Collagen is the main protein of connective tissue in animals and is the most abundant protein in mammals, making up about 25% of the total protein content. Hydroxyproline, Proline and Glycine make up approximately 50% of collagen. Hydroxyproline and Hydroxylysine are the 2 major cross-linking amino acids in collagen and contribute to collagen’s strength. This method is for accurate determination and quantification of amino acids in collagen.

Summary

Samples are digested using an acid-stable hydrolysis procedure to release the bound amino acids present in the protein of the sample. Samples are then diluted to an appropriate concentration based on the protein content of the sample. A solution of a deproteinizing solution (5-sulfosalicylic acid hydrate), internal standard (L-2-Amino-3-Guanidinopropionic Acid Hydrochloride), and sodium buffer is added to the sample to achieve the target dilution. The mixture is then filtered into an HPLC vial.

Collagen analysis is performed on Hitachi HPLC amino acid analyzers. Amino acids are separated by cation exchange chromatography using various sodium eluents. Absorbance is measured at 440 and 570 nm following post-column color development by Ninhydrin reagent. Appropriate standards are run to ensure each sequence is running properly and used in the quantification of samples using peak areas and applying an external standard technique.

Limits of detection are 0.3 to 1.0 micromoles per liter. There is no high-end limit as the sample can always be diluted down to an appropriate detection range.

Amino Acids Quantified

Hydroxyproline

Glutamic Acid

Alanine

Valine

Aspartic Acid

Proline

Isoleucine

Leucine

Threonine

Glycine

Tyrosine

Phenylalanine

Serine

Hydroxylysine

Lysine

Histidine

Arginine