Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Methods for the Quantification of Cefquinome, Ceftiofur, and Desfuroylceftiofuracetamide in Porcine Feces with Emphasis on Analyte Stability.

Cefquinome and ceftiofur are ß-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g-1 to 1000 ng g-1 for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g-1 to 2000 ng g-1 for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since ß-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL-1 tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix.

Effect of phenylacetamide isolated from lepidium apetalum on myocardial injury in spontaneously hypertensive rats and its possible mechanism.

Context: In the antihypertensive study of phenylacetamide (PA) on spontaneously hypertensive rats (SHR), it was occasionally found that PA prevents myocardial injury.Objective: Clarify the protective mechanism of PA on myocardial injury in SHR rats.Materials and methods: In vivo, SHR rats were treated with or without PA (15, 30, 45 mg/kg) for 3 weeks (12 per group). In vitro, H9c2 cells were treated with PA (1, 5, 10 µM) for 24 h, and then stimulated with H2O2 (300 µM) for 4 h. Molecular mechanisms were explored through cardiac pathology, cardiac function and biochemical markers.Results: In vivo, PA (15, 30, 45 mg/kg) reduced CVF from 14.8 ± 1.62 to 9.94 ± 1.56, 8.6 ± 1.33, 8.14 ± 1.45%; increased the LVEF relative level from 0.8 ± 0.06 to 0.83 ± 0.04, 0.86 ± 0.05, 0.9 ± 0.04. All three doses can improve the cardiac pathological structure and function (LVEDD, LVESD, LVFS, heart index, NT-proBNP, CKMB, SBP); however, 45 mg/kg works best. But different doses show different molecular mechanisms. PA (15 mg/kg) improves RAAS system (REN, ACE), inflammation (ET-1, IL-1ß) and MAPK pathway (p-ERK/ERK, p-JNK/JNK) better. PA (45 mg/kg) improves oxidative stress (SOD, NOX1) and TGF-ß pathway (Smad3) better. In vitro, PA improved cell viability, oxidative stress (SOD, NOX1) and Smad3 protein expression.Discussion and conclusions: PA regulates different mechanisms at different concentrations to improve myocardial injury, and high dose is the best. This experiment provides a theoretical basis for the development of new clinical drugs for cardiovascular disease.

Determination of 10 Haloacetamides in drinking water by gas chromatography with automated solid phase extraction.

Drinking water disinfection may result in the formation of different classes of toxic disinfection by-products (DBPs). Haloacetamides (HAcAms) are an emerging class of nitrogenous DBPs (N-DBPs), which are generally more prevalent at lower concentrations in disinfected water than carbonaceous DBPs. Herein a fast, convenient, and effective method of analyzing 10 HAcAms in drinking water samples was demonstrated. This method was developed using gas chromatography /electron capture detection (GC/ECD) supplemented with automated solid phase extraction (auto-SPE). The variables for automated SPE procedures were further optimized, including the selection of SPE sorbents, types and volumes of extraction solvents, SPE washing solvents and wash times. Under optimized conditions, the instrumental linearity range was 0.5-150 µg L-1 with correlation coefficients>0.9975. The limits of detection and quantification of this method were 0.002-0.003 µg L-1 and 0.005-0.010 µg L-1, respectively. The recovery values ranged from 72.4% to 108.5%, and the relative standard deviations ranged from 3.3% to 9.1%. Therefore, the auto-SPE-GC-ECD method showed acceptable linearity and repeatability and was subsequently validated and applied to analyze 10 HAcAms in drinking water.

Removal of haloacetamides and their precursors at water purification plants applying ozone/biological activated carbon treatment.

Haloacetamides (HAcAms) are nitrogenous disinfection byproducts in drinking water. The profiles of six HAcAms and their formation potentials (FPs) upon chlorination at water purification plant 1 (WPP-1) in September 2016 and at WPP-2 in September 2016 and January 2017 were investigated. HAcAms were removed effectively when they were formed via intermediate chlorination during water purification processes. Removal of total HAcAm-FPs ranged from 50% to 75%. Coagulation/flocculation/sand filtration showed the highest removal of total HAcAm-FPs. As for individual HAcAms, while chlorinated acetamide-FPs were removed, brominated acetamide-FPs, particularly 2,2-dibromoacetamide, remained. The bromine incorporation factors increased during all water purification processes except ozonation and the ozone/hydrogen peroxide process for diHAcAms (2,2-dichloroacetamide, 2-bromo-2-chloroacetamide, and 2,2-dibromoacetamide). The trends in relationships between DOM indices (fractions of dissolved organic matter, ultraviolet absorbance at 260 nm, and fluorescence intensities representing humic-like and tryptophan-like compounds) and total HAcAm-FPs during ozonation and ozone/hydrogen peroxide process were different from those during other processes.

