High-performance Liquid Chromatography System Research Articles

Liquid chromatography coupled to tandem mass spectrometry for the residue determination of ethylenethiourea (ETU) and propylenethiourea (PTU) in water

Ethylenethiourea (ETU) and propylenethiourea (PTU) are the main degradation products of dithiocarbamates fungicides, which are widely used in agriculture from several years ago. Their determination in water at low concentrations (e.g. sub-ppb levels) is highly problematic due to their polar character and low molecular size. In the present study, two analytical methodologies have been developed and compared for the selective and sensitive determination of ETU and PTU in various types of waters. Both approaches are based on liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) with electrospray ionization, using triple quadrupole analyzer. Whereas the first methodology used an on-line solid-phase extraction (SPE) step in order to reach the adequate sensitivity, the second one avoided sample treatment and was based on direct injection into an ultra high performance liquid chromatography (UHPLC–MS/MS) system, making use of a new-generation instrument in order to reach sub-ppb analyte levels in water. Strong matrix effects (typically leading to signal enhancement) were observed for most of the evaluated waters, especially when applying the on-line SPE method, surely due to the higher amount of sample injected into the system. The use of the own analyte (ETU-d4) as isotope-labelled internal standard (ILIS) allowed to compensate these effects and to achieve an accurate ETU quantification at low concentrations. Moreover, three simultaneous transitions, operating in selected reaction monitoring mode, were acquired for both ETU and ETU-d4. This fact together with the evaluation of their relative intensity ratios assured the reliable identification of the analyte in the water samples. The two optimized methodologies were validated by analysis of six different samples (two drinking water, two groundwater and two surface water), spiked at two levels (0.1 and 1.0μg/L), and analyzed each in quintuplicate. Satisfactory accuracy and precision, with recoveries ranging from 73 to 104% and RSDs lower than 20%, were obtained for ETU. Limits of detection for ETU were found to be 0.058μg/L and 0.027μg/L with direct injection and with the on-line methodology, respectively. No satisfactory recoveries were obtained, in general, for PTU despite using its own deuterium-labelled molecule for matrix effects correction. Notable differences in the chemical behaviour between PTU and PTU-d6 were observed, which lead to significant variation in their chromatographic retention time and ionization efficiency. Thus, no satisfactory correction of matrix effects could be reached illustrating that the use of deuterated ILIS can be problematic in some particular cases. Despite the poor correction, a semi-quantitative analysis would be feasible for PTU at sub-ppb levels in water. To the best of our knowledge, this is the first article reporting the use of LC–MS/MS for the trace level determination of these problematic analytes in water.

Characterization of resistant tomato mutants to bacterial canker disease

A small scale ethylmethanesulfonate (EMS) mutation was used to obtain resistant mutant plants to bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis isolate 2 (Cmm2). Susceptible EBR3 tomato line (200) seeds were mutagenised with the chemical EMS. Of the constructed M2 population, 450 seedlings were inoculated with Cmm2. Among the tested M2 plants, 15 M2 plants, not affected by the bacterial pathogen, produced M3 family seeds. 10 seeds from each M3 family were re-inoculated with the Cmm2, M3-9 and M3-15 family plant did not segregate for resistance confirming their resistant phenotypes. The M3-9 and M3-15 family mutant plants were analyzed for secondary metabolites especially phenolics; chlorogenic acid, caffeic acid, p-coumaric acid, rutin hydrate, ferulic acid, quercetin, naringenin and caempherol level in high performance liquid chromatography (HPLC) system. The chlorogenic acid and rutin hydrate levels were increased in M3-9 and M3-15 family mutant plants than susceptible original EBR3 plants. The increased levels of phenolic substances could be associated with resistance phenotypes in the M3-9 and M3-15 mutants’ plants. Keywords: Tomato, ethylmethanesulfonate mutagenesis, resistant mutants, bacterial canker

Survey of Residual Nitrite and Nitrate in Conventional and Organic/Natural/Uncured/Indirectly Cured Meats Available at Retail in the United States

