Analysis of hydroxyproline in urine by high-performance liquid chromatography after dabsyl-chloride derivatization

Abstract

In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4'-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l.

Selective Analysis of Secondary Amino Acids in Gelatin Using Pulsed Electrochemical Detection

“…Precolumn derivatization with reagents that produce fluorescent adducts has been used for determination of secondary amino acids in urine and tissue samples. − However, these reagents lack specificity for secondary amino acids and require a degree of kinetic control during derivatization to improve selectivity. Another technique commonly used in HPLC applications has been to block or “mask” primary amino acids by derivatization with o -phthalaldehyde (OPA) in the presence of a thiol followed by an additional derivatization with a reagent to produce fluorescent − or electrochemically active adducts . Although these approaches have been successful, stability of the amino acid derivatives remains an issue.…”