Simultaneous determination of 3-nitro tyrosine, o-, m-, and p-tyrosine in urine samples by liquid chromatography–ultraviolet absorbance detection with pre-column cloud point extraction

Abstract

Stable 3-nitro tyrosine (3-NO 2-Tyr), o-, m-, and p-tyrosine isomers induced by oxidation of tyrosine residues in protein were considered important biomarkers for the existence of toxic oxidizing agents peroxynitrite (ONOO −) and OH , which could lead to such diseases as acute lung injury, neurodegenerative disorders, atherosclerosis, cancers and many other diseases. Therefore, development of an accurate, simple and sensitive method to simultaneously detect o-, m-, and p-tyrosine and 3-NO 2-Tyr is necessary. Fluorescence detection is highly sensitive to o-, m-, and p-tyrosine, but it cannot be used to detect 3-NO 2-Tyr, due to the strong fluorescence-quenching characteristic of the NO 2 group. In this study, we developed a highly sensitive reversed HPLC–UV method, combined with pre-column cloud point extraction (CPE), to simultaneously determine o-, m-, and p-tyrosine and 3-NO 2-Tyr. The procedure included derivatization of a sample with 6-aminoquinolyl- N-hydroxy-succinimidyl carbomate (AccQ) at 0.20 mol/l borate buffer (pH 8.80) for 30 min at 70 °C, and pre-concentration with surfactant cloud point extraction. The surfactant-rich phase was then diluted with deionized water and injected directly into the to HPLC column for analysis. A C 18 column (3.9 mm i.d. × 300 mm) was used for gradient elution separation at 25 °C and the detection wavelength was at 254 nm. Nineteen general amino acids showed no interference. The detection limits of p-, o-, m-Tyr and 3-NO 2-Tyr were between 5 and 15 nmol/l. The linear range was from 0.05 to ∼100 μmol/l.