Journal of Chromatography B (v.827, #1)

Editorial response to author review in publishing science by R. Bischoff; G. Hopfgartner; H.T. Karnes; W. Lindner; D.K. Lloyd; T.M. Phillips (2).

Foreword by H. Thomas Karnes (3-4).

When biomolecules such as proteins, lipids, and DNA are subjected to oxidative attack by free radicals or other reactive species, a number of measurable biomarkers may be produced. The study of oxidative DNA damage is valuable in research concerning cancer and aging. The current review includes methodology involving various separation science techniques for the analysis of DNA oxidation biomarkers, mainly 8-hydroxy-2′-deoxyguanosine. This review will present recent analytical developments with respect to sample preparation and instrumental considerations, noting key outcomes and biological relevance where appropriate.
Keywords: Oxidative stress; 8-Hydroxy-2′-deoxyguanosine; DNA damage; Analysis;

A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis (CE) with electrochemical detection (CEEC) was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with R.S.D. values ≤5% and limit of detection of 0.5 nM for both analytes. Basal concentrations of 8oxoG in rat cerebral cortex microdialysate were determined to be 3.2 ± 0.6 nM. Actual 8oxoG concentration in the brain was estimated to be 5.5 ± 1.3 nM based on in vivo delivery probe calibration. 8OHdG was not detected under basal conditions in the rat cerebral cortex extracellular fluid (ECF). These results were confirmed by LC with tandem mass spectrometry.
Keywords: 8-Oxoguanine; 8-Hydroxy-2′-deoxyguanosine; Microdialysis sampling; Capillary electrophoresis with electrochemical detection; On capillary sample stacking;

Detection of chlorinated DNA and RNA nucleosides by HPLC coupled to tandem mass spectrometry as potential biomarkers of inflammation by Carine Badouard; Mitsuharu Masuda; Hoyoku Nishino; Jean Cadet; Alain Favier; Jean-Luc Ravanat (26-31).
Upon inflammation, activated neutrophils secrete myeloperoxidase, an enzyme able to generate hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. An analytical method, involving HPLC coupled to electrospray tandem mass spectrometry, has been set-up to detect low levels of HOCl-induced nucleic acids lesions, including both ribo and 2′-deoxyribonucleoside derivatives of 8-chloroguanine, 8-chloroadenine and 5-chlorocytosine. Validation of the developed method was achieved using isolated cells treated with HOCl. The method was found to be sensitive enough to allow the measurement of background levels of 5-chloro-2′-deoxycytidine in the DNA of human white blood cells isolated from 7 mL of blood.
Keywords: Oxidative stress; DNA damage; RNA damage; Hypochlorous acid; Tandem mass spectrometry; Human biomarkers;

Kinetic measurement by LC/MS of γ-glutamylcysteine ligase activity by Karim Chikh; Françoise Flourie; Khelifa Arab; Jean Paul Steghens (32-38).
γ-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l-2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the γ glutamylaminobutyrate formed is measured at m/z  = 233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.
Keywords: γ-Glutamylcysteine ligase; Glutathione metabolism; Oxidative stress; Catalytic activity; LC/MS;

Porous graphitic carbon chromatography–tandem mass spectrometry for the study of isoprostanes in human cerebrospinal fluid by Kristina Claeson Bohnstedt; Bo Karlberg; Hans Basun; Staffan Schmidt (39-43).
F2-isoprostanes are produced by the non-enzymatic peroxidation of arachidonic acid in membrane phospholipids. This paper describes a new method for the determination of all four classes of F2-isoprostanes in human cerebrospinal fluid (CSF) involving separation on a 1 mm × 150 mm porous graphitic carbon (PGC) column and detection by triple quadrupole mass spectrometry in negative-ion electrospray mode. The sample pre-treatment consisted of an ultrafiltration step, following which 300 μl of CSF sample could be injected directly onto a 1 mm × 10 mm PGC guard column functioning as a trap for the analytes. The loading solvent was Milli-Q water at 125 μl/min. After 3 min, the sample was switched into the separation column. The F2-isoprostanes were separated in 20 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5 and a flow of 50 μl/min The limit of detection (calculated as 3S/N) was approximately 40 pM (14 pg/ml). The assay was linear within the examined range (18–450 pg/ml), using CSF spiked with iPF2α-III standard (r 2  > 0.995). Repeatability data were calculated for CSF spiked to 90 pg/ml and the relative standard deviation (RSD) obtained was 3% (n  = 6).
Keywords: Isoprostanes; Porous graphitic carbon; Cerebrospinal fluid; Oxidative stress;

