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Sample prep:
1. Weigh 0.5 g sample to 15 mL vial.
2. Add 5 mL warm HPLC Water to 15 mL vial. Sonicate in heat to dissolve gummy.
3. Add 3 mL ACN to 15 mL. Vortex 1 minute.
4. Add 1 g MgSO4/ 0.25 g NaCl to 15 mL vial. Shake Vertically 1500 rpm for 3 minute. centrifuge 4400 rpm for 5 minutes.
5. Take 50 uL of the top layer into 1.5 mL vial with 950 uL (75/25 MeOH/H2O) dilution solution. vortex 1 minute.
6. Filter with 0.22 um syringe filter.
7.Take 25 uL of filtered supernatant into a 1 mL vial with 10 uL internal standard, 150 uL (75/25 MeOH/H2O) dilution solution.
I'm having issues determining the dilution factor from steps 1-4. i know step 5 is 50 uL supernatant to 1000 uL total so the dilution factor is 20. I know Step 7 is 25 uL supernatant to 185 uL total so dilution factor is 7.4. Theoretically if we assume 100% extraction, the same amount (ug) of an analyte would be present in steps 1-4.
Any advise?