Products Cited in Publications Worldwide Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources

Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources

Martina Lioi; Sara Tengattini; Roberto Gotti; Francesca Bagatin; Stefano Galliani; Gabriella Massolini; Simona Daly; Caterina Temporini

DOI:

Abstract

During collagen biosynthesis, is post-translationally converted to by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. has four isomers, depending on whether is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight isomers. After the chiral derivatization of the isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 μm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of abundances in different species (human, chicken) and sources (native, recombinant).

Keywords

Collagen ; Amino acid analysis ; isomers ; Recombinant collagen ; Reverse phase chromatography ; Mass spectrometry

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L-Proline

A110254147-85-395%

L-Proline

10.0/1mg

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H-Ile-OH

A15298173-32-597%

H-Ile-OH

10.0/250mg

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H-Leu-OH

A19711961-90-597%

H-Leu-OH

10.0/25g

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H-Hyp-OH

A33372851-35-497%

H-Hyp-OH

10.0/5g

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