Metabolite profiling and isolation of biologically active compounds from Scadoxus puniceus, a highly traded South African medicinal plant.

Scadoxus puniceus (Amaryllidaceae), a medicinal plant of high value in South Africa, is used as a component of a traditional herbal tonic prescribed to treat several ailments. Ultra-high performance liquid chromatography-tandem mass spectrometry quantified the phenolic compounds in different organs of S. puniceus. Gravity column chromatography was used to separate fractions and active compounds. The structure of these compounds was determined using 1D and 2D nuclear magnetic resonance and mass spectroscopic techniques. A microplate technique was used to determine the acetylcholinesterase inhibitory activity of the pure compounds. Metabolite profiling revealed a greater profusion of hydroxycinnamic acids (69.5%), as opposed to hydroxybenzoic acids (30.5%). Chlorogenic acid was the most abundant (49.6% of hydroxycinnamic acids) compound. In addition to chlorogenic acid, the study is the first to report the presence of sinapic, gallic, and m-hydroxybenzoic acids in the Amaryllidaceae. Chromatographic separation of S. puniceus led to the isolation of haemanthamine (1), haemanthidine (2), and a rare chlorinated amide, metolachlor (3), the natural occurrence of which is described for the first time. Haemanthamine, haemanthidine, and metolachlor displayed strong acetylcholinesterase inhibitory activity (IC50 ; 23.1, 23.7, and 11.5 µM, respectively). These results substantiate the frequent use of S. puniceus as a medicinal plant and hold much promise for further pharmaceutical development.

Selective solid phase extraction of chloroacetamide herbicides from environmental water samples by amphiphilic magnetic molecularly imprinted polymers.

In this study, a novel amphiphilic magnetic molecularly imprinted polymers (MMIPs) have been prepared by using Fe3O4 microspheres as the magnetic core, 4-vinyl pyridine (4-VP) and alkenyl glycosides glucose (AGG) as functional co-monomers. Fe3O4 microspheres were directly encapsulated by the polymer without any surface modification in the distillation-precipitation polymerization. The morphology and composition of MMIPs were characterized by X-ray diffraction (XRD), vibrating sample magnetometry (VSM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Binding property and magnetic separation ability were systematically investigated through the equilibrium binding experiments. The feasibility of magnetic molecular imprinted solid phase extraction (MMISPE) was investigated for the selective enrichment of chloroacetamide herbicides from environmental water samples. The developed MMISPE-HPLC method exhibited good linearity (0.1-200µgL-1), low limit of detection (0.03-0.06µgL-1), and good precision (RSD<7%) under the optimized conditions. The introduced MMISPE-HPLC method was successfully used to analyze chloroacetamide herbicides in environmental water samples. Spiked chloroacetamide herbicides recoveries in three water samples ranged from 82.1% to 102.9%. These results indicated that amphiphilic MMIPs were the promising sorbents for the selective enrichment of chloroacetamide herbicides at trace levels from real environmental water samples.

Removal of some common glycosylation by-products during reaction work-up.

With the aim of improving the general glycosylation protocol to facilitate easy product isolation it was shown that amide by-products from glycosylation with trichloroacetimidate and N-phenyl trifluoroacetimidate donors could be removed during reaction work-up by washing with a basic aqueous solution. Excess glycosyl acceptor or lactol originating from glycosyl donor hydrolysis could equally be removed from the reaction mixture by derivatization with a basic tag and washing with an acidic solution during reaction work-up.

Formation and speciation of haloacetamides and haloacetonitriles for chlorination, chloramination, and chlorination followed by chloramination.