A survey of residual nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in cured meats available at retail was conducted to verify concentrations in conventional (C) products and establish a baseline for organic/natural/uncured/indirectly cured (ONC) products. In this study, 470 cured meat products representing six major categories were taken from retail outlets in five major metropolitan cities across the United States. Random samples representing both C and ONC type products were analyzed for NO(2)(-) and NO(3)(-) content (ppm) using an ENO-20 high-performance liquid chromatography system equipped with a reverse phase column. Generally, there were no differences in NO(2)(-) concentrations between C and ONC meat categories, but a few ONC products surveyed in certain cities were lower in NO(3)(-) content. Pairwise comparisons between cities indicated that NO(2)(-) and NO(3)(-) contents of all C type products were not appreciably different, and the same was true for most ONC products. Numerical NO(2)(-) values were less variable than NO(3)(-) concentrations within each meat product category. NO(2)(-) concentrations were similar to those previously reported by Cassens ( Cassens , R. G. Residual nitrite in cured meat . Food Technol. 1997a , 51 , 53 - 55 ) in 1997. Residual NO(2)(-) and NO(3)(-) values in this study were numerically lower than those reported by NAS ( National Academy of Sciences . The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds ; National Academy Press : Washington, DC , 1981 ) in 1981. Data from this survey provide a benchmark of NO(2)(-) and NO(3)(-) concentrations for ONC products available at retail.

Stability of docetaxel infusions

BackgroundAccording to the manufacture, docetaxel infusions should be used within four h at room temperature (<25°C).PurposeTo determine the stability of intravenous mixtures (IVM) at a concentration of 0.7 mg/mL in...

Nanoencapsulation increases (−)‐epigallocatechin gallate stability and its cellular bioavailability in macrophages

Atherosclerotic cardiovascular disease is the leading cause of death in U.S. Macrophages are major cells responsible for atherosclerotic lesion development. (−)‐Epigallocatechin gallate (EGCG) abundant in green tea is valuable for the prevention and treatment of atherosclerosis. But low levels of stability and bioavailability limit its application in human. We encapsulated EGCG into biocompatible and biodegradable nanocarriers to increase its stability and cellular bioavailability. The stability of nanoencapsulated EGCG and native EGCG was measured at pH 7.2 at 4 °C and 25 °C. Nanoencapsulated EGCG was separated from nonencapsulated EGCG using a Sephadex™ G‐25 column. Total and nanoencapsulated EGCG was measured using a high‐performance liquid chromatography (HPLC) system. EGCG uptake by THP‐1 derived macrophages was measured using the same HPLC system. Nanoencapsulation significantly increased EGCG stability and its uptake by macrophages compared to native EGCG. This innovation portends a potential breakthrough in the prevention and treatment of atherosclerosis by using natural compounds with minimized immunogenicity and side‐effects.Grant Funding Source : NIH 1R15AT007013‐01

Vitamins at physiological levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA--alone or in synergism with metals

Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins' chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins' ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkali-labile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, β-carotene, folic acid, α-tocopherol, δ-tocopherol or γ-tocopherol did not induce oxidative damage to dG, while lycopene induced a weak dose-response increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins.

Antioxidants Release from Solvent-Cast PLA Film: Investigation of PLA Antioxidant-Active Packaging

The release study of natural antioxidants, i.e., ascorbyl palmitate and α-tocopherol, and synthetic phenolic antioxidants including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, and tert-butylhydroquinone (TBHQ) from polylactic acid (PLA) film into food simulants were accomplished. PLA antioxidant films were placed in contact with 95, 50, and 10 % ethanol at 40 and 20 °C. Released antioxidants were regularly measured by high-performance liquid chromatography system for 60 days. Ascorbyl palmitate was completely degraded during film preparation and is not a suitable antioxidant for PLA antioxidant-active packaging. The diffusion coefficient (D) and partition coefficient (K) of antioxidants were calculated based on obtained data. Diffusion of the antioxidants from PLA to the simulants showed a Fick’s behavior with the diffusion coefficient (D) value between 10−9 and 10−11 cm2 s−1 with 0–100 % of release. Faster and higher release of antioxidants occurred at 40 °C according to Arrhenius law. At 40 °C, TBHQ in 95 % ethanol decomposed to 2-tert-butyl-1,4-benzoquinone and other quinone derivatives, and α-tocopherol in 50 % ethanol decomposed to some unknown neoformed compounds. Antioxidants molecular weight, Log P, simulant polarity, and temperature were the most influencing factors on antioxidants release rate from PLA films in contact with food simulants. The D and K coefficients of studied antioxidants from PLA in three food simulants and two temperatures can be used to create PLA antioxidant-active packaging to continually control the oxidation reactions in diverse foodstuffs to ensure higher food qualities. The PLA antioxidant-active packaging approach also permits to reduce the amounts of directly added antioxidants in foods to provide safer foods.

