1. Prepare pet food samples.
2. For dry food, use mortar and pestle to disintegrate.
3. For wet food, homogenize by blender.

2. Prepare 10 ml screw-topped glass tubes (serum extraction tubes – no chipped opening).
3. Weigh each tube.
4. Add samples of food (1 gram for dry and 1.5 gram for wet) to tubes.
5. Reweigh each tube to get sample wet weight (subtract (a) from this weight).
6. Put in a vacuum oven at 40°C for drying overnight.
7. Reweigh each tube to determine sample dry weight.

3. 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.

4. Cool samples to room temperature (approx 25°C).
5. Transfer ~1.2 ml aliquots to microcentrifuge tubes and centrifuge for 5 minutes at 14,000 × g.
6. Transfer 1 mL of the supernatant to sample vials, add 20 µl 1 mg/ml quercetin methanol solution as internal standard, vortex.
7. 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.
8. Inject 5~10 µL aliquots to HPLC apparatus, perform HPLC.

5. 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

Gradient:

Time (min)                             B%

0                                        23

50                                         23

55                                         70

56                                         23

60                                         Stop

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