Preparation of extract for HPLC analysis. The extraction mixtures were centrifuged for 1 min in a tabletop microcentrifuge to pellet the cell debris. The resulting supernatant of each sample extract was filtered through a nonsterile 0.45-µm Nylon 66 syringe filter (Alltech Associates, Deerfield, IL, catalog number 2047*) into a 2-mL screw-capped autosampler vial that was compatible with the autosampler described below.
The chromatographic system used in this study consisted of dual Shimadzu (Kyoto, Japan) LC6A high-pressure pumps, a Shimadzu SIL6A automatic injector, a Shimadzu SCL6A integrated system controller, a Licrosorb C18 reverse-phase analytical column (25 × 0.4 cm, ODS 3, 5-µm particle size) (Phenominex Corp., Torrance, CA), and a Hewlett-Packard (HP) 1040A UV diode array detector with an attached HP analysis computer and data storage system. This system consisted of a HP 85B microcomputer, HP 9133 hard disk data storage device, HP inkjet printer, and 7470A HP plotter (Hewlett-Packard, Beaverton, OR).*
The gradient elution schedule consisted of an initial 2-min run of 80-percent 0.01 M phosphoric acid and 20-percent methanol followed by a linear gradient to 100-percent methanol over 55 min at a flow rate of 1 mL/min.
Usually, a trial run was performed on a new sample to determine the optimal volume of sample to be injected on the HPLC system for the best determination of the phenolic content; 25 µL of sample was routinely used for the initial run. The sample volume was then raised or lowered until the injection contained the equivalent of 5 µg of the most prevalent phenolic compound. Some samples required several HPLC runs to optimize the injection volume required to yield a good chromatographic trace. Typically, the injection volumes of leaf extract samples prepared according to this protocol were around 25 µL, while the injection volumes of extract samples prepared from fruit tissues were around 100 µL.
The injection of water as a blank failed to produce peaks in the resulting chromatogram. Furthermore, after injecting such quantities of flavonoid standards that the detector was swamped, a subsequent water blank chromatogram showed no traces of phenolic compounds. This indicates a high efficiency in flavonoid elution using this HPLC technique. It also indicates that traces of flavonoids with an unexpected glycosylation pattern did not result from contamination from previous runs. In most cases, samples from cultivars having unexpected patterns were reanalyzed to confirm the presence of unexpected or unusual flavonoids.
Peak area percentages and extinction coefficients were calculated from chromatograms of standards detected at 285 nm. This wavelength was chosen for monitoring because all phenolics examined in this study absorb at this wavelength and extinction coefficients were similar when calculated from several purified standards in the three different classes examined: flavonoids, substituted cinnamic acids, and psorolens. Peaks from chromatographic runs were then assigned to general phenolic classes or identified as specific flavonoids by the criteria given below. A typical chromatogram is shown in figure 1.
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Original posting: April 1, 1999.