Two-Dimensional Protein Separation by the HPLC System with a Monolithic Column

A two-dimensional high-performance liquid chromatography (2D-HPLC) system for protein separation was developed using an ion-exchange column in the first dimension and a reversed-phase monolithic column in the second dimension. The system demonstrated efficient separation of proteins in comparison with conventional systems. For proteomic analysis, proteins extracted from the cell surface of the yeast were separated by 2D-HPLC and evaluated.

Modeling Nonspecific Toxicity of Organic Compounds to the Fathead Minnow Fish by Means of Chromatographic Systems

The performance of chromatographic systems to mimic aquatic toxicity to the fathead minnow fish is evaluated taking into account the factors that contribute to the variance of biological-chromatographic correlations. These factors are the precision to measure the fathead minnow toxicity, the precision of the surrogate chromatographic system, and the error from the dissimilarity between the fathead minnow and chromatographic systems. The precisions are estimated through the characterization of the systems by the solvation parameter model. Several chromatographic systems as well as the common reference octanol-water partition system have been selected to test their ability to model the nonspecific toxicity to the fathead minnow by means of the proposed approach. Predictions and experimental tests show that the micellar electrokinetic chromatography system of sodium taurocholate and chromatographic measurements using an immobilized artificial membrane column provide the most precise estimations of this biopartitioning property. The octanol-water partition system, the conventional C18 high-performance liquid chromatography systems, and the micellar electrokinetic chromatography system of sodium dodecylsulfate show worse performances.

Importance of retention data from affinity and reverse-phase high-performance liquid chromatography on antitumor activity prediction of imidazoacridinones using QSAR strategy

Quantitative structure-activity relationships (QSAR) studies for prediction of cytotoxic and antitumor activity of imidazoacridinones (IA) based on experimentally obtained high-performance liquid chromatography (HPLC) retention data and calculated parameters using computational (molecular modeling) medicinal chemistry methods were proposed. The RP-HPLC and affinity-HPLC chromatographic techniques with four diversified HPLC systems applying columns with octadecylsilanes (C18), phosphatidylcholine (IAM), as well as α(1)-glycoprotein (AGP) and albumin (HSA) were used for the determination of the retention constants logk and logk(w) which characterize lipophilicity and protein affinity of IA. Moreover, molecular modeling studies were performed using HyperChem and Dragon software's, and structural descriptors were calculated and subsequently used. The QSAR equations using multiple linear regression (MLR) analysis method were derived which indicated that in vivo antileukemia activity of IA depends on cytotoxic activity against leukemia cells, whereas this cytotoxic activity depends on logk and logk(w) parameters obtained on all HPLC systems. Moreover, the QSRR equations were derived and indicated that logk and logk(w) parameters depend on calculated non-empirical structural parameters. The predictive power of obtained QSAR and QSRR equations allowed the prediction of cytotoxic and antitumor activity of IA and also their HPLC retention parameters. Finally, the equations can be used for prediction of antileukemia activity of IA without the necessity of carrying out experimental measurements.

An HPLC-DAD and LC-MS Study of Condensation Oscillations with S(+)-Ketoprofen Dissolved in Acetonitrile

In our earlier studies, a spontaneous chiral conversion of the selected low-molecular-weight carboxylic acids (i.e., amino acids, hydroxy acids, and profen drugs) dissolved in aqueous ethanol medium, running in vitro was described. Then it became clear that this spontaneous chiral conversion is accompanied by the spontaneous condensation of the discussed compounds. With several acids, it was established that this condensation is also oscillatory in nature. The theoretical models were developed aiming to give a rough explanation of the observed non-linear processes. In this paper, the results of these studies on the dynamics of condensation with S(+)-ketoprofen, a very popular profen drug, when stored for certain amount of time dissolved in a non-aqueous medium (i.e., acetonitrile) is presented. These investigations were carried out with the aid of two independent high-performance liquid chromatographic systems with the diode array detection and of a third high-performance liquid chromatographic system equipped with mass spectrometric detection. In one cycle of chromatographic measurements, it was possible to monitor condensation of S(+)-ketoprofen in 25-min intervals for 30 h, thus obtaining kinetic information on the progress of this process. Mass spectrometric detection confirmed the presence of new species in the stored solution with molecular weights much higher than that of S(+)-ketoprofen, which can be attributed to the condensation products. The obtained data show that condensation of S(+)-ketoprofen dissolved in acetonitrile progresses in a rapid manner, and that the observed oscillatory concentration changes with S(+)-ketoprofen and with the main condensation product characterize with an irregularity and shallow amplitudes. A theoretical model was referenced that jointly describes the oscillatory chiral conversion and the oscillatory condensation with the low-molecular-weight chiral carboxylic acids.