We have presently studied a dialdehydic reagent, i.e. naphthalene-2,3-dicarboxaldehyde (NDA), as a fluorogenic probe for the labeling of intracellular reduced glutathione (GSH), using a yeast strain Candida albicans as a cell model. Chemical reactivity of NDA with both amino and sulfhydryl groups of the GSH molecule leads to a highly selective detection. Moreover, fluorescence properties of the resulting adduct fit well with most of modern instruments adapted for in situ measurements, and equipped with an argon laser. After incubation of cells with 100 μM of NDA for 20 min, cells were harvested and corresponding lysates obtained after a freezing cycle, were suspended in 0.2 M borate buffer pH 9.2 and analysed with HPLC (column: Spherisorb ODS-2 (125 mm × 4.6 mm i.d.) 5 μm; mobile phase: methanol–0.01 M phosphate buffer pH 6.5 (20:80, v/v) at a flow rate of 0.8 mL min−1; spectrofluorimetric detection: λ exc  = 430 nm and λ em  = 530 nm). The GSH-NDA adduct was identified in the yeast strain extracts using the reported HPLC technique and quantified versus a calibration curve of NDA derivatized with an excess of GSH (linearity range: 9–230 nM). The cell loading step of the free probe NDA and the extraction efficiency of the resulting NDA-GSH adduct were optimized.
Keywords: Reduced glutathione; Naphthalene-2,3-dicarboxaldehyde; Yeast strain; Cellular staining;

Capillary electrophoresis (CE) has become a useful analytical tool for the analysis of microdialysis samples. However, CE with UV detection (CE-UV) does not provide detection limits sufficient to quantify glutathione (GSH) and glutathione disulfide (GSSG) in biological samples such as liver microdialysates, because of the small optical path length in the capillary. To overcome this limitation, an on-column preconcentration technique, pH-mediated base stacking, was used in this study to improve the sensitivity of CE-UV. This stacking technique allowed large volumes of high ionic strength sample injection without deterioration of the separation efficiency and resolution. A 26-fold increase in sensitivity was achieved for both GSH and GSSG using the pH-mediated base stacking, relative to normal injection without stacking. The limit of detection for GSH and GSSG was found to be 0.75 μM (S/N  = 6) and 0.25 μM (S/N  = 6), respectively. The developed method was used to analyze GSH and GSSG in liver microdialysates of anesthetized Sprague Dawley male rats. The basal concentrations of GSH and GSSG in the liver microdialysates of male rats were found to be 4.73 ± 2.08 μM (n  = 7) and 5.52 ± 3.66 μM (n  = 7), respectively.
Keywords: Capillary electrophoresis; Glutathione; Glutathione disulfide; pH-mediated stacking; On-column preconcentration; Microdialysis samples;

Thiol-dependent peroxidases catalyzing the reductive detoxification of lipid hydroperoxides (LOOHs) are crucial antioxidant components of mammalian cells. There is a growing interest in manipulating expression of such enzymes to better understand their biological roles. A new approach for determining their cellular activity is described, whereby LOOH reduction kinetics are tracked by high performance thin layer chromatography with peroxide-sensitive tetramethyl-p-phenylenediamine detection (HPTLC-TPD). The approach was tested on a tumor cell transfectant clone (7G4) over-expressing selenoperoxidase GPx4. Timed incubation of Triton-solubilized 7G4 cells with GSH and peroxidized phosphatidylcholine (PCOOH), followed by lipid extraction, HPTLC-TPD and densitometry revealed an exponential decay of PCOOH at a rate ∼80-times greater than that for GPx4-deficient controls (VC). A TPD-detectable cholesterol hydroperoxide (7α-OOH) was also reduced much faster by 7G4 than VC extracts. Spraying with H2SO4 after TPD revealed both 7α-OOH loss and resolved diol product (7α-OH) accumulation, the kinetics of which were identical. The approach described is relatively convenient, highly specific, and much more sensitive than conventional assays for cellular LOOH reducing enzymes.
Keywords: Peroxidase activity; Thin-layer chromatography; Phospholipid hydroperoxide; Cholestrol hydroperoxide;

