High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric
detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium
pratense L.) and related species has been developed. The separated isoflavones including
aglycones, glycosides and glycoside malonates, were individually analyzed and identified
by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS
and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones
were detected in red clover. Several isoflavones were also identified for the first
time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover)
and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone
aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin,
prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully
separated within 40 min and quantified individually by UV and MS detectors. For the
10 target compounds, the investigated concentrations ranged from approximately 24
to approximately 12500 ng/ml for UV detection and approximately 6 to approximately
3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99
for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability
(n = 10) were within 15% for these 10 compounds. This is the first method reported
that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover
and related species.