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

Stable 3-nitro tyrosine (3-NO2-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-NO2-Tyr is necessary. Fluorescence detection is highly sensitive to o-, m-, and p-tyrosine, but it cannot be used to detect 3-NO2-Tyr, due to the strong fluorescence-quenching characteristic of the NO2 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-NO2-Tyr. The procedure included derivatization of a sample with 6-aminoquinolyl-N-hydroxy-succinimidyl carbomate (AccQ) at 0.20mol/l borate buffer (pH 8.80) for 30min 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 C18 column (3.9mm i.d. × 300mm) was used for gradient elution separation at 25°C and the detection wavelength was at 254nm. Nineteen general amino acids showed no interference. The detection limits of p-, o-, m-Tyr and 3-NO2-Tyr were between 5 and 15nmol/l. The linear range was from 0.05 to ~100µmol/l