Vitamin AD&E analysis

[Deppizzymo]

We are working on attempting vitamin ADE analysis with an Agilent 1260/DAD detector/non-MS system. We are really hoping to find success following this very interesting study for rapid analysis but not so sure we're going to get it to work ok for us for vitamin D specifically.

https://pmc.ncbi.nlm.nih.gov/articles/PMC12096806/

We are struggling with recovery at the moment. Our current material is animal feed mineral premix with very broad inclusion rates on vitamins ranging from about 0.00132%-0.00250% vitamin D, 3.5-20% vitamin E, 0.02%-0.3% vitamin A. We decided focusing solely on vitamin D would be the best course of action because we figured if we could get good recovery from D, the rest would be easier to achieve in the end anyway. (using 265nm wavelength)

We created a cal curve that spans this range and our range is encapsulating the following method:
300mg material diluted in 1mL H2O and sonicated 20 mins and then added 5mL 3:7 DMSO (just like the method link) and sonicated 30 mins. Finished with centrifuging and syringe filtering. We weighed so much (in relation to the dilution) in order for a decent response. We are getting roughly 50% recovery following this and not much consistency between reps either. Our mobile phase is 100% methanol running at 1mL/min and the vitamin D is eluting around the 9 minute mark.

We are total newbies/blind leading the blind on this so I am mostly hoping for someone to come in who has experience with this and say "this is obviously not going to work" or at least give me some pointers as to things we might want to try before scrapping this method entirely? I know it seems like the old standard was a saponification and liquid liquid extraction but this current method is so nice we really hate to give it up.

Thanks a lot!!!

[Jensvd]

50 min sonication sounds like creating a recovery problem. We also have bad experience with DMSO in HPLC methods due to unstable baseline. I can share our method:
265 nm, lichrospher 60 RP-select-B, 125x4.6 mm x 5 um at 20°C. Gradient at 1ml/min with 80% methanol at start, to 100% at 8 min, flushing until 20 min then re-equalibrating. Mobile phase A = water.
Sample preparation: (pharmaceutical preparations). Weigh 6g sample (30mg retinylpalmitate, 10mg cholecalciferol, 5mg tocopherol acetate) in a 100ml vol. flask, add 2-propanol, add stir bar, stir until dissolved (10min). Filter if needed (check suitability! PTFE adsorbs a lot) and inject. If peak shape is unstable: dilute 1:1 with 80% ethanol in water.
Standard preparation is the same, but we weigh more and dilute 10ml to 100ml to achieve more robustness.
It's simple, but it works. Hope this helps.