Proceedings Volume 2010 (format .pdf) - SimpBTH

After the sample extraction, the second step in the protocol of carotenoiddetermination is the alkaline saponification, which is carried out in a KOHsolution: aqueous, ethanolic or methanolic solution and the concentration may varyfrom 10 to 60% [24]. The conditions under which the saponification is carried outmay be at ambient temperature overnight or under heating which reduces the timeof reaction [25]. After the saponification, the carotenoids are extracted with diethylether or hexane, and then the extract is washed several times with water until all theKOH is removed. In the case of dairy products, the saponification is requiredbecause of the high fat content (~45%) and carotenoids are contained in the lipidmatrix [26].The saponification procedure is usually used before the chromatographicanalysis, for removal of substances, like lipids and chlorophylls, which couldinterfere with the chromatographic detection.Generally, the carotenoids analysis is carried out by HPLC. Acetonitrile,methanol and their mixtures are the majority constituents used in the analysis ofcarotenoids. To optimize the separation of some carotenoids (for examplegeometrical isomers), the mobile phase is often modified by the addition of smallamounts of other organic solvents [27]. Methanol has been recommended inseveral reports [28] because it provides better recoveries than acetonitrile oracetonitrile-based solvents. Antioxidants, such as ascorbic acid or BHT are oftenadded to mobile phases to stabilize them.For the purpose of separation of either different carotenoids or cis /transisomers of a special carotenoid, a polymeric C 30 phase is necessary. This is moreeffective for the separation of non-polar geometric isomers than the C 18 phases[29]. An important factor that should be taken into account to achieve a satisfactoryseparation of carotenoids is the column temperature. Several authors [30] havereported that changes in ambient temperature cause significant changes in thechromatographic response of carotenoids; therefore, it is important to work atconstant temperature.An important analytical tool for the identification of carotenoids is theliquid chromatography-mass spectrometry technique (LC-MS), since it providesinformation about the structure and in addition, it is a very sensitive technique.The LC-MS methods developed for carotenoid analysis include mainlyatmospheric pressure ionization interfaces (APCI) [Error! Bookmark notdefined.] or electrospray ionization interfaces (EIS) [31].The high sensitivity and selectivity of mass spectrometry (MS) facilitatesthe identification and structural analysis of small quantities of carotenoids that aretypically obtained from samples such as plants, animals, or different products.Flavonoids are an important group of secondary metabolites, which aresynthesized by plants; structurally, they are phenolic compounds with two aromaticrings bonded by a pyran ring.293