The presence of pyridoxamine phosphate in extracts of freeze-dried raw and evaporated milks has been demonstrated by separation and identification of the vitamin B6 active compounds by chromatography and electrophoresis on paper. Its presence in the milk extracts is the cause of the higher values for vitamin B6 activity measured with Streptococcus faecalis as compared with those obtained with Str. faecium or Saccharomyces carlsbergensis. This is because Str. faecalis can utilize the phosphate for growth as readily as free pyridoxamine, whereas Str. faecium and S. carlsbergensis cannot. The mild acid treatment used for extracting the vitamin B6 active compounds from the milk samples for microbiological assay was found to be insufficient to hydrolyse the pyridoxamine phosphate. Further treatment of the acid extracts with intestinal phosphatase released the pyridoxamine from its phosphate and increased the vitamin B6 activity measured with S. carlsbergensis and Str.faecium so that the total vitamin B6 activities of the freeze-dried raw and evaporated milks measured microbiologiclly, were then in agreement with the values found in previous tests with chicks and rats.

Pyridoxamine phosphate could only be detected in small amounts in a sample of fresh milk. The possibility that more of it was formed during the processing and storage of the freeze-dried samples is discussed.