Different macroporous, monolithic capillary columns were prepared to separate various bile acid mixtures through capillary electrochromatography (CEC) at high efficiency. These columns are shown to be ideally suitable for coupling to an electrospray ionization/ion trap mass spectrometer. Detection and structural identification of different bile acid derivatives in either the positive- or negative-ion mode necessitated column technologies with different polarities and the capabilities of a reversed electroosmotic flow. High column efficiencies (610,000 theoretical plates/meter for glycocholic acid in normal-phase separation) were preserved in the coupling to mass spectrometry (MS), with the detection limits of approximately 40 femtomole (for cholic acid) and identification through CEC/MS/MS.

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Prostaglandins (PG) and isoprostanes (iso-PG) may be derived through cyclooxygenase or free radical pathways and are important signaling molecules that are also robust biomarkers of oxidative stress. Their quantification is important for understanding many biological processes where PG, iso-PG, or oxidative stress are involved. One of the common methods for PG and iso-PG quantifications is LC-MS/MS that allows a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the currently used LC-MS/MS methods require a multi-step extraction and a long (within an hour) LC separation to achieve simultaneous separation and analysis of the major iso-PG. The developed and validated for brain tissue analysis one-step extraction protocol and UPLC-MS/MS method significantly increases the recovery of the PG extraction up to 95%, and allows for a much faster (within 4 min) major iso-PGE2 and -PGD2 separation with 5 times narrower chromatographic peaks as compared to previously used methods. In addition, it decreases the time and cost of analysis due to one-step extraction approach performed in disposable centrifuge tubes. All together, this significantly increases the sensitivity, and the time and cost efficiency of the PG and iso-PG analysis.

Among the various reactions of lipid peroxidation products with proteins, 2-alkenals have been shown to react extensively with the epsilon-amino group of lysine residues [Zídek et al. (1997) Chem. Res. Toxicol. 10, 702-710]. To obtain additional information about the kinetic and mechanistic aspects of this modification, a model peptide (N-acetylglycyllysine O-methyl ester) was reacted with 2-hexenal. The reaction products were characterized through a combination of NMR and MS techniques. The structural elucidation efforts have shown the formation of pyridinium salts through the reaction of two or more alkenals with one amino group. Kinetic data were obtained using a continuous infusion of the reaction mixture into an electrospray ionization mass spectrometer. A mechanism is proposed that offers an alternative model for the formation of stable protein cross-links. The reaction progresses through a Schiff base intermediate to form a dihydropyridine species which can be alternatively reduced to form various 3,4- or 2,5-substituted pyridinium species or react with another Schiff base to form a trialkyl-substituted pyridinium structure. The stoichiometry of this structure (aldehyde/amine) is 3:2, in contrast to the widely accepted 1:2. Therefore, it represents another possible cross-linking mechanism for bifunctional products of lipid peroxidation.

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