Exploring the Possibilities of Cryogenic Cooling in Liquid Chromatography for Biological Applications: A Proof of Principle

The possibilities to use cryogenic cooling to trap components in liquid chromatography was investigated. In a first step, van 't Hoff plots were measured with a reversed-phase column using the temperature control unit of a conventional high performance liquid chromatography (HPLC) system to gain insight in the retention behavior of proteins at low temperatures. It was estimated that retention factors in the range of k = 10(4) could be achieved at T = -20 °C for lysozyme, indicating that temperature is a usable parameter to trap components in LC. In a next step, trapping experiments were carried out on a nano-LC system, equipped with a UV-detector, using a commercial reversed-phase column. An in-house built setup, allowing cooling of a segment of the column down to temperatures below T = -20 °C, was used to trap components. Experiments were conducted under isocratic and gradient conditions with methanol as organic solvent. It is demonstrated that, by thermally trapping and elution of components, an enhanced S/N ratio and decreased peak widths can be obtained. At the same time, a significant increase in pressure drop occurs during the cooling process. Limitations and benefits of the technique are further discussed.

Electrochemical Detection of Sugar-related Compounds Using Boron-doped Diamond Electrodes

Electrochemical detection of sugar-related compounds was conducted using a boron-doped diamond (BDD) electrode as a detector for flow-injection analysis (FIA). Sugar-related compounds oxidize at high applied potentials, for which the BDD electrode is suitable for electrochemical measurements. Conditions for an FIA system with a BDD detector were optimized, and the following detection limits were achieved for sugar-related compounds: monosaccharides, 25-100 pmol; sugar alcohols, 10 pmol; and oligosaccharides, 10 pmol. The detection limit for monosaccharide D-glucose (Glu) was 105 pmol (S/N = 3). A linear range was acquired from the detection limit to 50 nmol, and the relative standard deviation was 0.65% (20 nmol, n = 6). A high-performance liquid chromatography (HPLC) column was added to the system between the sample injector and the detector and detection limits to the picomole level were achieved, which is the same for the HPLC system and the FIA system. The electrochemical oxidation reaction of Glu was examined using cyclic voltammetry with the BDD detector. The reaction proved to be irreversible, and proceeded according to the following two-step mechanism: (1) application of a high potential (2.00 V vs. Ag/AgCl) to the electrode causes water to electrolyze on the electrode surface with the simultaneous generation of a hydroxyl radical on the surface, and (2) the hydroxyl radical indirectly oxidizes Glu. Thus, Glu can be detected by an increase in the oxidation current caused by reactions with hydroxy radicals.

Detection of N-glycans on small amounts of glycoproteins in tissue samples and sodium dodecyl sulfate–polyacrylamide gels

N-linked glycans harbored on glycoproteins profoundly affect the character of proteins by altering their structure or capacity to bind to other molecules. Specific knowledge of the role of N-glycans in these changes is limited due to difficulties in identifying precise carbohydrate structures on a given glycoprotein, which arises from the large amounts of glycoprotein required for N-glycan structural determination. Here, we refined a simple method to purify and detect trace amounts of N-glycans. During the N-glycan purification step, most contaminants were removed by two kinds of columns: a graphite carbon column and a cellulose column. N-Glycans were identified with a three-dimensional high-performance liquid chromatography (HPLC) system. Using our method, a global analysis of N-glycans from human muscle biopsy samples and mouse brain sections was possible. By combining sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with our method, we refined analytical procedures for N-glycans from SDS–PAGE gels using hydrazinolysis to achieve a high N-glycan recovery rate. N-Glycans on as little as 1μg of the target protein transferrin or immunoglobulin G (IgG) were easily detected. These methods allowed us to efficiently determine glycoprotein N-glycans at picomole (pmol) levels.

