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Quantitation of normal and formaldehyde‐modified deoxynucleosides by high‐performance liquid chromatography/UV detection
Author(s) -
Zhong Weiguang,
Hee Shane Que
Publication year - 2004
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.337
Subject(s) - chemistry , deoxyadenosine , chromatography , formaldehyde , reagent , centrifugation , deoxyguanosine , thymidine , ammonium acetate , hydroxymethyl , dna , high performance liquid chromatography , column chromatography , detection limit , stereochemistry , biochemistry , organic chemistry
A sensitive and selective method was developed for the rst time to quantify simultaneously the normal and formaldehyde (FA)‐modied bases in human placental DNA treated with 100 ppm FA for 20 h at 37°C. Digestion of DNA to deoxynucleosides with DNase I, phosphodiesterase and alkaline phosphatase occurred in that order with centrifugation steps. The normal and FA‐modied deoxynucleosides were then resolved from one another and reagent blank interferences to produce selective separation through high performance liquid chromatography–ultraviolet detection at 254 nm. A C 18 reversed‐phase column facilitated the resolution using 5 m m ammonium acetate and a gradient of 0–6% methanol at ow rates of 0.3–1.4 mL/min before column cleaning. The lower quantiable limits for deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, N 6 ‐hydroxymethyldeoxyadenosine (N 6 ‐dA), N 2 ‐hydroxymethyldeoxyguanosine (N 2 ‐dG) and N 4 ‐hydroxymethyldeoxycytidine (N 4 ‐dC) were 11, 7.6, 12, 15, 10, 10 and 22 pmol, respectively. The abundance order of the modied deoxynucleosides was N 6 ‐dA > N 2 ‐dG > N 4 ‐dC. dT did not form hydroxymethyl derivatives. The respective concentrations were about 6.0, 10.0 and 23 pmol of modied deoxynucleosides in 80 µg of human placental DNA after treatment with 100 µg/mL of formalin for 20 h at 37°C. The stabilities of N 6 ‐dA and N 2 ‐dG were much better at −20°C than at 25°C, where the respective halftimes were about 50.1 and 21.0 h. Copyright © 2004 John Wiley & Sons, Ltd.