Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry

Modification of collagen in vitro with respect to formation of Nɛ-carboxymethyllysine

“…2). The acid hydrolysates of the unmodified and carboxymethylated peptides were derivatized with l-FDVA and subjected to amino acid analysis, as we described recently for the analysis of different hydroxyproline isomers in collagen samples [19]. Both peptide samples contained the Ala-, Gly-, Phe-and Leu-derivatives detected at m/z 370.1, 356.1, 446.1 and 412.1, respectively, at the expected relative quantities.…”

“…Schematic view of the carboxymethylation reaction between collagen and glyoxalic acid by reductive alkylation (according to [20]). confirmed by the amino acid standard synthesized by direct l-FDVA-derivatization of -N-carboxymethyl-l-Lysine (NeoMPS SA, France) using the same conditions as for other amino acids and the hydrolysates [19]. Storage of this sample at room temperature for several days resulted in a slow degradation of the l-FDVA-CML(-l-FDVA) derivative and an increase of the Lys derivate content.…”

“…Gas phase hydrolysis and subsequent derivatization were performed as described previously in detail [19]. Briefly, 50 L of collagen solutions (9 mg/mL) were filled into small glass vial inlets (SUPELCO, Bellefonte, USA) and dried in vacuum.…”

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals

“…However, their applications were limited to abundant physiological fluids or large organs, because the amounts of d-Pro in mammalian tissues are too small in most cases. Concerning the determination of Hyp enantiomers in biological samples, an HPLC-MS method has been reported [28] to determine three Hyp isomers (trans-4-hydroxy-l-Pro, cis-4-hydroxy-l-Pro and cis-4-hydroxy-dPro) in the type I collagen hydrolysate. However, to our knowledge, there is no report on a two-dimensional method for the simultaneous determination of small amounts of d/l-Pro and all four isomers of 4-Hyp in mammalian tissues.…”

An Assay Method for Screening Inhibitors of Prolyl 4-hydroxylase in Immortalized Rat Hepatic Stellate HSC-T6 Cells

“…Hydroxyproline (HYP) in various tissues, plasma, and urine for the investigation of normal and pathological conditions of collagen metabolism is measured by colorimetric method (Kolar, 1990), HPLC (Langrock et al, 2007), GC/MS (Tredget et al, 1990) and enzymatic methods (Ono et al, 1999). The numerous assay procedures described before indicate the limits of each one with regarding to specificity, sensitivity, reproducibility, accuracy, and practical approach.…”