Sample Preparation for Phenolic Analysis
Fresh sample preparation and extraction. Mature fruit
and leaves used in this study were collected in late December
at the Citrus Variety Collection at the University of California,
Riverside. Fresh samples were stored frozen in sealed clear polyethylene
plastic bags (168 mm × 150 mm) (Glad-Lock zipper sandwich
bags, First Brands Corp., Danbury, CT*)
at –20 °C for up to 3 months until they could be processed
for extraction. Comparison of the chromatographic profiles of
extracts prepared from fresh samples and from the same samples
that had been stored frozen for over one year showed there was
little degradation of the phenolic content during freezing.
About 200 mg of thawed flavedo and albedo tissues, 100 mg of
thawed leaf tissue, or 500 mg of thawed juice vesicles were ground
or cut into small pieces, weighed, and placed in a 1.5-mL plastic
microfuge tube. One mL of a dimethylsulfoxide:methanol solution
(1:1) was added to each tube. The sample was thoroughly mashed
with a spatula, then allowed to soak at 25 °C for 2 hr or
longer. The sample was remashed with the spatula every 30 min
during the 2-hr soaking period. The extracts were then processed
for HPLC analysis as outlined below or returned to –20 °C
storage until they could be processed.
Dried leaf sample preparation and extraction. Collected
leaves were oven-dried at 60 °C in brown paper bags for at
least 24 hr. The dried leaves were then ground into a fine powder
by one of two methods. Samples of less than 10 leaves were ground
to a fine powder with a mortar and pestle with the addition of
a small amount of sand. Larger samples of more than 10 leaves
were ground to a powder in a small electrically driven coffee
bean mill. The dried, powdered samples could be stored indefinitely
in a refrigerator at 4 °C until time of extraction. Samples
(approximately 250 mg) were extracted with 1 mL of a 1:1 dimethylsulfoxide:methanol
mixture at 50 °C. Each extraction was allowed to soak for
at least 1 hour. After centrifugation, the supernatent was decanted,
the pellet re-extracted with an additional 1 mL of dimethylsulfoxide:methanol,
centrifuged, decanted, and extracted a third time. The three extractions
of each sample were combined and prepared for HPLC analysis.
United States
Department of Agriculture
Agricultural
Research Service
The material on this page is in the public
domain.
Original posting: April 1, 1999.
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