Optimized method for determination of 16 FDA polycyclic aromatic hydrocarbons (PAHs) in mainstream cigarette smoke by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method was validated for the determination of 16 polycyclic aromatic hydrocarbons (PAHs) from the FDA list of 93 harmful or potentially harmful constituents of mainstream cigarette smoke (MCS). Target analytes were extracted from total particulate matter using accelerated solvent extraction with a toluene/ethanol solvent mixture. Matrix artefacts were removed by two-step solid-phase extraction process. Three different GC-MS systems [GC-MS (single quadrupole), GC-MS/MS (triple quadrupole) and GC-HRMS (high resolution, magnetic sector)] using the same separation conditions were compared for the analysis of MCS of 3R4F Kentucky reference cigarettes generated under ISO and intense smoking regimes. The high mass resolution (m/∆m ≥ 10,000) and associated selectivity of detection by GC-HRMS provided the highest quality data for the target PAHs in MCS. Owing to the HR data acquisition mode enabling measurement of accurate mass, limits of quantification for PAHs were 5 to 15-fold lower for GC-HRMS than for GC-MS/MS and GC-MS. The presented study illustrates that the optimised sample preparation strategy followed by GC-HRMS analysis provides a fit-for-purpose and robust analytical approach allowing measurement of PAHs at (ultra)low concentrations in MCS. Furthermore, the study illustrates the importance and benefits of robust sample preparation and clean-up to compensate for limited selectivity when low-resolution MS is used.

The crystal structures of Lactococcus lactis MG1363 Dps proteins reveal the presence of an N-terminal helix that is required for DNA binding.

Dps proteins play a major role in the protection of bacterial DNA from damage by reactive oxygen species. Previous studies have implicated the extended lysine-containing N-terminal regions of Dps subunits in DNA binding, but this part of the structure has not previously been observed crystallographically. Here the structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis MG1363 reveal for the first time the presence of an N-terminal alpha helix that extends from the core of the Dps subunit. Consequently, the N-terminal helices are displayed in parallel pairs on the exterior of the dodecameric Dps assemblies. Both DpsA and DpsB bind DNA. Deletion of the DpsA N-terminal helix impaired DNA binding. The N-terminal Lys residues of Escherichia coli Dps have been implicated in DNA binding. Replacement of the lactococcal DpsA Lys residues 9, 15 and 16 by Glu did not inhibit DNA binding. However, DNA binding was inhibited by EDTA, suggesting a role for cations in DNA binding. In contrast to E. coli, Bacillus brevis and Mycobacterium smegmatis Dps:DNA complexes, in which DNA interacts with crystalline Dps phases, L. lactis DNA:Dps complexes appeared as non-crystalline aggregates of protein and DNA in electron micrographs.