Isoflavones analytical methods

Traditionally HPLC methods were used for isoflavone analysis from foods [Wang et al., 1990], and gas chromatography/mass spectrometry (GC/MS) to determine isoflavones and their metabolites in human biological fluids including urine [Adlercreutz et al., 1991 Kelly et al., 1993], plasma [Adlercreutz et al., 1993], and feces [Adlercreutz et al., 1995 Kurzer et al., 1995]. HPLC with photodiode array (PDA) detection was introduced in 1994 to measure these analytes in human urine [Franke and Custer, 1994 Xu et al., 1994]. Compared to GC/MS, HPLC methods require fewer steps for sample preparation and analysis and demand less technician time and less expensive instrumentation. 

One problem inherent in these earlier LC-NMR studies is that of analyte concentration. In on-flow mode, this may be too low for anything other than ID H spectra to be acquired. Use of the stopped-flow mode greatly increases the overall time of the LC-NMR experiment, especially where there are many components of interest. One solution is the so-called LC-SPE-NMR method, where the analytes of interest are concentrated by solid-phase extraction before transfer to the NMR flow-cell. Using this procedure, acquisition of 2D NMR datasets such as H— H COSY and inverse-detected H— C experiments (HSQC, HMBC) becomes more practicable. Lambert et al. (2005) have demonstrated the potential of this approach by using LC-SPE-NMR to identify 10 new and 7 known constituents present in extracts of Smimowia iranica. The new compounds comprised isoflavans, iso-flavanones, and isoflavones, many of which are prenylated. 

The ID-GC-MS-SIM method of Adlecreutz and coworkers (1993) is perhaps the most frequently used in clinical and medical studies, although intra- and total-assay variabilities of all analytes are considered to be high, probably reflecting low-analyte concentrations in biological fluids (Wilkinson et al., 2002). Other GC-MS methods report lower inter-assay variation of 6%-ll% isoflavones in serum. The latest method of measuring urinary excretion of isoflavones (daidzein, genistein, equol. 

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