Derivatization of thymine and thymine photodimers with 4-bromomethyl-7-methoxycoumarin for fluorescence detection in high-performance liquid chromatography.
Exposure of DNA to utraviolet radiation results in the formation of a number of photoproducts, including thymine photodimers. A sensitive and selective analytical method based on high-performance liquid chromatography (HPLC) and fluorescent labeling with 4-bromomethyl-7-methoxycoumarin has been developed to quantify both thymine and thymine photodimers. The identity of the thymine and thymine dimer derivatives were determined by HPLC-electrospray ionization mass spectrometry. The derivatization reaction yield was maximized by optimizing several reaction variables. The limit of detection for HPLC method was 1.0 pmol thymine and 0.4 pmol thymine dimer for S/N = 3.
Robinson
J K
JK
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309, USA.
Scott
W G
WG
Rowlen
K L
KL
Birks
J W
JW
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007202:Indicators and Reagents / Q000737:chemistry*; D010777:Photochemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013941:Thymine / Q000737:chemistry*; D014468:Umbelliferones / Q000737:chemistry*
D007202:Indicators and Reagents; D014468:Umbelliferones; C000317:4-bromomethyl-7-methoxycoumarin; D013941:Thymine