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!!!