Quantification of urinary etheno-DNA adducts by column-switching LC/APCI-MS/MS

Abstract

Lipid peroxidation induced etheno-DNA adducts are promutagenic and have been suggested to play a causal role in the development of human cancers. Therefore, human biomonitoring of etheno-DNA adducts in urine has been suggested as a potential marker for oxidative stress-related DNA damage. For quantitative determination, a column-switching LC/APCI-MS/MS method was developed for simultaneous analysis of Ade, dC, and dA in human urine. Quantitative validation parameters (precision, within-day repeatability, and betweenday reproducibility) yielded satisfactory results below 10%. Limit of quantification for Ade, dC, and dA was 5.3 fmol, 7.5 fmol, and 1.3 fmol on column, respectively. Mean urinary excretion rates of a six healthy volunteers were 45.8 pmol Ade/24 h, 96.8 pmol dC/24 h, and 18.1 pmol dA/24 h. The demonstrated levels of performance suggest a future applicability of this method to studies of cancer and other diseases related to oxidative stress in humans. To our knowledge, this is the first method described that allows simultaneous determination of Ade, dC, and dA in human urine samples. [2], are also generated endogenously by reactions of DNA with products derived from lipid peroxidation and oxidative stress [3]. These DNA adducts have miscoding potential and specific repair pathways supporting the hypothesis that -DNA adducts play a causal relationship in carcinogenesis [4]. Furthermore, several studies have found elevated -DNA adduct levels in disorders known as risk factors for cancers [5,6]. Therefore -DNA adducts have been proposed as biomarkers for human cancers associated with certain lifestyles or chronic inflammations [4]. Upon DNA repair, -DNA adducts are excreted in the urine and could, in that respect, be used to investigate the body burden of DNA damage.The analytical procedures for urinary DNA adducts are challenging indeed. The presence of many interfering substances in concentrations far greater than those of the DNA adducts demands high selectivity and low limit of quantitation for unequivocal identification and quantification. Methods using high-performance liquid chromatography (HPLC) with fluorescence detection have been used for dA determination [4,7] after immunoaffinity clean-up and a very tedious sample preparation. Furthermore, the internal standard was measured separately. Several etheno-DNA adducts have been determined in urine from healthy humans using gas chromatography-electron capture negative chemical ionization mass spectrometry (GC/EC-NCI/ MS) [8 -11]. Gas chromatography offers the advantage of greater peak resolution compared to conventional LC. However, the requirement for derivatization before analysis is a major limitation. Recently, LC-MS have been employed in determination of etheno adducts from human urine using electrospray ionization (ESI) [12][13][14]. Atmospheric pressure chemical ionization (APCI) has been used for analysis of dA and 1,N 2 -dG in vitro [15] and of dA in human urine [16].In this paper, we report that solid-phase extraction (SPE) for preconcent...

Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair

“…ɛdA and ɛdC excreted in a few milliliters of urine can be measured by an immunoenriched high-performance liquid chromatography fluorescence or 32 P-postlabeling TLC methods [26,27]. For quantifying ɛdA, ɛdC, ɛC, and ɛA in urine, mass spectrometric methods have also recently become available [28][29][30][31]. Several sensitive methods for analyzing M 1 dG in DNA and in urine were reported [32,33].…”

Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

“…Quantification of the standard is performed by UV spectroscopy using an extinction coefficient of 10,300 M -1 cm -1 at 260 nm. 42,43 dX and dO standards are synthesized by a modification of the method of Suzuki et al 45 Briefly, incubate 10 mM 2′-deoxyguanosine or [ 15 N 5 ]-2′-deoxyguanosine with 100 mM NaNO 2 in 0.3 M sodium acetate buffer (pH 3.7) at 37 °C for 6 h. dX and dO are purified by HPLC using a LUNA C18 reversed-phase column (250 × 3 mm, 5 μm particle size, 100 Å pore size; Phenomenex, Torrance, CA) with elution performed at a flow rate of 0.4 ml/min with 1% acetonitrile in 50 mM ammonium acetate (pH 7.4) for the first 5 min, followed by a linear gradient of 1-25% acetonitrile for 5 min; holding at 25% for 10 min; then a reversal of the gradient to 1% for 5 min; and finally eluting at 1% acetonitrile over the last 5 min. Fractions containing the products are dried under vacuum and redissolved in water followed by desalting on a second HPLC system consisting of a Haisil HL C18 reversed phase column (250 × 4.6 mm, 5 μm particle size, 100 Å pore size; Higgins Analytic Inc, Mountain View, CA) eluted with 5% acetonitrile in water at a flow rate of 0.4 ml/min, with elution confirmed using commercially available standards.…”

Trace LC/MS quantitative analysis of polypeptide biomarkers: Impact of 1‐D and 2‐D chromatography on matrix effects and sensitivity

“…Since urine is a matrix of highly variable composition [13], the present study was performed on human plasma prepurified by SPE. The composition of human plasma is much more consistent than urine.…”