The formation of haloacetamides (HAcAms) and haloacetonitriles (HANs) from a solution containing natural organic matter and a secondary effluent sample was evaluated for disinfection by chlorination, chloramination, and chlorination followed by chloramination (Cl2NH2Cl process). The use of preformed monochloramine (NH2Cl) produced higher concentrations of HAcAms and lower concentrations of HANs than chlorination, while the Cl2NH2Cl process produced the highest concentrations of HAcAms and HANs. These results indicate that the Cl2NH2Cl process, which inhibited the formation of regulated trihalomethanes compared with chlorination, enhanced the formation of HAcAms and HANs. For disinfection in the presence of bromide, brominated dihaloacetamides and dihaloacetonitriles were formed, and the trends were similar to those observed for chlorinated species in the absence of bromide. The degrees of bromine substitution of dihaloacetamides and dihaloacetonitriles were highest for chlorination, followed by the Cl2NH2Cl process and then by the NH2Cl process. For the Cl2NH2Cl process, HAN formation kept gradually increasing with prechlorination time increasing from 0 to 120 min, while HAcAm formation increased only until it reached a maximum at around 10-30 min. These results suggest that the prechlorination time could be reduced to control the formation of HAcAms and HANs. During chloramination, the formation of HAcAms and HANs was lower when using preformed NH2Cl than when chloramines were formed in situ, with higher formation of HAcAms and HANs when chlorine was added before ammonia than vice versa for the secondary effluent; this finding suggests that preformed NH2Cl could be used to inhibit the formation of HAcAms and HANs during chloramination.

Enantioseparation of four amide herbicide stereoisomers using high-performance liquid chromatography.

The chirality of herbicides has been the focus of research. However, there is little information on the enantioseparation of amide herbicides with different chiral elements. In this study, the need for different chiral stationary phases (CSPs), mobile phases, temperatures and flow rates for the separation of napropamide, acetochlor and propisochlor was discussed in detail and compared to metolachlor. Resolution of C-chiral enantiomers was easier than that of axial-chiral enantiomers. Metolachlor and acetochlor could achieve baseline separation only on AY-H and AS-H columns, respectively. Propisochlor had satisfactory separations on OD-H and AS-H columns. Napropamide was separated on OJ-H, AY-H and AS-H columns. Both the structures of the compounds and CSPs and the interactions between them played significant roles in the enantioseparations. Molecule dockings were also used to elucidate the separation mechanisms. C-chiral enantiomers had perfect symmetry in their optical properties, whereas the axial-chiral enantiomers did not. The elution order for napropamide, acetochlor and propisochlor, with a single chiral location, was R- prior to S-. These results were the first that compare the enantioseparations of four amide herbicides with different chirality, and they provided the absolute configurations for the herbicides. The paper also illustrated certain mechanisms for enantioseparations.

Metolachlor stereoisomers: Enantioseparation, identification and chiral stability.

Metolachlor is a chiral herbicide consisting of four stereoisomers, which is typically used as a racemic mixture or is enriched with the herbicidally active 1'S-isomers. Because studies on the enantioselective behavior of phyto-biochemical processes and the environmental fate of metolachlor have become significant, a practical method for analyzing and separating metolachlor stereoisomers must be developed. In the present study, the enantiomeric separation of metolachlor was achieved using OD-H, AS-H, OJ-H and AY-H chiral columns. The effects of different organic modifiers in an n-hexane-based mobile phase were investigated, and various temperatures and flow rates, which may influence metolachlor separation, were also explored. The optimal resolution was obtained using an AY-H column with n-hexane/EtOH (96/4) as the mobile phase at a rate and temperature of 0.6mLmin(-1) and 25°C, respectively. The absolute configuration of the four stereoisomers was identified as αSS, αRS, αSR, αRR using computed and experimentally measured ECD and VCD spectra. Thermal interconversion and solvent stability experiments were also performed. Pure metolachlor stereoisomers in different organic solvents and water at 4°C or 30°C were stable. These results were used to establish a sound method for analyzing, preparing, characterizing, and preserving individual metolachlor stereoisomers in most natural environments.

[A new phenylacetamide from the seeds of Lepidium apetalum Willd].

The chemical constituents of the seeds of Lepidium apetalum Willd. were investigated using chromatographic methods, including Diaion HP-20, Toyopearl HW-40, MCI Gel CHP-20, ODS, silica gel chromatography and semi-preparative-HPLC. Three compounds were isolated and their structures were elucidated by spectral data and physicochemical properties, which were identified as lepidiumamide A (1), cis-desulfoglucotropaeolin(2), trans-desulfoglucotropaeolin (3). Among those, compound 1 is a new phenylacetamide, compound 2 and 3 were isolated from this plant for the first time, and their configurations were also identified for the first time.

Two new amides from a halotolerant fungus, Myrothecium sp. GS-17.

