Development Of Ultrasensitive UPLC-MS/MS Methods For The Analysis Of Etheno-DNA Adducts In Human Samples

DNA is the most important genetic material, and it is of great significance for cell activity and physiological function to keep its stability and integrity of molecular structure. DNA oxidative damage was mainly caused by active oxygen free radicals and its metabolites which attack DNA. DNA is the main goal that endogenous and exogenous such as radiation and chemical oxidants of active oxygen free radicals attacked. One of the most common phenomenon is to combine with DNA. DNA adducts can be as biological markers to reflect the toxic substances that man contacted, therefore, there is a prerequisite to develop the ultrasensitive and high throughout methods for the analysis of DNA adducts to be used in human biomonrtoring and molecular studies.In this paper, we develop the ultrasensitive and high-throughout UPLC-MS/MS methods for the analysis of etheno-DNA (such as sdA and εdC) in human samples, the steps of methods development are following:1. Synthesis of standards:synthesis of3,N4-etheno-2’-deoxycytidine (sdC) to be a standard; synthesis of stable isotop lebelled [15N5]εdA and [15N3]εdC to be internal standards. Crude [15N5]-sdA and [15N3]sdC were purified by a reverse phase HPLC.Quantification of synthetic [15N5]εdA is performed by UV spectrometry using an extinction coefficient of10,300M"1cm-1at260nm as an internal standard. Quantification of synthetic [15N3]sdC is performed by UV spectrometry using an extinction coefficient of12,000M-1cm-1at272nm as an internal standard..2. Optimization of precedures for preparation of blood and urinary samples, micrococcal endonuclease, spleen phosphodiesterase and nuclease P1were used for DNA hydrolysis and dephosphorylation, a RP-HPLC method was used for enrichment of sdA and sdC adducts in human blood. C18OH solid-phase silica column conbined with RP-HPLC were used for enrichment and purification of urinary εdA and sdC. The recovery of εdA and sdC in human blood are83.7±7.1%and83.3±2.9%. The recovery of εdA and εdC in human urine are89.6±6.52%and88.9±7.7%. 3. Optimization of UPLC-MS/MS conditions for the analysis of εdA and εdC in human samples. An ACQUITY TQD mass spectrometer (Waters ACQUITY TQD), equipped with an ESI interface, Positive ions were acquired in the multiple reaction monitoring (MRM) mode Samples were quantified by comparing the areas of the εdA and EdC chromatogram peak to those of the [15N5]εdA and [15N3]εdC (I.S.) chromatogram peak. Additional [15N5]sdA and [15N3]εdC spiked into blood sample as internal standards.The limit of detection of εdA and εdC in human blood are0.65fmol/ml and0.83fmol/ml. The limit of detection of εdA and εdC in human urine are0.51fmol/ml and0.63fmol/ml.