Oxidative stress in brain aging, neurodegenerative and vascular diseases: An overview by E. Mariani; M.C. Polidori; A. Cherubini; P. Mecocci (65-75).
According to the free radical theory, aging can be considered as a progressive, inevitable process partially related to the accumulation of oxidative damage into biomolecules – nucleic acids, lipids, proteins or carbohydrates – due to an imbalance between prooxidants and antioxidants in favor of the former. More recently also the pathogenesis of several diseases has been linked to a condition of oxidative stress. In this review we focus our attention on the evidence of oxidative stress in aging brain, some of the most important neurodegenerative diseases – Alzheimer's disease (AD), mild cognitive impairment (MCI), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD) – and in two common and highly disabling vascular pathologies—stroke and cardiac failure. Particular attention will be given to the current knowledge about the biomarkers of oxidative stress that can be possibly used to monitor their severity and outcome.
Keywords: Oxidative stress; Vascular; Aging; Neurodegeneration; Brain;

A high-performance liquid chromatography (HPLC) method to determine malondialdehyde (MDA) as the 2,4-dinitrophenylhydrazine (DNPH) derivative was applied to biological samples (serum and liver homogenates). Since MDA is considered a presumptive biomarker for lipid peroxidation in live organisms, a model for nutritionally induced oxidative stress (hypercholesterolemic rats) was studied in comparison with normocholesterolemic animals. The effect of diet supplementation with fruits rich in antioxidant polyphenols was assessed. The proposed method showed to be precise and reproducible, as well as sensitive enough to reflect differences in the oxidative status in vivo. A significant decrease of serum and liver MDA concentrations in animals fed diets containing 0.3% of polyphenols from strawberry, cocoa or plum was observed in the normocholesterolemic groups. This reduction was especially noteworthy in the hypercholesterolemic animals, with increased MDA levels indicating enhanced lipid peroxidation in the controls, yet with values parallel to the normocholesterolemic groups in animals fed the polyphenol-rich diets. These results point out the beneficial effects of phenolic antioxidants from fruits in preventing oxidative damage in vivo.
Keywords: Malondialdehyde; HPLC; 2,4-Dinitrophenylhydrazine; Lipid peroxidation; Oxidative stress; Serum; Liver; Fruit antioxidant polyphenols;

8-Hydroxy-2′-deoxyguanosine (8OHdG) has been considered as an excellent marker of oxidative DNA damage associated with age-related diseases such as cancer. In this paper, two sensitive methods—capillary electrophoresis with electrochemical detection (CE-ECD) and gas chromatography/mass spectrometry (GC/MS) were developed for urinary 8OHdG analysis. The R.S.D. of the spiked recovery of the two methods for determining urinary 8OHdG was 4.03% and 8.25%, respectively, and the results from the two methods have a good consistency (r  = 0.999, P  < 0.01). The developed CE-ECD method was applied to investigate the urinary 8OHdG levels in different cancer patients and follow up the response of therapy. It was found that the excretion levels of urinary 8OHdG in cancer patients were significantly higher than those in healthy persons (35.26 ± 27.96 nM versus 13.51 ± 5.08 nM, P  < 0.05), and cancer patients receiving surgical therapy and chemotherapy showed a significant decrease in urinary 8OHdG.
Keywords: 8-Hydroxy-2′-deoxyguanosine; Cancer; Capillary electrophoresis; GC/MS;

Oxidative damage to brain is a featured shared by several destructive and degenerative diseases and is thought to contribute to disease pathogenesis. Two commonly proposed sources of the increased free radical stress that leads to oxidative damage in several of these diseases are excitotoxicity and activation of innate immunity, both of which are proposed pharmacologic targets. Here we used models of excitotoxicity, intracerebroventricular (ICV) kainate (KA), and innate immune activation, ICV lipopolysaccharide (LPS), to test the effectiveness of peripherally administered α-tocopherol (AT) and γ-tocopherol (GT) as neuroprotectants. We quantified murine cerebral oxidative damage by measuring F2-isoprostanes (IsoPs) and F4-neuroprostanes (NeuroPs) using stable isotope dilution methods followed by gas chromatography–mass spectrometry with selective ion monitoring. Our data showed that peripherally administered AT and GT were equally effective at suppressing acute oxidative damage from direct excitotoxicity caused by KA. In contrast, peripherally administered AT, but not GT, was effective at suppressing delayed neuronal oxidative damage from activated glial innate immune response. These data imply that AT may be more broadly protective of cerebrum from oxidative damage in different disease contexts.
Keywords: Brain; Oxidative damage; Tocopherol; Isoprostane; Neuroprostane;

