Survey of Phenolic Compounds Produced in Citrus
Experimental Methods

Specific Flavonoid Peak Assignments
and Quantification Procedures

The chromatographic peaks in each sample that were determined to be flavonoids by their spectral characteristics were then specifically identified by comparison to flavonoid standards and other information. Known flavonoid standards were chromatographed separately and as mixtures. These flavonoid standards were used to develop charts of relative retention times and to run nuclear magnetic resonance structural analysis for comparison to isolated flavonoid peaks from samples. The standards were added to tissue extracts to confirm relative retention times (Kanes et al. 1993). The identified flavonoids in each sample were then quantified on a percent weight basis and as milligrams per gram fresh weight of the sample to give a detailed flavonoid composition of the fruit and leaves for each of the cultivars examined (table 3 and table 4).

The flavanones and flavones in the sample analysis, which were previously identified by their characteristic spectral maxima under the general phenolic analysis procedure outlined above, were then specifically identified by their absolute retention times and retention times relative to other flavonoids in the same sample.

As a rule, each flavanone rutinoside eluted 0.7 to 1.1 min before its neohesperidosyl counterpart. This fact, as well as the preponderance of eriocitrin in lemons, was used to identify eriocitrin. The retention time difference relative to rhoifolin as well as its characteristic spectrum was used to identify isorhoifolin. In addition, all standards were visualized using thin-layer chromatography and their melting points were examined. Naringin–6"–malonate appears to be present in two forms in some samples, the first of which appears to be a closed lactone ring form of the malonate group (data not published). This may be an artifact of the extraction procedure and sample storage. The open ring form of naringin–6"–malonate may coelute with didymin.

Effects of polyploidy on flavonoid content. Leaf tissue from diploid and tetrapolid specimens of four Citrus paradisi Macf. (grapefruit) cultivars were analyzed for flavonoid content and quality-related characterisitics. The cultivars examined were 'Hall', 'Imperial', 'Royal', and 'Seedy Marsh'. Plant material was obtained from the Citrus Variety Collection at the University of California, Riverside. The samples were prepared for HPLC analysis as outlined above.

Quantification of the individual flavonoid compounds was based on integrated areas. An aliquot of juice was titrated potentiometrically for total acidity (Vandercook et al. 1975). The results were calculated as milligrams of anhydrous citric acid per milliliter. The Brix values were measured by refractometer and are uncorrected for acid.

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United States Department of Agriculture
Agricultural Research Service

The material on this page is in the public domain.

Original posting: April 1, 1999.