High-performance Liquid Chromatography System Research Articles

Integrated pharmacokinetics/pharmacodynamics parameters-based dosing guidelines of enrofloxacin in grass carp Ctenopharyngodon idella to minimize selection of drug resistance

BackgroundAntibiotic resistance has become a serious global problem and is steadily increasing worldwide in almost every bacterial species treated with antibiotics. In aquaculture, the therapeutic options for the treatment of A. hydrophila infection were only limited to several antibiotics, which contributed for the fast-speed emergence of drug tolerance. Accordingly, the aim of this study was to establish a medication regimen to prevent drug resistant bacteria. To determine a rational therapeutic guideline, integrated pharmacodynamics and pharmacokinetics parameters were based to predict dose and dosage interval of enrofloxacin in grass carp Ctenopharyngodon idella infected by a field-isolated A. hydrophila strain.ResultsThe pathogenic A. hydrophila strain (AH10) in grass carp was identified and found to be sensitive to enrofloxacin. The mutant selection window (MSW) of enrofloxacin on isolate AH10 was determined to be 0.5 - 3 μg/mL based on the mutant prevention concentration (MPC) and minimum inhibitory concentration (MIC) value. By using high-performance liquid chromatography (HPLC) system, the Pharmacokinetic (PK) parameters of enrofloxacin and its metabolite ciprofloxacin in grass carp were monitored after a single oral gavage of 10, 20, 30 μg enrofloxacin per g body weight. Dosing of 30 μg/g resulted in serum maximum concentration (Cmax) of 7.151 μg/mL, and concentration in serum was above MPC till 24 h post the single dose. Once-daily dosing of 30 μg/g was determined to be the rational choice for controlling AH10 infection and preventing mutant selection in grass carp. Data of mean residue time (MRT) and body clearance (CLz) indicated that both enrofloxacin and its metabolite ciprofloxacin present similar eliminating rate and pattern in serum, muscle and liver. A withdraw time of more than 32 d was suggested based on the drug eliminating rate and pharmacokinetic model described by a polyexponential equation.ConclusionsBased on integrated PK/PD parameters (AUC/MIC, Cmax/MIC, and T>MPC), the results of this study established a principle, for the first time, on drawing accurate dosing guideline for pharmacotherapy against A. hydrophila strain (AH10) for prevention of drug-resistant mutants. Our approach in combining PK data with PD parameters (including MPC and MSW) was the new effort in aquaculture to face the challenge of drug resistance by drawing a specific dosage guideline of antibiotics.

Application of high-performance liquid chromatography with charged aerosol detection for universal quantitation of undeclared phosphodiesterase-5 inhibitors in herbal dietary supplements

Incidents of detecting novel analogues of phosphodiesterase 5 (PDE-5) inhibitors in illicit dietary supplements for erectile dysfunction are constantly reported. However, little is known about their content in a single dose, mainly due to the poor availability or inaccessibility of pure reference standards. This study presents a new strategy of quantitative analysis of unknown and recently identified compounds. Charged aerosol detector (CAD), described as “universal detector”, combined with high-performance liquid chromatography (HPLC) system proved to be a useful tool for fast and simple quantitation of PDE-5 inhibitors’ analogues in a complex herbal matrix without individual reference standards available. Universal calibration was employed for calculations. Two easily obtainable reference materials – sildenafil and tadalafil – were selected as universal standards and the content of analogues was estimated with respect to their response. The error of proposed indirect determination was found to be ±3%, which is less than enough to obtain a reliable result of the content. The elaborated method was applied for quantitative analyses of PDE-5 inhibitors and 10 analogues detected in 22 illicit dietary supplements and two bulk powdered herbal materials. All target analogues were identified using time-of-flight mass spectrometry with electrospray ionization. Obtained results indicate that the quantity of PDE-5 inhibitors in all tested samples is considered to be pharmacologically relevant.

