Chromatography Forum

I'm working on method developement on HPLC UV for gummy candy. I'm trying to make sure my calculations are correct, doing the math so much I've just confused myself.
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?

Nominally, steps 1 to 4 result in a solution of the extracted analyte in 3 ml of ACN. You can carry out the steps with the standard instead of the sample and determine the resulting concentration using an independent standard solution prepared by simple dilutions.

Usually in similar cases, it is better to prepare the standard solutions in the same way as the sample solutions and use these standard solutions for the analysis of the samples. This allows you not to think about any dilution factors since they are the same for the standards and for the samples. Moreover, it is more advantageous here to add the internal standard in the first step of the procedure rather than in the final step.