To compare the HPLC profiles of FeSSIF-V2 prepared using a) FeSSIF-V2 Powder with FeSSIF-V2 Buffer Concentrate and b) FeSSIF-V2 Powder with buffer made from scratch, the following HPLC in-house method has been used throughout the different experiments.

Example of blank FeSSIF-V2 media

The chromatogram of FeSSIF-V2, using Buffer Concentrate (above left), shows a much cleaner HPLC profile avoiding the extra peak when using the Maleic acid buffer from scratch (above right).

HPLC Chromatograms of three drugs in improved FeSSIF-V2 media using Buffer Concentrate (in purple) compared with FeSSIF-V2 using buffer from scratch (in orange)

A peak attributable to the maleic acid is present in the medium FeSSIF-V2 using buffer from scratch but not present in the FeSSIF-V2 medium using the Buffer Concentrate.

Typical chromatographic parameters, showing the similarities of both media, are given in the table below. The samples have been diluted with the diluent 50% MeOH : 50% Phosphate Buffer pH 2.0 20mM at a 1:4 v/v ratio.