Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons

Abstract

LC-MS using electrospray ionization is currently the method of choice in bio-organic analysis covering a wide range of applications in a broad spectrum of biological media. The technique is noted for its high sensitivity but one major limitation which hinders achievement of its optimal sensitivity is the signal suppression due to matrix inferences introduced by the presence of coextracted compounds during the sample preparation procedure. The analysis of DNA adducts of common environmental carcinogens is particularly sensitive to such matrix effects as sample preparation is a multistep process which involves "contamination" of the sample due to the addition of enzymes and other reagents for digestion of the DNA in order to isolate the analyte(s). This problem is further exacerbated by the need to reach low levels of quantitation (LOQ in the ppb level) while also working with limited (2-5 μg) quantities of sample. We report here on the systematic investigation of ion signal suppression contributed by each individual step involved in the sample preparation associated with the analysis of DNA adducts of polycyclic aromatic hydrocarbon (PAH) using as model analyte dG-BaP, the deoxyguanosine adduct of benzo[a]pyrene (BaP). The individual matrix contribution of each one of these sources to analyte signal was systematically addressed as were any interactive effects. The information was used to develop a validated analytical protocol for the target biomarker at levels typically encountered in

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts

“…Moreover, many isobaric interferences and ion suppressive components are present in the DNA digestion matrix. Hence, we adapted the methodology developed by the Vouros laboratory, 39 and lowered the amount of DNase I, which was the primary source of background contamination; alternatively, DNase I was replaced with a recombinant endonuclease — Benzonase. 4-ABP-, B[ a ]P- and PhIP-modified CT DNA with known levels of adducts were used as controls to ensure that the enzymatic digestion efficiency and the recoveries of adducts were quantitative.…”

Differential Mobility Spectrometry-Mass Spectrometry (DMS-MS) in Radiation Biodosimetry: Rapid and High-Throughput Quantitation of Multiple Radiation Biomarkers in Nonhuman Primate Urine

“…Because of its very high concentration in human and NHP urine, creatinine could be quantified by direct dilution, since high dilution reduces matrix effects effectively [63–66]. The creatinine calibration curve was generated using a 2500-fold dilution in solvent (50% ACN + 0.1% formic acid) with IS addition.…”

“…Isobaric and isomeric compounds often can be separated with DMS, even when structural differences are minor [32–34]. 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 [33, 34] and biomarkers associated with DNA damage from carcinogens [35, 36]. …”

DNA adducts: Formation, biological effects, and new biospecimens for mass spectrometric measurements in humans

“…The measurement of DNA adducts is challenging in humans because of the low level of chemical modification of DNA. Sample preparation steps such as DNA isolation, DNA adduct cleanup/enrichment, choice of HPLC columns, LC solvents, even the choice of sample vials, can have a pivotal impact on the cleanliness of the extract, recovery of the DNA adduct, and the success of the analysis (section 3) …”