20 series

Plasma MDA were assessed by High Performance Liquid Chromatography (HPLC) using a modified method reported by Karatepe [28 ] against MDA standards. Briefly, MDA standards and plasma samples were precipitated with 5% (v/v) perchloric acid and the supernatant measured using a Shimadzu 20-series (Shimadzu Corporation, Kyoto, Japan) HPLC instrument equipped with a diode array detector. Calibration standards and samples were resolved using a Synergi™ 4 μM Polar-RP 80 Å column (Phenomenex®, Auckland, New Zealand) with a 95:5 (v/v) 30 mM monobasic potassium phosphate buffer (pH 3.6)-methanol mobile phase. The peak area and retention times of MDA in the standards and samples at 250 nm were evaluated using the Shimadzu LC solution software (Shimadzu Scientific Instruments, Auckland, New Zealand). MDA levels were calculated, against MDA standards and presented as μmol/L. All plasma samples were assayed in triplicate with the CV of replicate measures at < 10%.

Up to 12 different isoflavones have been isolated from soybean, with the same being possible from SMB [29 (link)]. This study aimed to quantify genistin, diadzin, and their aglycones, daidzein and genistein, using HPLC with a photodiode array detector (Shimadzu 20 series, UV–Vis detector) as described Montero, Günther [30 (link)], with modifications. In brief, the sample extracts were filtered through 0.2 µm syringe filter into HPLC vials. A reverse phase HPLC column (Agilent Raptor C18 column 4.6 × 150 mm with 5 µm particles (Restek Corporation, Bellefonte, PA, USA)) was used under column temperature of 30 °C, and auto-injection was applied with a 20µL injection volume. Mobile phase consisted of 0.2% formic acid solution (solvent A) and 100% acetonitrile (solvent B), with a solvent flow rate of 1 mL/min analysed at 254 nm. A solvent gradient was developed, with increasing concentration of solvent B as follows: 5 min 0% of solvent B increasing to 25% by 15 min, 40% after 25 min, 60% by 35 min, decreasing to 10% at 40 min and 0% at 45 min. Standard curves were developed for all isoflavones using the same procedure, from 500 µL/mL to 31.25 µL/mL, with retention time of 18.7 min, 19.8 min, 22.8 min and 26.0 min, for daidzin, genistin, daidzein and genistein respectively.