography, High Pressure Liquid/methods
KW - DNA/blood
KW - Guanine Nucleotides/analysis
KW - Humans
KW - Hydrogen-Ion Concentration
KW - Hydrolysis
KW - Leukocytes/chemistry
KW - Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood
KW - Quality Control
KW - Reproducibility of Results
KW - Sensitivity and Specificity
KW - Thioguanine/analogs & derivatives
KW - Thionucleotides/analysis
U2 - 10.1016/j.jchromb.2008.02.007
DO - 10.1016/j.jchromb.2008.02.007
M3 - Journal article
C2 - 18313997
SN - 1570-0232
VL - 864
SP - 149
EP - 155
JO - Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
IS - 1-2
ER -