Determination of LFER Descriptors of 30 Cations of Ionic Liquids—Progress in Understanding Their Molecular Interaction Potentials

In order to understand molecular interaction potentials of 30 cations of ionic liquids (ILs), the well-known linear free energy relationship concept (LFER) was applied. The LFER descriptors for the excess molar refractivity and the molar volume were calculated in silico and for hydrogen-bonding acidity and basicity, and the polarizability/dipolarity of IL cations were experimentally determined through high performance liquid chromatography (HPLC) measurements. For the study, three different columns (RP-select B, Cyan, and Diol) and buffered mobile phases, based on two organic solvents acetonitrile (ACN) and methanol (MeOH), were selectively combined to the HPLC separation systems RP-select B-ACN, RP-select B-MeOH, Cyan-MeOH, Diol-ACN, and Diol-MeOH. By measuring the retention factors of 45 neutral calibration compounds and calculating LFER descriptors of three cations in the HPLC systems, the system parameters, including an ionic z coefficient, were determined. Conversely, the LFER descriptors of 30 ionic liquid cations were determined, based on the parameters of five systems and their retention factors in the HPLC systems. The results showed that the type of head group, alkyl chain length and further substituents of the cation have a significant influence on the dipolarity/polarizability and the hydrogen-bonding acidity, and functionalized groups (hydroxyl, ether, and dimethylamino) lead to hydrogen-bonding basicity of the cation. The characterization of cationic LFER descriptors opens up the chance for a more quantitative understanding of molecular interaction potentials and physicochemical properties of ILs.

Preparative size-exclusion chromatography for separation and purification of water-stable Cd-based quantum dots

Size-exclusion chromatography (SEC) associated with High Performance Liquid Chromatography (HPLC) is a powerful tool to separate, purify and fractionalize materials based on size. In this paper, we present a SEC method that was developed for the separation of thiol-capped Cd(Se,S) quantum dots (QDs) synthesized in the aqueous phase. A HPLC system with a SEC column and a cascade of three detectors (UV/Vis, FLD and ELSD) was used to analyze the different size fractions of QDs. Nanocrystals–HPLC column interactions were suppressed using thioglycolic acid (TGA) as an ion pair agent. Five fractions, namely F1 to F5, of different sizes and tunable optical properties were isolated from the original QDs sample. The emission peaks for fractions F1 and F2 were red-shifted and the fractions F4 and F5 were blue-shifted compared with the original sample, which suggested the presence of nanocrystals having different sizes. Dynamic Light Scattering (DLS) confirmed that collected fractions exhibited different hydrodynamic diameters ranging from 98.2 nm (fraction F1) to 24.9 nm (fraction F5). Also, fractions F2 and F4 were functionalized with glutathione and analyzed by HPLC-SEC. Glutathione-capped Cd(Se,S) QDs showed an increase in their molecular weight, when compared to bare TGA-capped Cd(Se,S) QDs, without a remarkable change of their crystal size and optical properties. The developed SEC technique allows a fast and reproducible separation of water-stable Cd(Se,S) QDs. Collected fractions with tunable optical properties could have potential applications in the nanotechnology area such as bio-imaging and diagnostics, e.g. cell sorting.

HPLC method for Pharmacokinetics of cis and trans isomer of cefprozil diastereomers in human plasma

Using an high-performance liquid chromatography (HPLC) system and UV detection, a simple and precise analytical procedure was developed to quantify levels of cis and trans isomer of cefprozil diastereomers in human plasma. Cefprozil was extracted from plasma samples into 10% perchloric acid. The HPLC system included an acetonitrile-0.05M monopotassium phosphate water (adjust pH to 3.05 with glacial acetic acid) (12:88, v/v), flow rate of 1 mL/min, Ultimate TM XB-C18 column,(5m，4.6×150mm), and UV detection at 280 nm. The regression of the spiked calibrator curves were linear from 0.02–10 μg/mL and 0.02–1 μg/mL (r 2 =0.9951, 0.9950), respectively. The lower limits of quantification were 0.02 μg/mL, the inter- and intra-day precisions (RSD) were lower than 9.0%, and the extraction recoveries were all more than 90%. The cis and trans isomers of cefprozil were stable under a variety of storage and process conditions. To demonstrate utility, the pharmacokinetic parameters of cis and trans isomer of cefprozil were measured in twenty human after oral administration of 500mg cefprozil. The assay was sensitive, accurate and convenient, and can be used for the determination of cis and trans isomer of cefprozil in human plasma.