Two new amides, named N-acetyl-2,4,10,17-tetrahydroxyheptadecylamine (1) and N-acetyl-3,5,11,18-tetrahydroxyoctadecyl-2-amine (2), were isolated from a halotolerant fungus, Myrothecium sp. GS-17. Their structures were identified on the basis of spectroscopic characteristics. The cancer cell cytotoxicities of two compounds were evaluated, and compound 2 exhibited weak cytotoxicity in HL-60 cell line.

Enhanced detection for determination of enantiomeric purity of novel agomelatine analogs by EKC using single and dual cyclodextrin systems.

Performing CD-EKC, baseline separation of five agomelatine analogs, potential antidepressant compounds, was achieved. A method for the enantioresolution and determination of enantiomeric purity of these naphthalene derivatives was developed using capillaries dynamically coated with polyethylene oxide and anionic cyclodextrins (highly sulfated CD) as chiral selectors. Operational parameters such as the nature and concentration of the cyclodextrins were investigated. In a second step the implementation of a dual cyclodextrin system was found to strongly enhance the LOD of the analytes. After optimization, best conditions were a 25 mM phosphate buffer at pH 2.5 containing 5% w/v (i.e. 19.7 mM) of highly sulfated-γ-CD and 10 mM of 6-monodeoxy-6-monoamino-ß-CD dual system, leading to resolution of, at least, 3.6 in 35 min. A preliminary validation of the developed method was undertaken: linearity, precision, and LOD and LOQ were evaluated. The latest ones were found equal to 0.25 and 0.82 µM and to 0.31 and 0.96 µM respectively for the first and the second enantiomer of compound 1.

Quantification of linezolid in serum by LC-MS/MS using semi-automated sample preparation and isotope dilution internal standardization.

BACKGROUND: Linezolid serum concentrations have been shown to be highly variable in critically ill patients with often sub-therapeutic drug levels regarding minimal inhibitory concentrations for relevant pathogens. Consequently, therapeutic drug monitoring of linezolid must be considered, requiring a reliable and convenient analytical method. We therefore developed and validated an LC-MS/MS method applying isotope dilution internal standardization and on-line solid phase extraction for serum linezolid quantification. METHODS: Sample preparation was based on protein precipitation and on-line solid phase extraction with two-dimensional liquid chromatography and column switching. Three-fold deuterated linezolid was used as the internal standard. The method was validated involving two separate LC-MS/MS systems covering the concentration range of 0.13-32 mg/L. The run time was 4 min. RESULTS: Validation revealed good analytical performance, with inaccuracy <6% and imprecision of <7.3% (CV) for six quality control samples (0.38-16.0 mg/L). The method was found to be robust during the validation process and during a pharmacokinetic study so far involving 600 samples. Comparative measurements on two LC-MS/MS systems revealed close agreement. CONCLUSIONS: This LC-MS/MS assay described herein is a convenient, robust and reliable method for linezolid quantification in serum which can be routinely applied using different LC-MS/MS systems. The method can be used for clinical studies and subsequent TDM of linezolid.

Development of an SPE method for the determination of zaleplon and zopiclone in hemolyzed blood using fast GC with negative-ion chemical ionization MS.

An SPE procedure for the determination of zaleplon and zopiclone in low-volume human hemolyzed blood using fast GC with negative-ion chemical ionization MS has been developed and validated. Both analytes were well retained on Oasis MCX and HLB columns, and sufficient extraction efficiency was achieved at pH 9.0. For further study a hydrophilic-lipophilic sorbent Oasis HLB was selected due to the polarity of sorbent surface and its large surface area in order to achieve efficient extraction of both analytes in a single step. Special attention has been paid to choosing washing and eluting solvents, resulting in a particularly/extremely clean and moisture-free extract. The mean extraction efficiency was higher than 90.1% for zaleplon and 82.9% for zopiclone. The precision for zaleplon and zopiclone was between 3.04-10.58% and 4.08-9.52%, respectively. Whereas the accuracy was in the range from -5.73 to 6.00%, and from -7.00 to 6.32% for zaleplon and zopiclone, respectively. The results show that the developed method is accurate, selective, precise, and very fast with excellent recovery and low LOD and LOQ.

Coupling Fenton and biological oxidation for the removal of nitrochlorinated herbicides from water.