RP-HPLC Method Using One Marker for Quantification of Four Podophyllum Lignans in Medicinal Plants

A high-performance liquid chromatographic method using a single standard has been established for the quantitative analysis of four podophyllum lignans in Dysosma versipellis (Hance) M. Cheng and Podophyllum emodi Wall. Var. chinesis Sprague. The method involved the quantitative analysis of multiple components by a single marker. The chromatographic method was validated for linearity and range, limit of detection and qualification, precision, stability, reproducibility and robustness. Relative correcting factors were calculated and examined by five concentrations of four podophyllum lignans, two high-performance liquid chromatographic systems and three chromatographic columns. The method was applied to analyze 10 batches of samples. The quantitative results were compared with the results by an external standard method through intra-class coefficient, which indicated that the established method was reliable for the determination of the four podophyllum lignans in the two medicinal plants.

Selective enrichment of steroid and triterpenoid hydrocarbons from crude oil using gel permeation chromatography for stable carbon isotope analysis

Gel permeation chromatography (GPC) using a high performance liquid chromatography (HPLC) system was studied for the separation and enrichment of steroid and hopanoid hydrocarbons from crude oil for stable carbon isotope analysis. A crude oil sample was pretreated using silica gel chromatography and 5A molecular sieve to remove polycyclic aromatic hydrocarbons and n-alkanes. The GPC behavior of both the pretreated saturated hydrocarbon fraction of the oil and standard steroid [5α(H), 14α(H), 17α(H) C27–C29 steranes], hopanoid [17α(H) C27 trisnorhopane, 17α(H), 21β(H) C29–C32 hopanes] and triterpenoid [18α(H)-oleanane, gammacerane] mixtures were examined. The results indicate that 17α(H), 21β(H) hopanes as well as steranes could be enriched efficiently using GPC and that they could be obtained without removing n-alkanes from the oil saturated hydrocarbon fraction. The GPC behavior of steroid and triterpenoid hydrocarbons was controlled by molecular size and shape.

HPLC column-switching technique for sample preparation and fluorescence determination of propranolol in urine using fused-core columns in both dimensions

A new and fast high-performance liquid chromatography (HPLC) column-switching method using fused-core columns in both dimensions for sample preconcentration and determination of propranolol in human urine has been developed. On-line sample pretreatment and propranolol preconcentration were performed on an Ascentis Express RP-C-18 guard column (5 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water (5:95, v/v) at a flow rate of 2.0 mL min(-1) and at a temperature of 50 °C. Valve switch from pretreatment column to analytical column was set at 4.0 min in a back-flush mode. Separation of propranolol from other endogenous urine compounds was achieved on the fused-core column Ascentis Express RP-Amide (100 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water solution of 0.5% triethylamine, pH adjusted to 4.5 by means of glacial acetic acid (25:75, v/v), at a flow rate of 1.0 mL min(-1) and at a temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 229/338 nm. A volume of 1,500 μL of filtered urine sample solution was injected directly into the column-switching HPLC system. The total analysis time including on-line sample pretreatment was less than 8 min. The experimentally determined limit of detection of the method was found to be 0.015 ng mL(-1).

Two-dimensional high-performance liquid chromatographic determination of day–night variation of d-alanine in mammals and factors controlling the circadian changes

D-Alanine (D-Ala) is one of the naturally occurring D-amino acids in mammals, and its amount is known to have characteristic circadian changes. It is a candidate for a novel physiologically active substance and/or a biomarker, and the regulation mechanisms of the intrinsic amounts of D-Ala are expected to be clarified. In the present study, the effects of the possible factors controlling the D-Ala amounts, e.g., diet, D-amino acid oxidase (DAO) and intestinal bacteria, on the day-night changes in the intrinsic D-Ala amounts have been investigated using a highly sensitive and selective two-dimensional high-performance liquid chromatographic system combining a reversed-phase column and an enantioselective column. The circadian rhythm was not changed under fasting conditions. In the mice lacking D-amino acid oxidase activity (ddY/DAO(-) mice), clear day-night changes were still observed, suggesting that the factors controlling the D-Ala rhythm were not their food and DAO activity. On the other hand, in the germ-free mice, quite low amounts of D-Ala were detected compared with those in the control mice, indicating that the main origin of D-Ala in the mice is intestinal bacteria. Because the D-Ala amounts in the digesta containing intestinal bacteria did not show the day-night changes, the controlling factor of the circadian changes of the D-Ala amount was suggested to be the intestinal absorption.

