Thermo bds hypersil c18

Samples were analysed by liquid chromatography-mass spectrometry (LC-MS) alongside standard compounds to allow identification and quantification of constituents, particularly the phytoestrogens formononetin, coumestrol, daidzein, genistein and biochanin A.
For LC-MS/MS experiments, an Agilent 1100 series high-performance liquid chromatography (HPLC) system (Agilent, Waldbronn, Germany) equipped with a quaternary gradient pump, column heater, autosampler with sample cooler (maintained at 4ºC), and diode array detector, was coupled to a Thermo Scientific LTQ ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). 10 μL of neat extract was injected onto a 150 x 2.1 mm id., 3, Thermo BDS Hypersil C18 column (Thermo Fisher Scientific) maintained at 30 o C. The mobile phase consisted of two components: A (water with 0.1% v.v -1 formic acid) and B (acetonitrile with 0.1% v.v -1 formic acid); and was applied to the column with the following gradient: 0-45 min, 96% A to 16% A (0.2 mL.min -1 ); 46-51 min, 96% A (0.3 mL.min -1 ).

For the analysis of warfarin an MS/MS system: API 3200 (Applied Biosystems, MDS SCIEX, USA) attached to Agilent 1200 HPLC (Agilent Technologies, USA) controlled by Analyst 1.6.1 software, was utilised. For the extraction of warfarin from the samples, 100 µL of spiked/blank plasma were pipetted into previously labelled Eppendorf tube, 25 µL of the internal standard (IS) Fenofibric acid (FFA) from 100.0 µg FFA/mL IS solution was added to the tubes and vortexed for 30 sec. Afterwards, the precipitation solution, acetonitrile (400.0L) was added to the tube and vortexed for further 1 min. Samples were then centrifuged for 5 min at 14,000 rpm and the supernatant was collated and transferred into an auto-sampler micro vial for analysis. The mobile phase used for analysis comprised of (30:70) mixture of ammonium chloride 0.001M: acetonitrile respectively eluted at a flow rate of 0.7 mL/min through a Thermo BDS Hypersil C18 (50×2.1 mm, particle size 5 µm) column (Thermo Fisher Scientific, Germany) at the temperature 30˚C. The injection volume was 5 μL and the stop time was 0.7 min. The retention time of the drug was 0.3 min with a limit of detection of 10 ng/mL.