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.