Lysing activity of an indigenous algicidal bacterium Aeromonas sp. against Microcystis spp. isolated from Lake Taihu

This study aimed to isolate and characterize an indigenous algicidal bacterium named LTH-1 and its algae-lysing compounds active against three Microcystis aeruginosa strains (toxic TH1, nontoxic TH2 and standard FACHB 905). The LTH-1 isolated from Lake Taihu, near Wuxi City in China, was identified as Aeromonas sp. based on its morphological characteristic features and phylogenetic analysis by sequencing of 16S rDNA. Extracellular compounds produced by LTH-1 showed strong algae-lysing activity, and they were water-soluble and heat-tolerant, with a molecular mass lower than 2 kDa. Two algae-lysing compounds were isolated and purified from extracellular filtrate using silica gel column chromatography. One of these was identified as phenylalanine (C9H11NO2, m/z 166.0862) and the other (C8H16N2O3, m/z 189.1232) was unidentified by hybrid ion trap/time-of-flight mass spectrometry coupled with a high-performance liquid chromatography (LC/MS–IT–TOF) system. The half maximal effective concentration (EC50) of phenylalanine produced by LTH-1 against FACHB 905 was 68.2±8.2 μg mL−1 in 48 h. These results suggest that the algicidal Aeromonas sp. LTH-1 could play a role in controlling Microcystis blooms, and its extracellular compounds are also potentially useful for regulating blooms of the harmful M. aeruginosa.

Fast and quantitative analysis of branched-chain amino acids in biological samples using a pillar array column

In this study, a fast and quantitative determination method for branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, was developed using a pillar array column. A pillar array column with low-dispersion turns was fabricated on a 20 × 20-mm(2) microchip using multistep ultraviolet photolithography and deep reactive ion etching. The BCAAs were fluorescently labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), followed by reversed-phase separation on the pillar array column. The NBD derivatives of the three BCAAs and an internal standard (6-aminocaproic acid) were separated in 100 s. The calibration curves for the NBD-BCAAs had good linearity in the range of 0.4-20 μM, using an internal standard. The intra- and interday precisions were found to be in the ranges of 1.42-3.80 and 2.74-6.97%, respectively. The accuracies for the NBD-BCAA were from 90.2 to 99.1%. The method was used for the analysis of sports drink and human plasma samples. The concentrations of BCAAs determined by the developed method showed good agreements with those determined using a conventional high-performance liquid chromatography system. As BCAAs are important biomarkers of some diseases, these results showed that the developed method could be a potential diagnostic tool in clinical research.

Antidepressant-like effect of ethanol extract from Zuojin Pill, containing two herbal drugs of Rhizoma Coptidis and Fructus Evodiae, is explained by modulating the monoaminergic neurotransmitter system in mice

