University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Hydroxyproline

  • IUPAC Name: (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid
  • Molecular Formula: C5H9NO3
  • CAS Registry Number: 51-35-4
  • InChI: InChI=1S/C5H9NO3/c7-3-1-4(5(8)9)6-2-3/h3-4,6-7H,1-2H2,(H,8,9)/t3-,4+/m1/s1
  • InChI Key: PMMYEEVYMWASQN-DMTCNVIQSA-N

@ ChemSpider@ NIST@ PubChem

Citations 6

"Determination Of Hydroxyproline In Meat Products By Flow Injection Analysis"
Fresenius J. Anal. Chem. 1988 Volume 329, Issue 6 Pages 732-734
J. Möller and A. Sjödin

Abstract: Samples (10 g) were wrapped in tin foil and hydrolyzed (105°C, 15 h) with 30% H2SO4 (30 ml) or 6 M HCl (30 ml), then diluted to 500 mL. After filtration, the sample (100 µL) was injected into a carrier stream (hydrolysate blank, 0.6 mL min-1) and merged with oxidation reagent, aqueous 1.4% chloramine T - buffer (citrate - acetate, in aqueous 29% propanol; pH 6.0) (1:9; 0.6 mL min-1), and with color reagent, aqueous 10% dimethylaminobenzaldehyde - 70% HClO4 - propanol (2:1:1) (0.6 mL min-1). The reaction coil was operated at 80°C to 85°C and detection was at 550 nm. Sample throughput was 30 h-1 and the coefficient of variation (n = 20) was 1% for 50 mg L-1 of hydroxyproline.
Meat Spectrophotometry Heated reaction Tecator

"Improved High Performance Liquid Chromatography Method For Quantitation Of Proline And Hydroxyproline In Biological Materials"
J. Chromatogr. A 1986 Volume 356, Issue 1 Pages 163-170
K. Yaegaki, J. Tonzetich and A. S. K. Ng

Abstract: Test samples were derivatized with phthalaldehyde(I) to remove primary amino-acids and subjected to HPLC on a column (10 cm x 8 mm) of Nova-Pak C18 Radial-Pak with gradient elution with acetonitrile containing acetate buffer solution (pH 6.4) at 1.5 mL min-1. The secondary amino-acid-containing fractions were collected, freeze-dried and derivatized with phenyl isothiocyanate. The resulting mixture was evaporated to dryness, and the residue was dissolved in phosphate buffer and analyzed on the same column as before. Detection was at 254 nm. Proline(I) and hydroxyproline(II) were well resolved with good peaks; recovery was 93% provided that the analysis was initiated within 5 min of I derivatization. The method was evaluated by using human gingival fibroblast cultures and was found to be particularly useful for the analysis of low concentration. of I and II in the presence of large amounts of primary amino-acids. Compared with other methods, this technique has higher sensitivity and recovery, no interference and no need for post-column derivatization.
Gingival Human HPLC Spectrophotometry Interferences

"Rapid Microdetermination Of Hydroxyproline In Biomedical Samples By Flow Injection Analysis Using Cysteine As An Antioxidant"
Anal. Sci. 1990 Volume 6, Issue 1 Pages 39-44
K. UCHIDA, M. TOMODA, T. SHIBATA, S. IKEUCHI, T. HASEBE, T. MIWA, T. NOMOTO, K. FUKUSHIMA, S. SAITO and S. INAYAMA

Abstract: Tissue samples are hydrolyzed with 6 M HCl, the mixtures are freeze-dried, and the residues are dissolved in 0.01 M cysteine. Portions are injected into a stream of chloramine T solution (0.7 g L-1 in borate buffer of pH 8.7). Passage through a heating coil at 100°C causes hydroxyproline (I) to be oxidized and decarboxylated to pyrrole. Treatment with a solution of Ehrlich reagent (4-dimethylaminobenzaldehyde) in 10% H2SO4 and 10% Triton X-100 gives a colored product, which is detected at 560 nm. The calibration graph is rectilinear in the range 1 to 80 µg mL-1 of I. Optimization experiments are described especially for concentration. of chloramine T (oxidant) and cysteine (color stabilizer). The method, which avoids use of organic solvents, allows 100 samples to be analyzed in ~5 h. Recoveries of I are 94.8 to 103.7%. Results agreed well with those from a batch method.
Skin Spectrophotometry Buffer pH Triton X Calibration Optimization Heated reaction Surfactant

"Specific Proline And Hydroxyproline Detection Method By Post-column Derivatization For High Performance Liquid Chromatography"
Biosci. Biotechnol. Biochem. 1995 Volume 59, Issue 9 Pages 1764-1765
Ozaki, A.;Shibasaki, T.;Mori, H.

