H2O + Vitamin D3 (Internal Standards) + Vitamin C Solution (in MeOH) + Sodium Hydroxide. Water bath 30 min, extract with ethyl ester and evaporate to dry, and then dissolve in MeOH.
The peak @ rt=2.8 is the problem?
(1) Double check the contamination from the needles and HPLC volumetric flash, vials.
(2) If doesn't work, -> what is the concentration of the Sodium Hydroxide? 0.1 N? Do you make sure that the internal standard (Vitamin D3) would not discomposed with the present the anti-oxidant (Vitamin C) during the saponification process?
The peak @ rt=2.8 is the problem?
(1) Double check the contamination from the needles and HPLC volumetric flash, vials. (3) The final method is that follow the USP method to troubleshooting.
