Supercritical fluid extraction of isoflavones from biological samples with ultra‐fast high‐performance liquid chromatography/mass spectrometry

Abstract

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.

Current trends in isolation, separation, determination and identification of isoflavones: A review

“…HPLC column: Atlantis dC 18 (2062.1 mm, 3 lm), mobile phase: 0.3% v/v acetic acid and acetonitrile (solvent B), linear gradient elution (v,%): from 13 up to 20% B from start to 1.25 min, up to 30% B to 1.61 min, up to 35% B to 2.0 min, up to 40% B to 2.25 min, up to 50% B to 2.57 min, and followed by a negative gradient to 13% B to 3.86 min, flow rate: 0.68 mL/min, column temperature: 368C. For other details see [35]. Peak identification: 1 -daidzin, 2 -glycitin, 3 -genistin, 4 -ononin, 5 -daidzein, 6 -glycitein, 7 -sissotrin, 8 -genistein, 9 -formononetin, 10 -biochanin A.…”

“…A mixture of daidzin, glycitin, genistin, ononin, daidzein, glycitein, sissotrin, genistein, formononetin, and biochanin A was completely separated in less then 6 min [40] using optimized linear gradient elution with 0.2% formic acid and acetonitrile. Later [35], complete separation was accomplished in even less then 4 minutes by elution with a mobile phase consisting of 0.3% acetic acid and acetonitrile (Fig. 3).…”

“…From soy hypocotyls, isolates containing 640 mg of isoflavones/ 100 g of SPI were prepared at 35 MPa, and 458C during 120 min, and 83.7% of the isoflavone content was glycitin, daidzin, and genistin. SFE with continuous on-line modifier addition to the extraction phase was applied [35] to the extraction of daidzin, glycitin, genistein, ononin, daidzein, glycitein, sissotrin, formononetin, and biochanin A from red clover (chromatograms of real samples after SFE are shown in Fig. 3).…”

Comparison of various extraction techniques for isolation and determination of isoflavonoids in plants

“…The components in question can be isolated by simple solvent extraction (e. g. using a Soxhlet apparatus) or by using solid sorbents, e. g . matrix solid phase dispersion (MSPD) [6], but also by applying modern instrumental techniques, such as supercritical fluid extraction (SFE) [7,8], pressurized fluid extraction (PFE) [9 -11], SPE [12 -16], or ultrasonic extraction in an ultrasonic bath (USE) [17,18]. In these references one can find the basic principles of the individual extraction techniques.…”

Supercritical fluid extraction of amino acids from birch (Betula pendula Roth) leaves and their liquid chromatographic determination with fluorimetric detection

“…For the troublesome lyophilized leaves matrix [18,19], it was necessary: (i) to find appropriate extraction conditions for SFE of AAs; (ii) to avoid the prospective matrix effect and verify the method robustness for various leaf samples; (iii) to confirm that amino acid enrichment of samples cannot occur during the extraction, either due to contamination by human touch or by hydrolysis of proteins or any other amino acids producing or releasing mechanism. Common validation and reproducibility tests were also performed.…”