Cyclodextrin-Modified Capillary Zone Electrophoresis for the Chiral Analysis of Proline and Hydroxyproline Stereoisomers in Chicken Collagen Hydrolysates

Abstract

The stability of collagen, the most abundant protein in humans and many animals, is related to the hydroxylation of L-proline, a post-translational modification occurring at carbon 3 and 4 on its pyrrolidine ring. Collagens of different origins have shown different proline hydroxylation levels, making hydroxyprolines useful biomarkers in structure characterizations. The presence of two chiral carbon atoms, 3-hydroxyproline and 4-hydroxyproline, results in eight stereoisomers (four pairs of enantiomers) whose quantitation in collagen hydrolysates requires enantioselective analytical methods. Capillary electrophoresis was applied for the separation and quantitation of the eight stereoisomers of 3- and 4-hydroxyproline and D,L-proline in collagen hydrolysates. The developed method is based on the derivatization with the chiral reagent (R)-(-)-4-(3-Isothiocyanatopyrrolidin-yl)-7-nitro-2,1,3-benzoxadiazole, enabling the use of a light-emitting diode-induced fluorescence detector for high sensitivity. The separation of the considered compounds was accomplished in less than 10 min, using a 500 mM acetate buffer pH 3.5 supplemented with 5 mM of heptakis(2,6-di-O-methyl)-β-cyclodextrin as the chiral selector. The method was fully validated and showed the adequate sensitivity for the application to samples of collagen hydrolysates. The analysis of samples extracted from chicken Pectoralis major muscles affected by growth-related myopathies showed different stereoisomer patterns compared to those from the unaffected control samples.

Keywords: capillary electrophoresis; chiral separations; collagen; cyclodextrins; derivatization; fast-growing chickens; fluorescent detection; hydroxyprolines; spaghetti meat; wooden breast.

Figure 1

The structure of proline and hydroxyproline stereoisomers and the related abbreviations used in this paper.

Figure 2

The proposed structure of the thiourea derivative [19] obtained by the reaction of the derivatization reagent (R)-NCS (black moiety) with a generic 4-hydroxyproline (red moiety).

Figure 3

Electropherograms of the separation of D,L-Pro and hydroxyproline amino acids derivatized with (R)-NCS using a BGE composed of 500 mM acetate buffer with a pH of 3.5 (A), supplemented with DM-βCD at 2.5 mM (B) and 5 mM (C). Other conditions: a fused-silica capillary with a 55.5 cm total length (34 cm, length to the detector); 50 μm i.d.; voltage 30 kV; temperature 25 °C; hydrodynamic injection at 50 mbar × 10 s; and LEDIF detection (480 nm excitation wavelength). Symbols: Reagent excess (R); the peaks are the thiourea derivatives of the compounds whose abbreviations are given in Figure 1. In the electropherograms (A,B) the peak of the reagent excess (R) has been erased.

Figure 4

The electropherogram of the separation of D,L-Pro and hydroxyproline amino acids derivatized with (R)-NCS using a BGE composed of a 500 mM acetate buffer with a pH of 3.5 supplemented with βCD at a 5 mM concentration. Other conditions and symbols as in Figure 3.

Figure 5

The scheme of the two-step derivatization and proposed structures [19,21] of the obtained derivatives. Abbreviations and symbols: OPA: ortho-phthalaldehyde, ME: 2-mercaptoethanol; and TEA: triethylamine. A generic 4-hydroxyproline molecule is depicted in red.

Figure 6

The electropherogram of a collagen type III hydrolysate extracted from chicken Pectoralis major muscle affected by a Wooden Breast abnormality; the insets provide magnified details of the electropherogram, highlighting the areas corresponding to the target analytes. Conditions and symbols as in Figure 3C.