Ethnopharmacological relevanceZuojin Pill (ZJP), a traditional Chinese medicinal decoction, contains two herbal drugs: Coptis chinensis Franch. and Evodia rutaecarpa (Juss.) Benth. in the ratio of 6:1 (w/w). Previous pharmacological studies have shown that two herbs in ZJP have the antagonistic effects on catecholamine secretion in bovine adrenal medullary cells. Furthermore, the alkaloids from the two herbs in ZJP may provide a protective effect for depression in individuals with a low expressing 5-HTT allele by increasing receptor concentration in serotonergic neurons. However, antidepressant effect has not been reported before and has not been fully clarified. Aim of the studyThe present study aimed to investigate the antidepressant potential of ethanol extract from ZJP and its monoaminergic mechanism in mice. Materials and methodsSeven alkaloids were determined from the ethanol extract of ZJP using High Performance Liquid Chromatography (HPLC) with the gradient mobile phase. The ethanol extract from ZJP was used to evaluate the antidepressant potential in mice. Mouse models of depression including the tail suspension test (TST) and the forced swim test (FST) were used to evaluate the effects of the ethanol extract from ZJP. A possible mechanism was explored in the tests of antagonism of reserpine-induced ptosis and hypothermia, and 5-HTP induced head twitch response in mice. The contents of monoamine neurotransmitters including norepinephrine (NE), serotonin (5-hydroxytryptamine or 5-HT) in hippocampus of mice and NE, 5-HT, dopamine (DA) in striatum of mice were determined by HPLC system with Electrochemical Detector (ECD). ResultsThe results showed that intragastric administration of the ethanol extract from ZJP (5, 10, 20mg/kg) or fluoxetine (7.5mg/kg) significantly reduced the duration of immobility in TST and FST. However, the effect was not dose-dependent. Ethanol extract from ZJP (5, 10, 20mg/kg) also increased the accumulative number of the 5-HTP-induced head twitch response in mice. The mice were treated with the ethanol extract from ZJP (5, 10, 20mg/kg) or fluoxetine (7.5mg/kg), which could antagonize reserpine-induced ptosis and hypothermia, moreover, both of them could elevate the contents of NE, 5-HT in hippocampus as well as NE, 5-HT, DA in striatum significantly. ConclusionThese results indicate that the ethanol extract from ZJP produced antidepressant-like effect and the possible mechanism, at least in part, is via the central monoaminergic neurotransmitter system and 5-HT plays a major role.

Automation Highlights from the Literature

Automation Highlights from the Literature

Physiologically relevant changes in serotonin resolved by fast microdialysis.

Online microdialysis is a sampling and detection method that enables continuous interrogation of extracellular molecules in freely moving subjects under behaviorally relevant conditions. A majority of recent publications using brain microdialysis in rodents report sample collection times of 20-30 min. These long sampling times are due, in part, to limitations in the detection sensitivity of high performance liquid chromatography (HPLC). By optimizing separation and detection conditions, we decreased the retention time of serotonin to 2.5 min and the detection threshold to 0.8 fmol. Sampling times were consequently reduced from 20 to 3 min per sample for online detection of serotonin (and dopamine) in brain dialysates using a commercial HPLC system. We developed a strategy to collect and to analyze dialysate samples continuously from two animals in tandem using the same instrument. Improvements in temporal resolution enabled elucidation of rapid changes in extracellular serotonin levels associated with mild stress and circadian rhythms. These dynamics would be difficult or impossible to differentiate using conventional microdialysis sampling rates.

Evaluation of the suitability of chromatographic systems to predict human skin permeation of neutral compounds

Several chromatographic systems (three systems of high-performance liquid chromatography and two micellar electrokinetic chromatography systems) besides the reference octanol–water partition system are evaluated by a systematic procedure previously proposed in order to know their ability to model human skin permeation. The precision achieved when skin-water permeability coefficients are correlated against chromatographic retention factors is predicted within the framework of the solvation parameter model. It consists in estimating the contribution of error due to the biological and chromatographic data, as well as the error coming from the dissimilarity between the human skin permeation and the chromatographic systems. Both predictions and experimental tests show that all correlations are greatly affected by the considerable uncertainty of the skin permeability data and the error associated to the dissimilarity between the systems. Correlations with much better predictive abilities are achieved when the volume of the solute is used as additional variable, which illustrates the main roles of both lipophilicity and size of the solute to penetrate through the skin. In this way, the considered systems are able to give precise estimations of human skin permeability coefficients. In particular, the HPLC systems with common C18 columns provide the best performances in emulating the permeation of neutral compounds from aqueous solution through the human skin. As a result, a methodology based on easy, fast, and economical HPLC measurements in a common C18 column has been developed. After a validation based on training and test sets, the method has been applied with good results to the estimation of skin permeation of several hormones and pesticides.