The air pollutant 3-nitrobenzanthrone (3-NBA), emitted in diesel exhaust, is a potent mutagen and genotoxin. 3-NBA can isomerise to 2-nitrobenzanthrone (2-NBA), which can become more than 70-fold higher in concentration in ambient air. In this study, three independent methods have been employed to evaluate the oxidative stress and genotoxicity of 2-NBA compared to 3-NBA in the human A549 lung cell line. HPLC–EC/UV was applied for measurements of oxidative damage in the form of 8-oxo-2′-deoxyguanosine (8-oxodG), 32P-HPLC for measurements of lipophilic DNA-adducts, and the Comet assay to measure a variety of DNA lesions, including oxidative stress. No significant oxidative damage from either isomer was found regarding formation of 8-oxodG analysed using HPLC–EC/UV. However, the Comet assay (with FPG-treatment), which is more sensitive and detects more types of damages compared to HPLC–EC/UV, showed a significant effect from both 3-NBA and 2-NBA. 32P-HPLC revealed a strong DNA-adduct formation from both 3-NBA and 2-NBA, and also a significant difference between both isomers compared to negative control. These results clearly show that 2-NBA has a genotoxic potential. Even if the DNA-adduct forming capacity and the amount of DNA lesions measured with the 32P-HPLC and Comet assay is about one third of 3-NBA, the high abundance of 2-NBA in ambient air calls for further investigation and evaluation of its health hazard.
Keywords: HPLC–EC/UV; 32P-HPLC; Comet assay; 2-NBA; 3-NBA; Genotoxicity; 8-oxodG; Oxidation; Oxidative stress; DNA-adducts; DNA lesions; FPG-enzyme;

We recently described an isotope dilution reversed-phase liquid chromatography–atmospheric pressure chemical ionization–ion-trap-tandem mass spectrometry (HPLC–APCI–MS/MS) method for the quantitative determination of oxidized amino acids in human urine, including o,o-dityrosine, a specific marker of protein oxidation. In the present study, we investigated the possibility to use a triple quadrupole instrument for the analysis of this biomarker in urine. The two instruments were compared in terms of sensitivity, specificity and reproducibility. Results showed that the triple quadrupole instrument reaches 2.5-fold higher sensitivity (LOD = 0.01 μM) compared to the previously used ion-trap instrument. Precision of the present assay is as follows: in-day variation is 4.6% and inter-day variation is 17%. The currently developed method was applied to a group of smoker urine samples. The mean urinary o,o-dityrosine concentration was 0.08 ± 0.01 μM. Expressed per urinary creatinine concentration, this corresponds to 10.1 ± 0.4 μmol/mol creatinine. This is comparable to the previously reported values of 5.8 ± 0.3 μmol/mol creatinine in non-smokers night-time urines, and 12.3 ± 5 μmol/mol creatinine in day-time urines measured by the ion-trap instrument.
Keywords: o,o′-Dityrosine; Atmospheric pressure chemical ionisation; Triple quadrupole tandem mass spectrometry; Isotope dilution; Biomarker; Smoker;

A sensitive, selective, specific and rapid liquid chromatographic–electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination in skeletal muscle of the Michael adducts between 4-hydroxy-trans-2-nonenal (HNE), one of the most reactive lipid peroxidation-driven unsaturated aldehyde, and glutathione (GSH) and the endogenous histidine-containing dipeptides carnosine (CAR) and anserine (ANS), with the final aim to use conjugated adducts as specific and unequivocal markers of lipid peroxidation. Samples (skeletal muscle homogenates from male rats) were prepared by protein precipitation with 1 vol. of a HClO4 solution (4.2%; w/v) containing H-Tyr-His-OH as internal standard. The supernatant, diluted (1:1, v/v) in mobile phase, was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water–acetonitrile–heptafluorobutyric acid (9:1:0.01, v/v/v) at a flow rate of 0.2 ml/min, with a run time of 12 min. Detection was on a triple quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were in multiple reaction monitoring (MRM) mode using the following precursor → product ion combinations: H-Tyr-His-OH (IS): m/z 319.2 → 156.5 + 301.6; GS-HNE: m/z 464.3 → 179.1 + 308.0; CAR-HNE: m/z 383.1 → 110.1 + 266.6; ANS-HNE: m/z 397.2 → 109.1 + 126.1. The method was validated over the concentration ranges 1.5–90 (GS-HNE) and 0.4–40 (CAR-HNE, ANS-HNE) nmoles/g wet tissue, and the LLOQ were 1.25 and 0.33 pmoles injected respectively. The intra- and inter-day precisions (CV%) were <7.38% (≤10.90% at the LLOQs); intra- and inter-assay accuracy (RE%) was within ± 7.0% for all the concentrations (≤18% at the LLOQs). The method was applied to quantitate peptide-HNE Michael adducts in rat skeletal muscles exposed to oxidative stress to endogenously generate HNE, and the results indicate that CAR-HNE can be considered as an early, specific and stable marker of lipid peroxidation in excitable tissues.
Keywords: Glutathione; Histidine-containing peptides; Carnosine; Anserine; 4-Hydroxy-trans-2-nonenal; Michael adducts; LC–ESI-MS/MS; Oxidative stress markers; Rat skeletal muscle;

