Journal of Chromatography B, 803 (2004) 321–329 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 Ming Du, Wei Wu, Nuran Ercal, Yinfa Ma ∗ Department of Chemistry, University of Missouri-Rolla, 1870 Miner Circle, Rolla, MO 65409, USA Received 28 October 2003; received in revised form 6 January 2004; accepted 8 January 2004 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 extrac- tion (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 mol/l. © 2004 Elsevier B.V. All rights reserved. Keywords: Tyrosine; 3-Nitrotyrosine 1. Introduction Nitric oxide (NO or NO • ) is a ubiquitous gaseous free radical which is important in regulating numerous biologi- cal processes [1–4]. A NO group can react with superoxide anions to yield toxic oxidizing agents called reactive nitro- gen species (RNS). For example, peroxynitrite (ONOO - ) [5–7] reacts with tyrosine residues in proteins to form 3-nitro-tyrosine (3-NO 2 -Tyr) [8], which results in such dis- eases as acute lung injury, neurodegeneration, atherosclero- sis and some cancers. Since peroxynitrite has a very short half-life at physiological pH, the stable 3-nitro-tyrosine (3-NO 2 -Tyr), induced by oxidation of tyrosine residues in protein, was considered an important biomarker of RNS pro- duction in various tissues. In addition, the levels of ortho- ∗ Corresponding author. Tel.: +1-573-341-6220; fax: +1-573-341-6033. E-mail address: yinfa@umr.edu (Y. Ma). and meta-tyrosine have been reported to be endogenous biomarkers of oxidative damage by oxygen radicals OH • [9–11]. Therefore, the development of an accurate, simple and sensitive method to simultaneously detect ortho-, meta-, para-tyrosine (o-, m- and p-Tyr) and 3-NO 2 -Tyr is neces- sary. In the past, several analytical methods were developed to measure them, including GC [8,12,13], GC–MS [14–20], HPLC with electrochemical detection (HPLC–ECD) [14–21], HPLC–UV [8,22], and HPLC with fluorescence de- tection [23–25]. However, these methods cannot be used to simultaneously determine all of these biomarkers. GC–MS and HPLC–ECD are the most sensitive methods for assess- ing these compounds. However, high cost is associated with GC–MS because tedious sample pretreatment is required, and electrode poisoning is often encountered in ECD caused by other biological compounds in the sample which may result in loss of detection sensitivity. Even though a fluores- cence detector can be used to detect the other three tyrosine isomers, it cannot be directly used to detect of 3-NO 2 -Tyr 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.01.027