Analysis of N7‐(benzo[a]pyrene‐6‐yl)guanine in urine using two‐step solid‐phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Abstract

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.

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

“…The column‐switching system has been described previously 20. In brief, it consisted of a switching valve (two‐position microelectric actuator from Valco, Houston, TX, USA) and a Nucleosil NH2 cartridge (35 × 4.6 mm i.d., 10 µm).…”

“…Recently, liquid chromatography with tandem mass spectrometry (LC/MS/MS) has become a powerful technology to overcome the sensitivity and specificity issues in analysis of DNA adducts. Accurate quantitation of extremely low concentrations of adducted bases has frequently relied on the use of non‐radioactive isotope‐labeled standards to compensate for loss of analyte during sample preparation, which has been the most critical step to eliminate the matrix effect for analysis of modified bases by mass spectrometry 19, 20. Moreover, on‐line sample extraction with a column‐switching device is an extremely useful technique to automate the preparation of biological samples for LC/MS methods 21.…”

Quantitation of DNA Adducts by Stable Isotope Dilution Mass Spectrometry

“…This method allows for urinary BP-guanine adducts to be analyzed as a biomarker of risk in workers exposed to BP. 118 …”

Ultrahigh‐pressure dual online solid phase extraction/capillary reverse‐phase liquid chromatography/tandem mass spectrometry (DO‐SPE/cRPLC/MS/MS): A versatile separation platform for high‐throughput and highly sensitive proteomic analyses

“…Online SPE/cRPLC has been applied widely for high-efficiency and desalting affinity analyses [10,[23][24][25]; the online coupling of the SPE method to an LC systems provides an indispensable technology for modern proteomics. Among its various advantages, the ability to effectively separate trace amounts of biological analytes [26] and the wide analytesolid phase interaction range [10] enable researchers to identify low-abundance proteins with great efficiency when using bottom-up approaches.…”