Abstract: Samples (10 l) were applied to a Sumiciral OA-5000 column (25 cm x 4.6 mm i.d.) at 38°C and eluted (1 ml/min) with 1 mM aqueous copper sulfate. The eluate was mixed with 25 mM EDTA in 0.3 M borate buffer of pH 9.6 at a flow rate of 0.2 ml/min. Derivatization was carried out by adding 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole in methanol (1 g/l) at a flow rate of 0.5 ml/min at 60°C. Detection was at 541 nm (excitation at 503 nm). Calibration graphs were linear from 7.6-7600 pmol-trans-4-hydroxy-L-proline. Other stereoisomers were detected by this method (results tabulated). This technique can be applied to the analysis of microbial hydroxyproline with good results.
HPLC Post-column derivatization

"New Convenient Method For Microdetermination Of Hydroxyproline By Flow Injection Analysis"
Chem. Pharm. Bull. 1986 Volume 34, Issue 6 Pages 2649-2652
Uchida, K.;Shibata, T.;Tomoda, M.;Saito, S.;Inayama, S.

Abstract: The flow injection analysis modification of the conventional KISO method is based on the color reaction of pyrrole, produced from hydroxyproline(I) by oxidation and decarboxylation, with Ehrlich reagent. The hydrolysate solution (~20 µg of I in 0.5 to 0.75 ml) prepared from the tissue sample was injected into a stream of the buffer (KCl 18.0 g l-1, H3BO3 4.95 g l-1, and KOH, pH 8.7) and oxidant solution (chloramine T, 0.1408 g l-1); after oxidation, the sample was decarboxylated at 100°C. The resulting stream was mixed 1:1 with Ehrlich reagent, and the absorbance of the colored product was measured 560 nm. Response was rectilinearly related to I concentration. up to 80 µg mL-1. The time required for one measurement was ~3.5 min, and the coefficient of variation at 100 µg mL-1 was 1.05% (n = 24).
Biological tissue Spectrophotometry Heated reaction

"Measurement Of Free And Total Hydroxyproline By Automated Flow Injection Of Serum Or Urine Samples From Maintenance Hemodialysis Patients With Renal Osteodystrophy"
J. Clin. Lab. Anal. 1994 Volume 8, Issue 5 Pages 267-272
Uji Y, Karmen A, Okabe H, Hata K, Miura M, Ozaki K, Minamizaki M, Shibata T, Inayama S

Abstract: For the determination of total hydroxyproline, 100 µL of plasma or 1 mL of urine was mixed with 200 µL of 12 M HCl and autoclaved for 3 h at 120°C. The hydrolysate was neutralized with 1 mL of 12 M KOH and 1 mL of 1 M L-cysteine. A 100 µL portion was injected into a carrier stream in a FIA system of chloramine T in borate buffer and KCl adjusted to pH 8.7 with 1 M KOH. The mixture was heated at 120°C in a mixing coil (24 m x 1 mm i.d.) in an Al block. The reaction mixture was merged with a stream of Ehrlich's reagent (1:1), the resulting solution passed through a reaction coil (10 m x 1 mm i.d.) to a double beam photometer for detection at 560 nm. Free hydroxyproline was determined as above but omitting the hydrolysis step and filtering the serum. Calibration graphs were linear up to 1.22 mM with a detection limit of 3.8 µM. The within-run RSD was 2.34, 2.25 and 2.53% for 76, 38 and 19 µM, respectively. The recovery of 10^-50 µM of hydroxyproline in urine was 92-104%. Results agreed well with those obtained by HPLC.
Blood Plasma Urine Serum Human Spectrophotometry HPLC Clinical analysis Dialysis