What are possible reasons for low recovery of histidine after acid hydrolysis of a pure protein API? - WKB190630

ENVIRONMENT

• Amino acid analysis (AAA)
• Histidine
• Acid hydrolysis
• AccQ•Tag
• Pico•Tag

ANSWER

• Constant boiling phenol was not added to the hydrolysis tubes.
• Histidine cleavage is much slower than other amino acids and can be inhibited by amine-containing buffers like TRIS. Prior to hydrolysis, ensure that the protein is free from amine-containing buffers. 
• An internal standard is not being used.

ADDITIONAL INFORMATION

For additional information:

Does waters have a recommended protein hydrolysis method for AccQ.Tag? 

When should the internal standard be added in amino acid analysis?

Fountoulakis, Michael, Lahm, Hans-Werner, "Hydrolysis and amino acid composition analysis of proteins," Journal of Chromatography A, 826, 109-134 (1998).