HPLC isoflavones

COWARD L, KIRK M, ALBIN N and BARNES s (1996) Analysis of plasma isoflavones by reversed-phase HPLC-multiple reaction ion monitoring-mass spectrometry. Clin Chim Acta. 247 (1-2) 121-42. 

A similar RP-HPLC method has been developed for the determination of isoflavones in soybean powder. Ground soybean powder (100 g) was extracted four times with 300 ml of hexane, then the defatted powder was extracted with six solvents or solvent mixtures such as acetone, methanol-water (4 1, v/v), methanol-ethyl acetate-acetone (1 1 1, v/v), acetone-0.1 M HC1 (5 1, v/v), methanol-water (4 1, v/v) plus 0.1 M HC1 (5 1, v/v), and methanol-ethyl acetate-acetone (1 1 1, v/v) plus 0.1 M HC1 (5 1, v/v). An aliquot of 0.5 g defatted or non-defatted soybean powder was shaken for 2h or sonificated for lh with 5 ml... 

For the analytical HPLC of a given subclass of flavonoids (flavones, flavonols, isoflavones, anthocyanins, etc.), the stationary phase, solvent, and gradient have to be optimized. 

Isoflavones, chiefly found in the Leguininosae (such as soy, Medicago sativa, and red clover. Trifolium pratense) in the plant kingdom, are also successfully analyzed by HPLC on Ci8 columns. ... 

MALDI-TOF-MS has been used to identify and quantify other anthocyanins in foods.When the anthocyanin content of highbush blueberries at different stages of anthocyanin formation were analyzed by both HPLC and MALDI-TOF-MS, it was found that both techniques provided comparable quantitative anthocyanin profiles. While HPLC could distinguish anthocyanin isomers, MALDI-TOF-MS proved to be more rapid. MALDI-TOF-MS has also been used to identify the isoflavones in soy samples. In a comparison of several matrices, 2, 4, 6 -trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid... 

Barnes, S., Kirk, M., and Coward, L., Isoflavones and their conjugates in soy foods extraction conditions and analysis by HPLC-mass spectrometry, J. Agric. Food Chem., 42, 2466, 1994. 

Support Protocol 2 Sample Preparation for HPLC Analysis Support Protocol 3 Converting Glycosidic Isoflavones to Their Aglycones 11.6.10... 

Figure 11.6.3 Gradient HPLC separation of isoflavone standards (see Basic Protocol 3). Peaks 1, daidzin 2, glycitin 3, genistin 4, malonyldaidzin 5, malonylglycitin 6, acetyldaidzin 7, acetylglycitin 8, malonylgenistin 9, daidzein 10, glycitein 11, acetylgenistin 12, genistein. Conditions Waters Nova-Pak C18 reversed-phase column (150 x 3.9 mm 4-pm i.d. 60 A pore size) mobile phase 1% acetic acid in water (solvent A) and acetonitrile (solvent B) flow rate 0.60 ml/min UV detector 260 nm column temperature 25°C. The dotted line represents the gradient of solvent B.

Soy foods are complex matrices (see Table 11.6.1). After initial aqueous extraction, the crude extract containing isoflavones is concentrated but may contain components that are immiscible with the HPLC mobile phase for direct injection. A clean-up step is necessary to prevent the introduction of particles and precipitates that may block the frit and foul the column. 

HPLC is the method of choice for the analysis of isoflavones in soy products. HPLC is fast, reproducible, requires small sample sizes, and can be used for both qualitative and quantitative analysis as well as for separation purposes. 

Quantification. For accurate quantification, a standard curve of peak area versus concentration should be constructed for each standard using the same chromatographic conditions (e.g., wavelength and solvent) as for the samples under analysis. The concentration range of standard curves should be determined according to both the isoflavone level of soy food samples and dilution factors during sample preparation such that the UV absorbance of the injected sample is within a range of 0 to 1. The appropriate standard curve can then be used to calculate the quantity of isoflavones represented by each HPLC peak in the sample. 

Many parameters are critical to the successful and reproducible separation of isoflavones using HPLC. 

The extraction protocol typically recovers 96% to 108% of isoflavones. The HPLC protocol typically recovers isoflavones in a range between 97% and 105% when tested using spiked reference standards. During reversed-phase HPLC separation, all isoflavones elute from the column in the order of polar to nonpolar and are separated within 50 min of run time. 

Table 9 Gradient HPLC Analysis of Isoflavones in Soybean Products HPLC condition...

Isoflavone derivatives along with nonvolatile components in soy sauce were also analyzed by gradient reversed-phase HPLC. This HPLC profile of soy sauce was further utilized by a pattern recognition program to understand the quality differences of soy sauces (132), and three isoflavone derivatives (esters of tartaric acid with daidzein, genistein, and 8-hydroxygenistein) were found to contribute significantly to the differentiation in fermented soy sauce. Fermented... 

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. 

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... 

Barnes S, Coward L, Kirk M, Sfakianos J. 1998. HPLC-mass spectrometry analysis of isoflavones. Proc Soc Exp Biol Med 217 254-262. 

Klejdus B, Vacek J, Benesova L, Kopecky J, Lapcik O, Kuban V. 2007. Rapid-resolution HPLC with spectrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts. Anal Bioanal Chem 389 2277-2285. 

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