Detection of Iron Ligands in Seawater and Marine Cyanobacteria Cultures by High-Performance Liquid Chromatography–Inductively Coupled Plasma-Mass Spectrometry

Organic ligands dominate the speciation of iron in the ocean. Little is known, however, about the chemical composition and distribution of these compounds. Here, we describe a method to detect low concentrations of organic Fe ligands using reverse-phase high-performance liquid chromatography (HPLC) tandem multicollector inductively coupled plasma mass spectrometry. This technique can be used to screen seawater and marine cultures for target compounds that can be isolated and structurally characterized. Sensitive detection (<1 picomole Fe) is achieved using an iron-free HPLC system to reduce background Fe levels, minimizing (40)Ar(16)O(+) interferences on (56)Fe with a hexapole collision cell, and introducing oxygen into the sample carrier gas to prevent the formation of reduced carbon deposits that decrease sensitivity. This method was tested with a chromatographic separation of five trace metal complexes that represent the polarity range likely found in seawater. Good separation was achieved with a 20 min water/methanol gradient, although sensitivity decreased by a factor of 2 at high organic solvent concentrations. Finally, Fe ligand complexes were detected from the organic extract of surface South Pacific seawater and from culture media of the siderophore producing cyanobacteria Synechococcus sp. PCC 7002.

Homocysteine and MTHFR C677T polymorphism in children and adolescents with psychotic and mood disorders

Background : High level of homocysteine (Hcy) is risk factor of schizophrenia and mood disorders. Aim: The aim was to detect a serum level of Hcy, examine the associations between the level of Hcy, methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism and clinical properties for patients with schizophrenia, mood disorders and in a control group. Material and methods: There were 88 patients with schizophrenia, 28 with affective disorders and 94 from the control group. The Hamilton Anxiety Scale (HAM-A) was performed to study anxiety, the Hamilton Depression Scale (HAM-D) to study depression and the Brief Psychiatric Rating Scale (BPRS) to study severity of schizophrenia. The level of Hcy was stated by isocratic high-performance liquid chromatography (HPLC) system with fluorometric detection. DNA isolation from venous blood was performed by the phenol–chloroform method. Results: The levels of B12 vitamin and folic acid were within normal limits for all the patients. The average level of Hcy was 11.94 ± 5.6 μmol/l for patients with schizophrenia and 11.65 ± 3.3 μmol/l for patients with affective disorders, vs. 6.80 ± 2.93 μmol/l in a control group. The highest level of Hcy has been observed in patients with an episodic–recurrent course of schizophrenia, paranoid schizophrenia—continuous, particularly in patients with CT genotype (r = − 0.58; P < 0.01). In the given diagnostic groups, the highest level of anxiety was observed. Conclusions: The level of Hcy is higher in the blood of a heterozygotic person who becomes ill with schizophrenia, and the process of the illness is more serious and with more typical affective disorders.

Kynurenic Acid in Synovial Fluid and Serum of Patients with Rheumatoid Arthritis, Spondyloarthropathy, and Osteoarthritis

Previously we demonstrated that kynurenic acid (KYNA), an endogenous metabolite of kynurenine, is present in the synovial fluid of patients with rheumatoid arthritis (RA). KYNA inhibits proliferation of synoviocytes in vitro. The goal of our study was to compare KYNA concentrations in synovial fluid and blood of patients with RA, inflammatory spondyloarthropathies (SpA), and osteoarthritis (OA). Serum and synovial fluid samples were obtained from 189 patients with RA, 56 patients with SpA, and 32 patients with OA. KYNA was separated using a high-performance liquid chromatography system and measured fluorometrically. KYNA concentration in synovial fluid obtained from patients with RA and SpA was significantly lower than that in patients with OA (p < 0.05). The concentration of KYNA in serum of patients with RA, SpA, and OA did not differ among all groups studied. The positive correlation between KYNA content in synovial fluid and serum was found in patients with RA (p < 0.05). Univariate linear regression analysis demonstrated that fibrinogen was significantly associated with KYNA in synovial fluid (p < 0.05), and red blood cell counts, morning stiffness, and pain scores were significantly associated with KYNA level in serum (all p < 0.05). Multivariate regression analysis revealed correlation between the following independent variables: hemoglobin level, hematocrit, red blood cell count in conjunction with age and KYNA content in synovial fluid. A lack of correlation was observed between KYNA content in synovial fluid of patients with RA and other clinical and laboratory measures of disease activity. Our data show a local deficit of KYNA in inflammatory states.

