Quali-quantitative evaluation of chemopreventive coumarins in citrus fruits by HPLC-UV/F/MS: comparison of original analytical methods

The Citrus genus includes fruits belonging to several species and hybrids commonly used for food and industrial purposes (lemon, sweet and bitter orange, tangerine, grapefruit, lime, bergamot orange, kumquat). In addition to being rich in vitamin C, folate and fibre, some Citrus fruits contain auraptene and umbelliferone, coumarin-based molecules that can increase the potential of these fruits as functional foods. Auraptene (7-((E)-3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one) seems to be promising as an adjuvant for cancer prevention: in particular against melanoma, gastro-intestinal, liver and mammary cancers. Due to its strong UV absorbance, umbelliferone (7-hydroxychromen-2-one) is employed in cosmetic and sunscreen preparations. Both auraptene and umbelliferone are also reported to have antioxidant properties. Therefore it is very useful to develop innovative analytical methods able to provide a reliable measurement of the content of these molecules in Citrus fruits. Three methods have been developed for this purpose based on HPLC with spectrophotometric (UV), spectrofluorimetric (F) and tandem-mass (MS/MS) detection. The sample pre-treatment is carried out by means of original miniaturized extraction procedures. The HPLC-UV system has the great advantage of being simple and inexpensive, thus a good choice for preliminary assays. At the same time, HPLC-F takes advantage of the native fluorescence possessed by the analytes to achieve very high sensitivity. Finally, LC-MS/MS system allows analyte identification through mass spectra and provides a very accurate and selective quantification. The comparison of the developed analytical methods gives an useful overview about the content of chemopreventive coumarins in different types of Citrus, as well as in the same fruits coming from several cultivars and at different ripening levels. Moreover, coumarin levels are being also investigated in various parts of the fruits (exocarp, mesocarp, endocarp and seeds) to gather information both from a nutraceutical point of view, and for the possibility of use as raw materials for nutritional supplements