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-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 degrees 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. x 300 mm) was used for gradient elution separation at 25 degrees 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 approximately 100 micromol/l.
Du
Ming
M
Department of Chemistry, University of Missouri-Rolla, 1870 Miner Circle, Rolla, MO 65409, USA.
Wu
Wei
W
Ercal
Nuran
N
Ma
Yinfa
Y
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007536:Isomerism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014443:Tyrosine / Q000737:chemistry / Q000652:urine*
D014443:Tyrosine