Within the group of DNA alkylation products, phosphotriesters (PTE) ar
e among the most stable lesions. Hence, alkyl PTE are attractive bioma
rkers for DNA alkylation monitoring purposes, We have developed a P-32
-postlabelling method for the analysis of both methyl and ethyl PTE in
DNA, Since PTE bonds are not cleaved by any known DNA degrading enzym
e, they are easily obtainable as PTE dinucleoside monophospates. A pur
ification step, separating the PTE dinucleoside monophosphates from in
terfering compounds, such as mono- or oligonucleotides resulting from
incomplete digestion of DNA, was developed using Waters C-18 Sep-Pak c
artridges, Phosphotriester dinucleoside monophosphates themselves are
not a substrate for phosphorylation by polynucleotide kinase, Polynucl
eotide kinase probably requires a negative charge on the phosphate clo
sest to the 5'-end, Therefore, prior to the postlabelling step they ha
ve to be converted into either phosphodiester dinucleoside monophospha
tes or 3'-phosphate alkylated mononucleotides by treatment with alkali
. For analysis of the labelled compounds we developed a two-step proce
dure, combining TLC and HPLC, that gave very straightforward informati
on on the composition of the rather complex mixture. The detection lim
it is similar to 20 fmol PTE.