The combination of Fenton and biological oxidation for the removal of the nitrochlorinated herbicides alachlor, atrazine and diuron in aqueous solution has been studied. The H2O2 dose was varied from 20 to 100% of the stoichiometric amount related to the initial chemical oxygen demand (COD). The effluents from Fenton oxidation were analyzed for ecotoxicity, biodegradability, total organic carbon (TOC), COD and intermediate byproducts. The chemical step resulted in a significant improvement of the biodegradability in spite of its negligible or even slightly negative effect on the ecotoxicity. Working at 60% of the stoichiometric H2O2 dose allowed obtaining highly biodegradable effluents in the cases of alachlor and atrazine. That dose was even lower (40% of the stoichiometric) for diuron. The subsequent biological treatment was carried out in a sequencing batch reactor (SBR) and the combined Fenton-biological treatment allowed up to around 80% of COD reduction.

JBIR-107, a new metabolite from the marine-sponge-derived actinomycete, Streptomyces tateyamensis NBRC 105047.

As part of our chemical screening program for new microbial secondary metabolites, we discovered a new compound, JBIR-107 (1), from the culture of Streptomyces tateyamensis NBRC 105047 isolated from a marine sponge sample. Extensive NMR and MS spectroscopic data enabled the structure of 1 to be determined as 5-acetamido-6-(4-(methyl(2-oxo-3-phenylpropyl)amino)phenyl)-4-oxohexanoic acid.

Determination of alachlor residues in pepper and pepper leaf using gas chromatography and confirmed via mass spectrometry with matrix protection.

Alachlor residues were determined in pepper and pepper leaf, after 49 days of manufacturer-recommended single- and double-dose application to the soil and plant. The samples were extracted with acetonitrile, partitioned with n-hexane, and purified through solid-phase extraction, and finally detected with a gas chromatography-microelectron capture detector. The linearity of the analytical response across the studied range of concentrations (0.05-4.0 µg/mL) was excellent, obtaining coefficients of determination (r(2) ) of 0.999. Recovery studies were carried out on spiked pepper and pepper leaf samples, at two concentrations levels (0.2 and 1.0 mg/kg), with three replicates performed at each level. Mean recoveries of 73.1-109.0% with relative standard deviations of 1.3-2.3% were obtained. The method was successfully applied to field samples, and alachlor residue was found in pepper (0.02 mg/kg) and pepper leaf (0.03 mg/kg), at levels lower than the maximum residue limits (0.2 mg/kg) set by the Korea Food and Drug Administration. The field-detected residues were further confirmed with gas chromatography-mass spectrometry with the help of pepper leaf matrix protection.

Application of dispersive liquid-liquid microextraction based on solidification of floating organic drop for simultaneous determination of alachlor and atrazine in aqueous samples.

Dispersive liquid-liquid microextraction based on solidification of floating organic drop coupled with HPLC-UV detection as a fast and inexpensive technique was applied to the simultaneous extraction and determination of traces of two common herbicides, alachlor and atrazine, in aqueous samples. The critical experimental parameters, including type of the extraction and disperser solvents as well as their volumes, sample pH, salt addition, and extraction time were investigated and optimized. Under the optimum conditions, the calibration graphs found to be linear in the range of 0.1-200 µg/L with LOD in the range of 0.02-0.05 µg/L. The RSDs were in the range of 4.2-5.3% (n = 5). The relative recoveries of well, tap, and river water samples which have been spiked with different levels of herbicides were 94.0-106.0, 99.0-105.0, and 88.5-97.0%, respectively.

Removal of alachlor in anoxic soil slurries and related alteration of the active communities.

Despite the implication of anaerobic soil communities in important functions related to C and N biogeochemical cycles, their responses to pesticides are rarely assessed. This study focused on the impact of alachlor, a chloroacetanilide herbicide, on two agricultural soils differing in their land use (fallow and corn-cultivated) in order to investigate the potential adaptation of anaerobic or facultative anaerobic soil microorganisms from fields with long history of herbicide use. The experiment was performed by developing slurries in anoxic conditions over 47 days. Changes in the community structure assessed through terminal restriction fragment length polymorphism analysis of 16S rRNA genes clearly showed a shift in the bacterial community of the cultivated soil, whereas the modification of the microbial community of the fallow soil was delayed. In addition, the analysis of alachlor degradation capacities of the two anaerobic communities indicated that 99 % of alachlor was removed in anoxic slurries of cultivated soil. Both these results suggested the preexistence of microorganisms in the cultivated soil able to respond promptly to the pesticide exposure. The composition of the anaerobic active community determined by 16S rRNA transcript analysis was mainly composed of strictly anaerobic Clostridia and the facultative anaerobe Bacilli classes. Some genera, described for their role in herbicide biodegradation were active in alachlor-treated slurries, whereas others were no longer detected. Finally, this study highlights, when triggered, the important diversity of the anaerobic community in soil.