Web Release Date: June 24,
Evaluation of Isoflavone Aglycon and Glycoside Distribution in Soy Plants and Soybeans by Fast Column High-Performance Liquid Chromatography Coupled with a Diode-Array Detector
ivoj Klejdus,
il,
ch Adam,
and
*
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry,
Zemdlská 1, CZ-613 00 Brno, Czech Republic, Department of Analytical Chemistry,
Masaryk University Faculty of Science, Kotláská 2, CZ-611 37 Brno, Czech Republic, and
Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030,
CZ-128 40 Prague 2, Czech Republic
Received for review February 5, 2005. Revised manuscript received
May 16, 2005. Accepted May 21, 2005. This work was supported by
grants the Grant Agency of the Czech Republic GA 
R 525/03/0352
and 525/04/P132 and by a grant from the IGA MZLU 3/2004.
Abstract:
An ultrafast HPLC/UV-vis DAD method working at 254 nm was applied for the determination of
isoflavone aglycons and glycosides (genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin,
formononetin, sissotrin, and biochanin A) in roots, stems, leaves, and soy pods of soy plants and in
soybeans of five varieties (Korada, Quito, Rita, OAC Erin, and OAC Vison). An Atlantis dC18 ultrafast
RP chromatographic column (20 mm × 2.1 mm, 3 
m particle size) was applied for separation of the
isoflavone aglycons and glycosides. A flow rate of the mobile phase (0.1% (v/v) acetic acid, pH 3.75-solvent A and methanol-solvent B) was 0.35 mL min-1, and the column temperature was 36 
C. A
linear gradient profile from 13 up to 22% B (v/v) from zero to 2.5 min, up to 30% B to 3.21 min, up
to 35% B to 4 min, up to 40% B to 4.5 min, up to 50% B to 5.14 min, and followed by negative
gradient up to 13% B to 7.71 min was used. The absolute limits of detection per sample injection (5
L) were the highest for biochanin A (166.2 fmol) and the lowest for genistin (17.0 fmol), respectively.
An accelerated solvent extraction (ASE) in combination with sonication was applied for isolation of
biologically active compounds. A solid-phase extraction procedure was used to purify the extracts in
the case of analysis of soy plants parts. The recoveries of 96-106% were obtained for the different
concentrations of the isoflavone aglycons and glycosides and the different matrixes (overall RSDs
2-9%). The highest isoflavone concentrations were found in roots (12.5 g g-1 dry weight), while
the amounts were about 3-1100 g g-1 fresh weight in different varieties of soybeans.
Keywords: Isoflavones; liquid chromatography; UV-vis DAD; accelerated solvent extraction; ultrasonic extraction; solid-phase extraction; soy plants; roots; leaves; stems; pods
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