Rapid and sensitive quantification of urinary N7‐methylguanine by isotope‐dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on‐line solid‐phase extraction

Abstract

A rapid, highly specific and sensitive isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to measure N7-methylguanine (N7-MeG) in urine. 15N5-Labeled N7-MeG was synthesized to serve as an internal standard, and an on-line SPE cartridge was used for on-line sample cleanup and enrichment. The urine sample can be directly analyzed within 15 min without prior sample purification. The detection limit for this method was estimated as 8.0 pg/mL (4.8 pmol) on-column. This method was further applied to study exposure to methylating agents arising from cigarette smoke. Sixty-seven volunteers were recruited, including 32 regular smokers and 35 nonsmokers. Urinary cotinine, a major metabolite of nicotine, was also determined using an isotope-dilution LC/MS/MS method. The results showed that urinary levels of N7-MeG observed in smokers (4215 +/- 1739 ng/mg creatinine) were significantly (P < 0.01) higher than those in nonsmokers (3035 +/- 720 ng/mg creatinine). It was further noted that the urinary level of N7-MeG was found to be correlated with that of cotinine for smokers, implying that cigarette smoking resulted in increased DNA methylation, followed by depurination and excretion of N7-MeG in urine. As a result of the on-line extraction system, this method is capable of routine high-throughput analysis and accurate quantitation of N7-MeG, and could be a useful tool for health surveillance of methylating agent exposure.

Repeated Measurements of Urinary Methylated/Oxidative DNA Lesions, Acute Toxicity, and Mutagenicity in Coke Oven Workers

“…Urinary N7-MeG has been previously measured in smokers for assessing the exposure of tobacco-specific nitrosamines. We previously measured urinary N7-MeG among smokers and nonsmokers using LC-MS/MS and reported the mean urinary levels of 4,215 and 3,035 ng/mg creatinine for smokers and nonsmokers, respectively (16). Ichiba et al (44) also reported a mean urinary N7-MeG level of 4,600 ng/mg creatinine among smokers, a level that was found to be significantly decreased by 46% (2,500 ng/mg creatinine) after smoking cessation.…”

“…Urinary N7-MeG concentrations were measured using a published method of LC-MS/MS with on-line solid-phase extraction (16). Fifty microliters of urine were diluted 1,000 times with 96% acetonitrile containing 0.1% formic acid.…”

“…If not repaired, apurinic sites can potentially cause G to T transversions (14). Previously, urinary excretion of N7-MeG has been found to be increased in laboratory animals after exposure to methylating agents (15), and the N7-MeG in urine has been shown to be higher among smokers than among nonsmokers, probably resulting from exposure to tobaccospecific nitrosamines (16,17). Although nitrosamines do exist in coke oven emissions, no information was available on the urinary N7-MeG levels of workers related to the occupational exposure to coke oven emissions.…”

Clinical-Scale High-Throughput Analysis of Urinary 8-Oxo-7,8-Dihydro-2′-Deoxyguanosine by Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry with On-Line Solid-Phase Extraction

“…Accurate quantification of adducted bases at extremely low concentrations has frequently relied on the use of nonradioactive isotope-labeled standards to compensate for the loss of analyte during sample preparation, which has been the most critical step in eliminating the matrix effect for analysis of modified bases by MS (19 ). Furthermore, on-line sample extraction with a column-switching device is an extremely useful technique for automating the preparation of biological samples for LC-MS methods (20,21 ). Its advantages include less ion suppression and relatively short run times as well as higher sensitivity and selectivity, especially for urine samples containing a considerable amount of coeluting interferents.…”

Progress in Sample Pretreatment for Analysis of Estrogens with Liquid Chromatography-Mass Spectrometry

“…In order to seek more effective, environmental friendly, and economic enrichment materials for the application of on-line SPE analysis, many scholars pay attention on the development of new materials, including amino column [65] , hydrosulfonyl column [66] , copper isonicotinate coordination polymer [67] , -cyclodextrin silica gel [68] , polymer columns [69,70] , immunoaffinity column [71,72] , expanded graphite [73] , multi-walled carbon nanotube [74] , and some application even use cotton [75] and cigarette filter [76,77] for on-line adsorption of organic pollutants and perfect results could be obtained. The properties of these new adsorbents are low cost, easy producibility, some of which can be used repeatedly.…”