IAC-SULF- Operation Manual by Clover Technology Group,Inc.

1. IAC-SULF- Operation Manualby Clover Technology Group,Inc.

2. 1.0 IAC-SULF Test procedure for animal derived food overview SAMPLE EXTRACTION Mix 5.0 g of muscle tissue homogenate and 20 ml ethanol/water(80:20,v/v) vortexed high speed for 2 minutes, and then shaken for 30min. centrifugation at 3000RPMfor 5min DILUTION Dilute 5 ml extract with 45ml distilled water. Mix well. AFFINITY CHROMATOGRAPHY Pass 20mL filtered diluted extract through column.  Wash column by passing 10mL distilled water through the column.  Elute citrinin by passing 2mL Eluting Solution through column. Collect eluate. MEASURE Dry down eluate under an Nitrogen stream at 50℃. Reconstitute with 400µL mobile phase. injected into HPLC or LC-MS

3. 1.1 SAMPLE EXTRACTION ① Place 5.0 g of muscle tissue homogenate into 50mL polypropylene centrifuge tube ② Add to tube 20 ml ethanol/water(80:20,v/v).

4. ③ vortexed high speed for 2 minutes ④ shaken for 30min with an orbital shaker

5. ⑤ After centrifugation at 3000RPMfor 5min 1.2DILUTION ① Dilute 5 ml extract with 45ml distilled water. Mix well. ②Filter diluted extract through glass microfibre filter and collect filtrate in a clean container.

6. 1.3 AFFINITY CHROMATOGRAPHY ① Remove two end caps from IAC

7. ② Pass 20 ml of filtered extract (20 ml = 0.5g sample equivalent) through the column at a steady slow flow rate of about 1 drops per second ③ After extract has completely passed through column, pass 10 ml distilled water through column at about 1-2 drops per second flow rate.

8. ④ Elute SULF column at flow rate of 1 drops per second with 2.0 ml HPLC grade methanol and collect in a clean glass cuvette. ⑤ Dry down eluate under an Nitrogen stream at 50℃. Reconstitute with 500µL mobile phase.

9. 2.0 IAC-SULF Test procedure for Blood Plasma overview SAMPLE EXTRACTION Mix 10 mL of Blood Plasma and10 ml acetonitrile vortexed high speed for 2 minutes,and then shaken for 30min. centrifugation at 3000RPMfor 5min DILUTION Dilute 5 ml extract with 45ml distilled water. Mix well. AFFINITY CHROMATOGRAPHY Pass 10mL filtered diluted extract through column.  Wash column by passing 10mL distilled water through the column.  Elute citrinin by passing 2mL Eluting Solution through column. Collect eluate. MEASURE Dry down eluate under an Nitrogen stream at 50°C. Reconstitute with 500µL mobile phase.injected into HPLC or LC-MS

10. 1.1 SAMPLE EXTRACTION ① Place 10 mL of Blood Plasma into 50mL polypropylene centrifuge tube ② Add to tube 10 ml acetonitrile

11. ③ vortexed high speed for 2 minutes ④ shaken for 30min with an orbital shaker

12. ⑤ After centrifugation at 3000RPMfor 5min 1.2DILUTION ① Dilute 5 ml extract with 45ml distilled water. Mix well. ②Filter diluted extract through glass microfibre filter and collect filtrate in a clean container.

13. 1.3 AFFINITY CHROMATOGRAPHY ① Remove two end caps from IAC

14. ② Pass 20 ml of filtered extract (20 ml = 0.5g sample equivalent) through the column at a steady slow flow rate of about 1 drops per second ③ After extract has completely passed through column, pass 10 ml distilled water through column at about 1-2 drops per second flow rate.

15. ④ Elute SULF column at flow rate of 1 drops per second with 2.0 ml HPLC grade methanol and collect in a clean glass cuvette. ⑤ Dry down eluate under an Nitrogen stream at 50℃. Reconstitute with 500µL mobile phase.

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