- Prepare pet food samples.
- For dry food, use mortar and pestle to disintegrate.
- For wet food, homogenize by blender.
- Prepare 10 ml screw-topped glass tubes (serum extraction tubes – no chipped opening).
- Weigh each tube.
- Add samples of food (1 gram for dry and 1.5 gram for wet) to tubes.
- Reweigh each tube to get sample wet weight (subtract (a) from this weight).
- Put in a vacuum oven at 40°C for drying overnight.
- Reweigh each tube to determine sample dry weight.
- Add 9 ml methanol and 1 ml concentrated hydrochloric acid to each tube, tightly cap (make sure good seal and no chipped openings to tubes), vortex, and place in a boiling water bath for 5 hours with hourly mixing by inversion.
- Cool samples to room temperature (approx 25°C).
- Transfer ~1.2 ml aliquots to microcentrifuge tubes and centrifuge for 5 minutes at 14,000 × g.
- Transfer 1 mL of the supernatant to sample vials, add 20 µl 1 mg/ml quercetin methanol solution as internal standard, vortex.
- For standards, dry down different amounts of standards (10 – 300 uL of 0.1 mg/ml of each isoflavone – daidzein, genistein and glycitein) in HPLC tubes, then add 1 mL acidified methanol and 20 – 50 uL 1 mg/mL quercetin and vortex.
- Inject 5~10 µL aliquots to HPLC apparatus, perform HPLC.
- HPLC Method
Column: Phenomenex® Synergi 10µ Hydro-Rp, 250×4.60 mm
Column Temperature: 25°C
Flow Rate: 2 ml/min
Inject Vol: 5 µL
UV Detection: 260 nm
Run Time: 60 min
Mobile Phase A: 50 mM KH2PO4 Buffer solution (pH 2.2)
Mobile Phase B: 95% Acetonitrile
Time (min) B%