Determination of N7-(2-hydroxyethyl)guanine by HPLC with electrochemical detection.
A method has been developed for the determination of N7-(2-hydroxyethyl)-guanine (N7-EtOHGua) via HPLC with electrochemical detection (EC). N7-EtOHGua is the major base adduct formed in DNA upon exposure to ethylene oxide. N7-EtOHGua, released from DNA, was separated from the unmodified nucleobases by chromatography on a reversed-phase column. For electrochemical detection, an amperometric detector cell was used with a glassy carbon working electrode, set at 1.35 V relative to an Ag/AgCl reference electrode. With purified N7-EtOHGua a linear dose-response relation was observed in the range between 0.11 and 13 pmol. The signal-to-noise ratio during analysis of 0.11 pmol N7-EtOHGua was about 8 to 1. Determination of adducts in a series of DNA samples treated with 0.16-10 mM ethylene oxide showed a linear dose-dependent increase in the level of N7-modifications. For DNA samples, the detection limit of this HPLC-EC analysis is 1 N7-EtOHGua per 6 x 10(6) nucleotides.
van Delft
J H
JH
TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
van Weert
E J
EJ
van Winden
M J
MJ
Baan
R A
RA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005027:Ethylene Oxide / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis; D012474:Salmon
C035018:N(7)-hydroxyethylguanine; D006147:Guanine; D004247:DNA; D005027:Ethylene Oxide