SOP1: Validation ring testing protocol for organic small

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Develonutri
Validation ring testing protocol for organic small molecular weight (<2000 amu)
molecules
1. Five 100mg aliquots for each sample are weighted out and placed into independent
tubes/vessels.
2. Methanol (1ml) is added the suspension mixed (vortex) 3 times for 3 second bursts.
3. Water (1ml) is added the suspension mixed (vortex) 3 times for 3 second bursts.
4. Chloroform (3ml) added the suspension mixed (vortex) 3 times for 3 second bursts.
5. The suspension is incubated at 4oC for 30 mins.
6. The mixture centrifuged at 3,000 rpm for 5 min.
7. Aqueous (hyperphase) removed and placed in a clean tube/vessel for subsequent
analyse of polar compounds.
8. Chloroform (hypophase) removed and placed into a clean tube/vessel for subsequent
analyse of non-polar compounds.
9. Non-polar chloroform fraction is dried and stored at -20oC (in aliquots if appropriate).
10. Stored aqueous phase at -20oC (in aliquots if appropriate).
11. The polar and non-polar fractions are then analysed on your analytical/derivatisation
platforms available.
12. Determine only the predominant bio-active or anti-nutrients on each system (to keep
everything simple) if you have isomers just the predominant isomer.
13. Determine the amounts of the predominant compounds firstly by (i) dose-response
curve, (ii) relative to an internal standard ADDED TO THE EXTRACT after extraction
and prior to running on the analytical platforms/ derivatisation.
-1), not
14. Report the values as micro grams per milligram dry weight
as an average but individually.
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