Acute L-Arginine supplementation does not increase nitric oxide production in healthy subjects

Dietary supplements containing L-arginine have been marketed with the purpose of increasing vasodilatation, and thus, blood and oxygen supply to the exercising muscle. The present study evaluated the acute effect of L-arginine supplementation on indicators of NO production, nitrite (NO2-) + nitrate (NO3-) (NOx), in healthy subjects. Plasma concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have also been addressed. Seventeen healthy males participated in a randomized, double-blind, placebo-controlled study. Blood samples were drawn from a left antecubital vein at baseline (T0). Afterwards, subjects were randomly submittedto 6 g of oral L-arginine supplementation (as L-arginine hydrochloride) or placebo (as corn starch); afterwards, the subjects remained at rest in supine position and blood samples were drawn again at 30 (T1), 60 (T2), 90 (T3) and 120 minutes (T4) after supplementation. To analyze NO production, NO3- was converted to NO2- by nitrate reductase, followed by the derivatization of NO2- with 2,3-diaminonaphthalene. NOx, ADMA and SDMA were analyzed using a high-performance liquid chromatography system and monitored with a fluorescence detector. Two-way ANOVA with repeated measures showed no significant changes in NOx concentrations on the L-arginine group as compared to placebo group at any of the fivetime points (T0: 17.6 ± 3.9 vs 14.6 ± 2.3 μmol/L; T1: 15.8 ± 2.4 vs 14.3 ± 1.7 μmol/L; T2: 16.8 ± 4.9 vs 13.7 ± 2.7 μmol/L; T3: 16.7 ± 3.9 vs 14.6 ± 2.1 μmol/L; T4: 15.1 ± 2.8 vs 13.5 ± 3.5 μmol/L). Furthermore, plasma levels of ADMA and SDMA were not statistically significant between the L-arginine and placebo groups at T0 (0.43 ± 0.19 vs 0.39 ± 0.15 μmol/L and 1.83 ± 1.13 vs 1.70 ± 0.62 μmol/L), respectively. In conclusion, acute L-arginine supplementation does not increase plasma concentration of NOx in healthy individuals with normal plasma concentrations of ADMA.

Development and Validation of a Reversed-Phase Liquid Chromatography Method for the Simultaneous Determination of Indole-3-Acetic Acid, Indole-3-Pyruvic Acid, and Abscisic Acid in Barley (Hordeum vulgare L.).

A simple, sensitive, precise, and specific reverse HPLC method was developed and validated for the determination of plant hormones in barley (Hordeum vulgare L.). The method includes extraction in aqueous organic solvent followed by solid-phase extraction, sample evaporation, and reversed-phase HPLC analysis in a general purpose UV-visible (abscisic acid (ABA)) and fluorescence detection (indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPA)), high-performance liquid chromatography system. The separation was carried out on Zorbax Eclipse XDB C8 column (150 × 4.6 mm I.D) with a mobile phase composed of methanol and 1% acetic acid (60 : 40 v/v) in isocratic mode at a flow rate of 1 ml min−1. The detection was monitored at 270 nm (ABA) and at 282 nm (Ex) and 360 nm (Em) (IAA, IPA). The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification, and robustness. The determined validation parameters are in the commonly acceptable ranges for that kind of analysis.

Screening of Marketed Pork for Ceftiofur residues by HPLC And UV-Vis Spectrophotometer

In the present study pork samples from markets of Assam, India were collected and analyzed to detect the presence of Ceftiofur residues using a High Performance liquid Chromatography (HPLC) System and a UV-Vis Spectrophotometer. 300 nos. of representative pork samples were collected from different pork markets of Assam. The samples after collection were preserved at -20°C. Analyses of the samples using High Performance Liquid Chromatography with UV-Vis Detector were done as per the method of Oka et al, 1985 while analyses of the same samples using UV-Vis Spectrophotometer were done as per the method of Annapurna et al. ,2009. Ceftiofur residues were extracted with Mc-IIvaine buffer. Solid phase extraction clean up was done with Sep-pak C18 cartridge. Recovery ranged from 83-95% (HPLC) and 5780%(Spectrophotometer).Out of the tested samples, 10 nos of the screened samples were detected to be positive for trace residues of Ceftiofur using Spectrophotometer while 14 nos .of same samples were detected for ceftiofur residue using HPLC method which were well below the MRL value.