Ionic liquid based dispersive liquid–liquid microextraction coupled with micro-solid phase extraction of antidepressant drugs from environmental water samples

Ionic liquid-dispersive liquid–liquid microextraction combined with micro-solid phase extraction (IL-DLLME-μ-SPE), and high-performance liquid chromatography (HPLC) was developed for the determination of tricyclic antidepressants (TCAs) in water samples. Two hundred microliters of an organic solvent (as disperser solvent) and 20μl of 1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate were injected into a 5.0ml sample for sonication-assisted DLLME. After this, a μ-SPE device, containing a novel material zeolite imidazolate framework 4 (ZIF-4), was added into the sample solution and 1min of vortex-assisted extraction was performed. After 5min of sonication-assisted desorption, 10μl of desorption solvent was injected into a HPLC system for analysis. A characteristic property of DLLME-VA-μ-SPE is that any organic solvent and solid sorbent immiscible with water can be used. Special apparatus, or conical-bottom test tubes, and tedious procedures conventionally associated with DLLME such as centrifugation, or refrigeration of solvent are not necessary in the present approach. A novel material, ZIF-4 was employed as μ-SPE sorbent. Under the optimized conditions, the calibration curves were linear in the range of 1–1000μg/L. The relative standard deviations and the limits of detection were in the range of 1.5% and 7.8% and 0.3 and 1μg/L, respectively. The relative recoveries of canal water samples, spiked with drugs, were in the range of 94.3% and 114.7%. The results showed that IL-DLLME-μ-SPE was suitable for the determination of TCAs in water samples.

Fast HPLC Method for Determination of Fenoxycarb and Permethrin in Antiparasitic Veterinary Shampoo Using Fused-Core Column

A new and fast high-performance liquid chromatography (HPLC) method using technology of fused-core columns for separation of fenoxycarb and cis-, trans-permethrin has been developed and used for their determination in antiparasitic veterinary shampoo. Separation of insecticides and internal standard sudan II was achieved on the fused-core column Ascentis Express RP-Amide (100 × 3.0 mm), particle size 2.7 μm, with mobile phase acetonitrile/water (55:45, v/v) at a flow rate of 1.0 mL min−1 and at temperature 60 °C. The detection wavelength of detector was set at 225 nm for both compounds and internal standard sudan II. Under the optimum chromatographic conditions standard calibration curves were measured with good linearity [r 2 = 0.99991 for fenoxycarb, r 2 = 0.99987 for trans-permethrin, and r 2 = 0.99984 for cis-permethrin (n = 8)]. Commercial samples of antiparasitic veterinary shampoo were extracted with ethanol in ultrasound bath for 5 min. A 2-μL sample volume of the filtered solution was directly injected into the HPLC system. Accuracy of the method defined as a mean recovery of insecticides from shampoo matrix was in the range 100.43–103.85 % for both insecticides.

