Development and validation of an ultra high performance liquid chromatography-electrospray tandem mass spectrometry method using selective derivatisation, for the quantification of two reactive aldehydes produced by lipid peroxidation, HNE (4-hydroxy-2(E)-nonenal) and HHE (4-hydroxy-2(E)-hexenal) in faecal water.

Red or processed meat rich diets have been shown to be associated with an elevated risk of colorectal cancer (CRC). One major hypothesis involves dietary heme iron which induces lipid peroxidation. The quantification of the resulting reactive aldehydes (e.g. HNE and HHE) in the colon lumen is therefore of great concern since these compounds are known for their cytotoxic and genotoxic properties. UHPLC-ESI-MS/MS method has been developed and validated for HNE and HHE quantification in rat faeces. Samples were derivatised using a brominated reagent (BBHA) in presence of pre-synthesized deuterated internal standards (HNE-d11/HHE-d5), extracted by solid phase extraction, and then analysed by LC-positive ESI-MS/MS (MRM) on a TSQ Vantage mass spectrometer. The use of BBHA allowed the efficient stabilisation of the unstable and reactive hydroxy-alkenals HNE and HHE. The MRM method allowed selective detection of HNE and HHE on the basis of characteristic transitions monitored from both the 79 and 81 bromine isotopic peaks. This method was validated according to the European Medicines Agency (EMEA) guidelines, by determining selectivity, sensitivity, linearity, carry-over effect, recovery, matrix effect, repeatability, trueness and intermediate precision. The performance of the method enabled the quantification of HNE and HHE in concentrations 0.10-0.15 µM in faecal water. Results are presented on the application to the quantification of HNE and HHE in different faecal waters obtained from faeces of rats fed diets with various fatty acid compositions thus corresponding to different pro-oxidative features.

Liquid chromatography/electrospray ionisation mass spectrometric tracking of 4-hydroxy-2(E)-nonenal biotransformations by mouse colon epithelial cells using [1,2-13C2]-4-hydroxy-2(E)-nonenal as stable isotope tracer.

4-Hydroxy-2(E)-nonenal (HNE), a product of lipid peroxidation, has been extensively studied in several areas, including metabolism with radio-isotopes and quantification in various matrices with deuterium-labelled HNE as standard. The aim of this work was to evaluate the relevance of (13)C-labelled HNE in biotransformation studies to discriminate metabolites from endogens by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). (13)C-Labelled HNE was synthesised in improved overall yield (20%), with the incorporation of two labels in the molecule. Immortalised mouse colon epithelial cells were incubated with 2:3 molar amounts of HNE/(13)C-HNE in order to gain information on the detection of metabolites in complex media. Our results demonstrated that the stable isotope m/z values determined by mass spectrometry were relevant in distinguishing metabolites from endogens, and that metabolite structures could be deduced. Six conjugate metabolites and 4-hydroxy-2(E)-nonenoic acid were identified, together with an incompletely identified metabolite. Stable-isotope-labelled HNE has already been used for quantification purposes. However, this is the first report on the use of (13)C-labelled HNE as a tracer for in vitro metabolism. (13)C-Labelled HNE could also be of benefit for in vivo studies.

Chemistry and biochemistry of lipid peroxidation products.

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as 'second messengers' of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.

Investigation of the regio- and stereo-selectivity of deoxyguanosine linkage to deuterated 2-hydroxyestradiol by using liquid chromatography/ESI-ion trap mass spectrometry.

From previous studies on the reactivity of estradiol 2,3-quinone towards deoxyribonucleosides, it was demonstrated that several isomeric adducts were formed. Although adduction on steroid ring A or B has been evidenced using sequential MS(n) experiments, in some cases attachment positions are difficult to identify unambiguously. In this work, 2-hydroxyestradiol labeled with deuterium at various positions [6beta (1); 6alpha-7alpha (2); 6alpha-6beta-7alpha (3)] have been used. Isomeric adduct differentiation could be achieved using LC-ESI-MS(n). The m/z shift of the quasi-molecular ions as well as the fragmentation pathways suggested that adduction could occur on both C6 and C9 sites of the steroid B ring: Nucleophilic attack of the base on the C6 position of the steroid led to major adducts and addition of the base on the activated C9 site gave minor adducts that were found to be unstable. LC-MS(n) experiments carried out under deuterated medium provided information about some fragmentation processes by studying the m/z shift of fragment ions: (1) the loss of deoxyribose from the quasi-molecular ions took place according to a process involving a deuterium transfer from the deoxyribose alcohol function; (2) the cleavage of the steroid-base linkage involved a deuterium transfer from the hydroxy group of the catechol and likely occurred via the formation of an ion-dipole complex. The model studies conducted in this work provide new information on the fragmentation mechanisms of covalent adducts formed from estrogen quinones and deoxyguanosine, the most reactive DNA base. Besides, the first unequivocal characterization of adducts involving the steroid C9 position is shown by using deuterium labeled estrogen quinones.

Oligonucleotide covalent modifications by estrogen quinones evidenced by use of liquid chromatography coupled to negative electrospray ionization tandem mass spectrometry.

Liquid chromatography coupled to tandem mass spectrometry has been used for the detection and the structural characterization of T-rich model oligonucleotides covalently modified by estradiol-2,3-quinone. After separation by gradient elution, adducts were analyzed by negative electrospray mass spectrometry, enabling to evidence and localize the modifications in the oligonucleotide sequence. Modifications by one molecule of estrogen were evidenced on purines (A, G) whereas no reaction was observed on pyrimidic bases (T). Isomeric adducts were differentiated using tandem mass spectrometry, and energy resolved mass spectrometry allowed to underline differences in the behavior of the adducts towards collisional excitation into an ion trap device.