On the other hand, 3,7,11,11-tetramethyl-10,15-dioxo-hexadec-2,4,6,8-tetra-enal was identified as two different hydrazones: one of them resulting from the reaction between DNPHi and one carbonyl group and the other from the reaction between DNPHi and two carbonyl groups of the CC. This fact shall be related to differences in the reaction rates between DNPHi and each different CC structure. BLU9931 mouse The factors which can interfere with these rates include the electrophilic character of the carbonylic carbons, the steric conditions in the molecules (for example, for aldehydes the carbonyl group is located in the molecule’s extremity, making easier the approximation of a reagent than in ketones), the pH of the media

and the DNPHi concentration Metabolism inhibitor (Bicking and Cooke, 1998). The time elapsed between the sampling and the elution of the cartridge for analysis is also a critical factor, depending on the type of compound reacting with the derivatisation agent (Van Leeuwen, Hendriksen, & Karst, 2004). Some authors (Zwiener, Glauner,

& Frimmel, 2002) have demonstrated that a minimum of 1 h of contact is necessary for the compounds which have high reaction rates with DNPHi, such as monocarbonyl compounds. However, for the dicarbonyl and hydroxicarbonyl compounds, a minimum of 12 h may be necessary to carry out the reaction in the experimental conditions evaluated. In this study, as stated in section 2.5, the total elapsed time between the sampling and the elution of the cartridges was seven hours. Although the formation of some of the compounds highlighted in this work (e.g., 5,6-epoxy-12´-apo-β-carotenal, 7-apo-β-caroten-7-al (β-cyclocitral), 12´-apo-β-carotenal, amongst others) as oxidation products of β-carotene is well documented, there is not a well-defined mechanism in the literature for their formation yet (Rodriguez and Rodriguez-Amaya,

2007, Waché, Bosser-Deratuld, LY, & Belin, 2002). In fact, it is inadequate to propose just one mechanism for all of the products obtained, considering that the precursor molecule is highly unsaturated and offers several possibilities for the initial ozone attack. In addition, the products that are initially formed may also react with ozone themselves, giving rise to new products as described previously. Casein kinase 1 When a double bond is broken, several different compounds may be formed; for example, when the double bond localised inside the ring is broken, epoxyde and secocarotenoids are formed. Fig. 2 shows the mechanism proposed for the formation of 2-methyl-buten-2-dial, beginning with the ozone attack on the double bonds between C12´–C11´ and C8´–C7´. A highly unstable ozonide is then formed, followed by the dienal and two Criegee’s biradicals. Fig. 3 shows, on the other way, the proposed mechanism for the formation of an epoxycarotenoid, which is originally based on that suggested by other authors for the ozonolysis of alkenes (Bailey, Mann, & Maittlis, 1975).