Quercetin encapsulated nanocarriers: effects on breast cancer cell growth, apoptosis, and uptake in vitro and bioavailability in vivo

BackgroundQuercetin, a natural flavonoid, has a potential against many breast cancer cells, but its low solubility and bioavailability in the body make administering it in therapeutic dose unrealistic. We have successfully synthesized quercetin encapsulated nanocarriers (NanoQ). Our hypothesis is that NanoQ can enhance quercetin stability and solubility, increase quercetin bioavailability in vitro and in vivo, decrease the viability of breast cancer cells, and induce their apoptosis.MethodsThe stability, solubility and cellular uptake of quercetin in MCF7 and MDA‐MB‐231 breast cancer cells were measured using a high performance liquid chromatography system. The viability and apoptosis of breast cancer cells were measured using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and Annexin‐V/propidium iodide assay, respectively. The pharmacokinetics of NanoQ was measured in SD rats.ResultsNanoQ were about 30 nm in diameter. Nanoencapsulation significantly increased the stability and solubility of quercetin, and increased quercetin cellular content 3.9 times and 9.3 times in MCF7 and MDA‐MB‐231 cells, respectively. NanoQ also significantly lowered the viability of both breast cancer cells and induced their apoptosis as compared to free quercetin at the same concentrations.ConclusionNanoQ is a promising approach for the prevention and treatment of breast cancer.Grant Funding Source: TTU research incentive funding

Evaluation Of Antioxidant Defense In Patients Undergoing Minor Elective Surgery With Balanced Anesthesia Maintained With The Inhaled Isoflurane

It is suggested that invasive surgical procedure can induce oxidative stress. However, only a few data regarding elective surgeries and the adjuvant effect of anesthesia are available. Fifteen patients without comorbidities, aged 18–45 years old, and scheduled for septoplasty or tympanoplasty, received general anesthesia maintained with the volatile isoflurane anesthetic. Blood samples were collected before and at the end of anesthesia. Total antioxidant performance (TAP), that measures the oxidizability of both aqueous and lipid compartments of plasma, was evaluated by fluorometry and plasma antioxidants carotenoids, tocopherols and retinol were determined by a high performance liquid chromatography (HPLC) system. Most of the carotenoids significantly decreased as well as alpha‐tocopherol and retinol (p&lt;0.001), whereas TAP was increased (p&lt;0.002) at the end of anesthesia. In vitro experiment revealed that this anesthetic showed an antioxidant potential in the equivalent concentration of blood during anesthesia in dose‐dependent manner. Thus, the current study indicates that despite of the decrease of some lipophilic endogenous antioxidants during balanced anesthesia with isoflurane in patients undergoing minor elective surgeries, the antioxidant activity of isoflurane itself may be resulted in increases of plasma TAP in these patients.Supported by FAPESP, Brazil #2010/05611–0 and USDA/USA #58–1950‐7–07

Single-Stage Dual-Column HPLC Technique for Separation and Determination of Lanthanides in Uranium Matrix: Application to Burnup Measurement on Nuclear Reactor Fuel

A single-stage dual-column chromatographic technique has been developed in this study for separation and determination of lanthanides in a uranium matrix. A 5-cm-length reversed-phase column coated with tri-n-octylphosphine oxide (TOPO) was connected in series to a 10-cm-length reversed-phase monolithic column (dynamically modified into a cation exchange column) to accomplish individual isolation of lanthanides from the uranium matrix. The proposed technique eliminates the step of uranium matrix removal for the determination of lanthanides. Samples with a lanthanide-to-uranium ratio (1 part lanthanide to 105 parts uranium) were directly injected into the dual column for the quantitative determination of lanthanides without uranium matrix removal. In some studies, samples of lanthanides in the uranium matrix could be injected as much as 45 times consecutively into a high-performance liquid chromatography system for determination of lanthanides without any uranium elution. The retention behavior of Pu(IV), Pu(III), Am(III), and fission products was also investigated on the TOPO-coated support. The single-stage dual-column chromatographic technique was demonstrated for the determination of fission products such as La and Nd in the dissolver solution of pressurized heavy water reactor spent fuel for the measurement of atom percent fission burnup. The technique can also be employed to estimate lanthanide impurities in samples of UO2 (1 part lanthanide to 106 parts uranium) without removal of the uranium matrix.