Scientific Publications by FDA Staff

Anal Bioanal Chem 2015 Jan;407(3):749-59

Modern analytics for naturally derived complex drug substances: NMR and MS tests for protamine sulfate from chum salmon.

Gucinski AC, Boyne MT 2nd, Keire DA

This work describes orthogonal NMR and MS tests for the structure and composition of the drug protamine sulfate derived from chum salmon. The spectral response pattern obtained by 1D-1H-NMR and MS methods from salmon protamine, a mixture of four predominant peptide chains is dependent on the amino acid sequence and abundance of each peptide. Thus, an assay was developed based on the ratios of alanine, glycine and arginine amino acid residue NMR peaks (relative to the arginine CdeltaH proton signal) in this mixture that are unique to the salmon source. In addition, MS analysis provided sensitive sequence determination and impurity analysis based on shifts from exact masses. Spectra from protamine sulfate active pharmaceutical ingredient (API) suppliers and from a formulated drug product purchased from the US market were examined. Based on these marketplace survey data, NMR acceptance criteria for chum salmon derived protamine sulfate could be based on the absence of aromatic amino acid signals and on ratios of Ala betaH/Arg deltaH, Gly alphaH/Arg deltaH and Arg alphaH/Arg deltaH integrated areas of 2.4 +/- 1 %, 9.4 +/- 3 % and 50 +/- 5 %, respectively. For MS, acceptance criteria based on the presence of specific mass to charge (m/z) ratio peaks (m/z = +8 of 530.455, 540.841, 532.208 and 508.950) could be used for the four major peptides present in the mixture with relative abundances of 17 +/- 1 %, 31 % +/- 2 %, 27 +/- 1 % and 25 % +/- 3 %, respectively. The specificity of the combined NMR and MS assay was tested by comparison to data obtained from herring protamine which contains a different mixture of peptides with related amino acid sequences. Both assays were able to clearly distinguish protamine derived from these different natural sources.