Isoflavone analyses

Higher sensitivity and selectivity for HPLC analyses can be achieved by detection with MS, which has gained extreme attention due to ease of use through electrospray ionization [Barnes et al., 1998 Franke et ah, 2002 Twaddle et al., 2002]. For food isoflavone analyses we favor PDA monitoring... 

Table 11.6.6 Mobile Phase Gradients for Isoflavone Analysis Using a Reversed-Phase C18 Column...

Figure 11.6.4 A flow diagram for isoflavone analysis using enzymatic hydrolysis.

Column equilibration ( 10 column vol. recommended) ensures baseline stability, good peak shape, and reproducible retention times. For the specific column chosen in this unit for isoflavone analysis, at least 18 ml total is needed to equilibrate the system with mobile phase. The common practice is to purge the pump system and connect the inlet end of the column to the injector outlet. The initial pump flow should be set at 0.1 ml/min and increased to 0.6 ml/min in 0.1 ml/min increments. Once a steady backpressure and baseline have been achieved, the column is ready to use. Before injecting samples, it is suggested to run a blank gradient first to clean the column and help check for the possibility of impurity peaks. 

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. 

Table 3.6 Methodological Overview of Isoflavone Analysis from Foods and Biological Samples...

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