Profiling isoflavonoids found in legume root extracts using capillary electrophoresis.
Legumes such as alfalfa (Medicago sativa L.), barrel medic (Medicago truncatula), white sweet clover (Melilotus alba) and fenugreek (Trigonella graecum), normally accumulate (-)-medicarpin and its malonated glucose conjugate as natural inhibitors of fungal pathogens. These plants also accumulate the biosynthetic precursor formononetin as well as the malonated glycoside. We were interested in developing a robust high-throughput method to quantitate the levels of these two isoflavonoids, both free and conjugated, in legume root extracts, for use in screening for mutant plants accumulating altered levels of these compounds. Capillary electrophoresis was examined as an alternative to current high-performance liquid chromatography (HPLC) methods to generate isoflavonoid profiles. The developed assay used micellar electrokinetic capillary chromatography (MEKC) to provide the required selectivity in complex root extracts. The addition of 1,2-hexanediol to the sodium dodecyl sulfate (SDS) electrolyte provided improved resolution of adjacent isoflavonoids. We examined the role of several factors including sample preparation, buffer composition, buffer pH, and organic component in the injected sample. The use of capillaries with longer path lengths were also examined to increase sensitivity. A comparison of results obtained using MEKC and HPLC showed good correlation in the relative amounts of the isoflavonoids studied.
Department of Physical Sciences, Southeastern Oklahoma State University, P.O. Box 4025-Campus, Durant, OK 74701-0609, USA.
Brandi R
BR
Baggett
John D
JD
Cooper
Eric T
ET
Hogan
Jason
J
Carper
Nancy L
NL
Paiva
Joel T
JT
Smith
D002851:Chromatography, High Pressure Liquid; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods / Q000592:standards; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D007887:Fabaceae / Q000737:chemistry*; D006018:Glycols; D006586:Hexanes; D006863:Hydrogen-Ion Concentration; D007529:Isoflavones / Q000032:analysis / Q000302:isolation & purification*; D010936:Plant Extracts / Q000032:analysis / Q000302:isolation & purification; D018517:Plant Roots / Q000737:chemistry
D006018:Glycols; D006586:Hexanes; D007529:Isoflavones; D010936:Plant Extracts; C119102:1,2-hexanediol