Susceptibility of actin to modification by 4-hydroxy-2-nonenal by Munetaka Ozeki; Aya Miyagawa-Hayashino; Shinya Akatsuka; Tomoyuki Shirase; Wen-hua Lee; Koji Uchida; Shinya Toyokuni (119-126).
4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3–5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced.
Keywords: 4-Hydroxy-2-nonenal; Monoclonal antibody; Oxidative stress; Protein modification; Kidney; Iron; Carcinogenesis; Michael adducts;

Ulam raja (Cosmos caudatus) is used traditionally for improving blood circulation. In this study, it was found that ulam raja had extremely high antioxidant capacity of about 2400 mg l-ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh sample. Antioxidant peaks in extract of ulam raja were firstly characterized using free radical spiking test through high performance liquid chromatography coupled with mass spectrometry (MS). Upon reaction with 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, intensities of antioxidant peaks will be significantly reduced. HPLC/MS n was further applied to elucidate the chemical structures of antioxidant peaks characterized in the spiking test. More than twenty antioxidants were identified in ulam raja, and their chemical structures were proposed. The major antioxidants in ulam raja were attributed to a number of proanthocyanidins that existed as dimers through hexamers, quercetin glycosides, chlorogenic, neo-chlorogenic, crypto-chlorogenic acid and (+)-catching. High content of antioxidants antioxidants contained in ulam raja could be partly responsible for its ability to reduce oxidative stress.
Keywords: Ulam raja; Antioxidants; Phenolic compounds; HPLC/MS;

Ascorbylated 4-hydroxy-2-nonenal as a potential biomarker of oxidative stress response by John Sowell; Heather M. Conway; Richard S. Bruno; Maret G. Traber; Balz Frei; Jan F. Stevens (139-145).
Oxidative stress, resulting from the generation of reactive oxygen species, contributes to the development of a multitude of age-related diseases. Current methods of assessing oxidative stress levels range from the detection of lipid peroxidation products, such as F2-isoprostanes and malondialdehyde, to monitoring the redox status of glutathione. While useful, traditional biomarkers of oxidative stress are not without their drawbacks, including low in vitro concentrations and possible artifact formation. In the present study, we utilize liquid chromatography coupled with tandem mass spectrometry for investigation into the use of a novel compound, ascorbylated 4-hydroxy-2-nonenal, as a potential biomarker of oxidative stress.
Keywords: Oxidative stress; Biomarker; 4-Hydroxy-2-nonenal; Vitamin C; Ascorbic acid; Lipid peroxidation; Mass spectrometry;

Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography–tandem mass spectrometry and evaluation of the excretion after oral intake by Dimitrios Tsikas; Anja Mitschke; Maria-Theresia Suchy; Frank-Mathias Gutzki; Dirk O. Stichtenoth (146-156).
3-Nitrotyrosine (NO2Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)–tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[2H3]tyrosine, and GC–tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5 ± 7.2) 3-nitrotyrosine levels were determined to be 8.4 ± 10.4 nM (range, 1.6–33.2 nM) or 0.46 ± 0.49 nmol/mmol creatinine (range, 0.05–1.30 nmol/mmol creatinine). The present GC–tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 μg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.
Keywords: Reactive-nitrogen species; 3-Nitrotyrosine; Human; Urine; Biomarker;

Isoprostanes are isomers of prostaglandins that are generated from free radical-initiated autoxidation of arachidonic acid. Quantification of F2-isoprostanes is regarded as the “gold standard” to assess oxidative stress in various human diseases. There are 32 possible racemic isoprostane isomers that exist as four sets of regioisomers. Each regioisomer is composed of eight diastereomers. We report liquid chromatographic/mass spectrometric methods to separate and identify F2-isoprostane stereoisomers. These methods have been applied to the analysis of F2-isoprostanes derived from tissues of rats exposed to an oxidative stress and are useful to assess the relative formation of various regioisomers and stereoisomers generated in vitro and in vivo. The delineation of the more abundant isomers formed will allow for studies to examine the biological relevance of selected compounds in vivo.
Keywords: Isoprostanes; Lipid peroxidation; Liquid chromatography; Mass spectrometry; Free radical;