An improved liquid chromatography–tandem mass spectrometry method for the quantification of 4-aminobiphenyl DNA adducts in urinary bladder cells and tissues

Abstract

Exposure to 4-aminobiphenyl (4-ABP), an environmental and tobacco smoke carcinogen that targets the bladder urothelium, leads to DNA adduct formation and cancer development [1]. Two major analytical challenges in DNA adduct analysis of human samples have been limited sample availability and the need to reach detection limits approaching the part-per-billion threshold. By operating at nano-flow rates and incorporating a capillary analytical column in addition to an online sample enrichment step, we have developed a sensitive and quantitative HPLC–MS/MS method appropriate for the analysis of such samples. This assay for the deoxyguanosine adduct of 4-ABP (dG-C8-4-ABP) gave mass detection limits of 20 amol in 1.25 μg of DNA (5 adducts in 109 nucleosides) with a linear range of 70 amol to 70 fmol. 4-ABP-exposed human bladder cells and rat bladder tissue were analyzed in triplicate, and higher dose concentrations led to increased numbers of detected adducts. It was subsequently established that sample requirements could be further reduced to 1 μg digestions and the equivalent of 250 ng DNA per injection for the detection of low levels of dG-C8-4-ABP in a matrix of exfoliated human urothelial cell DNA. This method is appropriate for the characterization and quantification of DNA adducts in human samples and can lead to a greater understanding of their role in carcinogenesis and also facilitate evaluation of chemopreventive agents.

Mass Spectrometric Mapping of the DNA Adductome as a Means to Study Genotoxin Exposure, Metabolism, and Effect

“…69 Unfortunately, none of the above-mentioned techniques have been applied for untargeted DNA adductome mapping, merely leaving us with the promise of a giant leap forward in this field of research. It has, at least in part, been demonstrated for targeted DNA adduct analysis, but still needs to be established and confirmed for DNA adductomics purposes.…”

Isolation and Detection of Carcinogenic Nucleic Acid Adducts

“…For example, an LOD of ~5 adducts per 10 9 nucleosides with a linear dynamic range between 70 amol and 70 fmol has recently been reported. 53 …”

Application of liquid chromatography-mass spectrometry and differential mobility spectrometry-mass spectrometry for detection and quantitation of biomarkers in complex biological matrices

“…However, in order to achieve the same sensitivity, the amount of DNA required for LC-MS was still very high (500 µg) compared to 32 P-postlabelling (30 µg) [76]. The requirement for the large amount of sample quantity was at least in part due to the extensive offline cleanup [77] which involved multiple steps such as SPE, liquidliquid extraction, and protein precipitation [72,74,[78][79][80][81]. Nevertheless, the advances made with the use of micro-LC mentioned above inspired the incorporation of online cleanup/online SPE methods in combination with column switching which mitigated the sample requirement problem dramatically by reducing sample losses.…”

“…DMS therefore offers orthogonal separation and serves as a prefilter to MS [34,70]. DMS can also reduce the background noise and interferences and thus improve signal to noise (S/N) ratio, which will give better limit of detection (LOD) and limit of quantitation (LOQ), and also prevents contamination of the MS. Isobaric and isomeric compounds can be well separated with DMS, even when structural differences are minor [23,71,72]. Moreover, most significantly, the time required for separation in DMS is of the order of milliseconds compared to the minutes required in LC and thus provides ideal conditions for high-throughput analysis.…”

“…Moreover, most significantly, the time required for separation in DMS is of the order of milliseconds compared to the minutes required in LC and thus provides ideal conditions for high-throughput analysis. Our laboratory has pioneered the use of DMS-MS as a high-throughput alternative to LC-MS for quantitative analysis in a variety of areas, including forensics [23,72] and biomarkers associated with DNA damage from carcinogens [73,74]. In a previous study with a smaller scope, we applied DMS-MS to the analysis of two small molecule water-soluble analytes in NHP urine as a function of dose (0 Gy to 10 Gy).…”