Enzymatic rotating biosensor for cysteine and glutathione determination in a FIA system

The high sensitivity that can be attained using an enzymatic system and mediated by catechols has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP, [EC 1.11.1.7], immobilized on a rotating disk, in presence of hydrogen peroxide catalyzed the oxidation of catechols, whose back electrochemical reduction was detected on glassy carbon electrode surface at −150 mV. Thus, when l-cysteine (Cys) or glutathione (GSH) was added to the solution, these thiol-containing compounds participate in Michael addition reactions with catechols to form the corresponding thioquinone derivatives, decreasing the peak current obtained proportionally to the increase of its concentration. Cys was used as the model thiol-containing compound for the study. The highest response for Cys was obtained around pH 7. This method could be used to determine Cys concentration in the range 0.05-90 μM (r = 0.998) and GSH concentration in the range 0.04-90 μM (r = 0.999). The determination of Cys and GSH were possible with a limit of detection of 0.7 and 0.3 nM, respectively, in the processing of as many as 25 samples per hour. Current response of the HRP-rotating biosensor is not affected by the oxidized form of GSH and Cys (glutathione disulfide, GSSG, and l-cystine, respectively), by sulfur-containing and alkyl-amino compounds such as methionine and lysine, respectively. The interferences from easily oxidizable species such as ascorbic acid and uric acid are lowest.     

Sensitive monitoring of monoterpene metabolites in human urine using two-step derivatisation and positive chemical ionisation-tandem mass spectrometry

A gas chromatographic–positive chemical ionisation-tandem mass spectrometric (GC–PCI-MS/MS) method for the simultaneous determination of 10 oxidative metabolites of the monoterpenoid hydrocarbons α-pinene, (R)-limonene, and Δ³-carene ((+)-3-carene) in human urine was developed and tested for the monoterpene biomonitoring of the general population (n = 36). The method involves enzymatic cleavage of the glucuronides followed by solid-supported liquid–liquid extraction and derivatisation using a two-step reaction with N,O-bis(trimethylsilyl)-trifluoroacetamide and N-(trimethylsilyl)imidazole. The method proved to be both sensitive and reliable with detection limits ranging from 0.1 to 0.3 μg L⁻¹. In contrast to the frequent and distinct quantities of (1S,2S,4R)-limonene-1,2-diol, the (1R,2R,4R)-stereoisomer could not be detected. The expected metabolite of (+)-3-carene, 3-caren-10-ol was not detected in any of the samples. All other metabolites were detected in almost all urine samples.     

Facile, sensitive and selective fluorescence turn-on detection of HSA/BSA in aqueous solution utilizing 2,4-dihydroxyl-3-iodo salicylaldehyde azine

A novel fluorescence turn-on detection method of human serum albumin (HSA) and bovine serum albumin (BSA) in aqueous solution is investigated using 2,4-dihydroxyl-3-iodo salicylaldehyde azine (DISA). Upon the addition of DISA to HSA/BSA solution, a fluorescence turn-on effect at 529 nm can be observed with a large stokes shift of ∼129 nm based on hydrophobic binding-mode between protein and dye. Under the optimal condition, the linear ranges of fluorescence intensity for HSA and BSA are 0.1-30 μg mL⁻¹ with the relative correlation coefficient of R² = 0.991 (n = 10) and 0.3-50 μg mL⁻¹ with R² = 0.997 (n = 10); and the detection limits for HSA and BSA based on IUPAC (C_DL = 3S_b/m) are 20 ng mL⁻¹ and 50 ng mL⁻¹, respectively.     

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples

In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λ_ext/λ_em = 340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L⁻¹ CH₃COONH₄ (pH = 2.5)/CH₃CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25–5.0 μmol L⁻¹ and the LOD was 19 nmol L⁻¹. No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.     

Second-order calibration of excitation-emission matrix fluorescence spectra for determination of glutathione in human plasma

A rapid non-separative spectroflourimetric method based on the second-order calibration of the excitation-emission data matrix was proposed for the determination of glutathione (GSH) in human plasma. In the phosphate buffer solution of pH 8.0 GSH reacts with ortho-phthaldehyde (OPA) to yield a fluorescent adduct with maximum fluorescence intensity at about 420 nm. To handle the interfering effects of the OPA adducts with aminothiols other than GSH in plasma as well as intrinsic fluorescence of human plasma, parallel factor (PARAFAC) analysis as an efficient three-way calibration method was employed. In addition, to model the indirect interfering effect of the plasma matrix, PARAFAC was coupled with standard addition method. The two-component PARAFAC modeling of the excitation-emission matrix fluorescence spectra accurately resolved the excitation and emission spectra of GSH, plasma (or plasma constituents). The concentration-related PARAFAC score of GSH represented a linear correlation with the concentration of added GSH, similar to that is obtained in simple standard addition method. Using this standard addition curve, the GSH level in plasma was found to be 6.10 ± 1.37 μmol L⁻¹. The accuracy of the method was investigated by analysis of the plasma samples spiked with 1.0 μmol L⁻¹ of GSH and a recovery of 97.5% was obtained.     

Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry

For the first time a liquid chromatography method with high resolution mass spectrometric detection has been developed for the simultaneous determination all key metabolites of the sulfur pathway in yeast, including all thiolic (cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), γ-glutamyl-cysteine (Glu-Cys)) and non-thiolic compounds (methionine (Met), s-adenosyl-methionine (AdoMet), s-adenosyl-homocysteine (AdoHcy), and cystathionine (Cysta)). The developed assay also permits the speciation and selective determination of reduced, oxidized and protein bound fractions of all of the five thiols. Iodoacetic acid (IAA) was chosen as the derivatizing reagent. Thiols were extracted from sub-mg quantities of yeast using hot 75% ethanol. The detection limits were in the range of 1–12 nmol L⁻¹ for standard solution (high femotomole, absolute), except AdoMet (116 nmol L⁻¹), which was unstable. In freshly harvested yeast, most of the thiols were in the reduced forms and low levels of protein-bound GSH and Glu-Cys were found. In a selenium enriched yeast, the thiols were mainly in the oxidized forms, and a significant amount of protein-bound Cys, HCys, GSH, Cys-Gly and Glu-Cys were found. The method was also applied to the metabolic study of the adaptive response of Saccharomyces cerevisiae to hydrogen peroxide, cadmium, and arsenite, and the change in concentration of thiols in the sulfur pathway was monitored over a period of 4 h.     

Analysis of salicylic acid based on the fluorescence enhancement of the As(III)-salicylic acid system

A new, simple and sensitive spectrofluorimetric method for the determination of salicylic acid (λ_ex = 315 nm, λ_em = 408 nm) using As(III) as a sensitizing reagent has been investigated by measuring the increase of fluorescence intensity of salicylic acid due to the complexation of As(III)-salicylic acid in presence of sodium dodecyl sulfate (SDS) 10⁻³ M. Under optimum conditions, a significant relationship was obtained between the fluorescence intensity and salicylic acid concentration. A linear calibration curve was obtained in the range 13.8-13812 μg l⁻¹ with product-moment correlation coefficient (R) 0.99985 and detection limit 4.2 μg l⁻¹. The R.S.D. is 2.35% (n = 5).The method was applied successfully to the determination of salicylic acid in human serum.     

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

Membrane electrodes for the determination of glutathione

Four glutathione (GSH)-selective electrodes were developed with different techniques and in different polymeric matrices. Precipitation-based technique with bathophenanthroline-ferrous as cationic exchanger in polyvinyl chloride (PVC) matrix was used for sensor 1 fabrication. β-Cyclodextrin (β-CD)-based technique with either tetrakis(4-chlorophenyl)borate (TpClPB) or bathophenanthroline-ferrous as fixed anionic and cationic sites in PVC matrix was used for fabrication of sensors 2 and 3, respectively.β-CD-based technique with TpClPB as fixed anionic site in polyurethane (Tecoflex) matrix was used for sensor 4 fabrication. Linear responses of 1 × 10⁻⁵ to 1 × 10⁻⁴ M and 1 × 10⁻⁶ to 1 × 10⁻³ M with slopes of 37.5 and 32.0 mV/decade within pH 7-8 were obtained by using electrodes 1 and 3, respectively. On the other hand, linear responses of 1 × 10⁻⁵ to 1 × 10⁻² and 1 × 10⁻⁵ to 1 × 10⁻³ M with slopes of 47.9 and 54.3 mV/decade within pH 5-6 were obtained by using electrodes 2 and 4, respectively. The percentage recoveries for determination of GSH by the four proposed GSH-selective electrodes were 100 ± 1, 100.5 ± 0.7, 100 ± 1 and 99.0 ± 0.8% for sensors 1, 2, 3 and 4, respectively. Determination of GSH in capsules by the proposed electrodes revealed their applicability for determination of GSH in its pharmaceutical formulations. Also, they were used to determine GSH selectively in presence of its oxidized form (GSSG). Sensor 4 was successfully applied for determination of glutathione in plasma with average recovery of 100.4 ± 1.11%. The proposed method was compared with a reported one. No significant difference for both accuracy and precision was observed.     

Rapid monitoring of grapevine reserves using ATR–FT-IR and chemometrics

Predictions of grapevine yield and the management of sugar accumulation and secondary metabolite production during berry ripening may be improved by monitoring nitrogen and starch reserves in the perennial parts of the vine. The standard method for determining nitrogen concentration in plant tissue is by combustion analysis, while enzymatic hydrolysis followed by glucose quantification is commonly used for starch. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR–FT-IR) combined with chemometric modelling offers a rapid means for the determination of a range of analytes in powdered or ground samples. ATR–FT-IR offers significant advantages over combustion or enzymatic analysis of samples due to the simplicity of instrument operation, reproducibility and speed of data collection. In the present investigation, 1880 root and wood samples were collected from Shiraz, Semillon and Riesling vineyards in Australia and Germany. Nitrogen and starch concentrations were determined using standard analytical methods, and ATR–FT-IR spectra collected for each sample using a Bruker Alpha instrument. Samples were randomly assigned to either calibration or test data sets representing two thirds and one third of the samples respectively. Signal preprocessing included extended multiplicative scatter correction for water and carbon dioxide vapour, standard normal variate scaling with second derivative and variable selection prior to regression. Excellent predictive models for percent dry weight (DW) of nitrogen (range: 0.10–2.65% DW, median: 0.45% DW) and starch (range: 0.25–42.82% DW, median: 7.77% DW) using partial least squares (PLS) or support vector machine (SVM) analysis for linear and nonlinear regression respectively, were constructed and cross validated with low root mean square errors of prediction (RMSEP). Calibrations employing SVM-regression provided the optimum predictive models for nitrogen (R² = 0.98 and RMSEP = 0.07% DW) compared to PLS regression (R² = 0.97 and RMSEP = 0.08% DW). The best predictive models for starch was obtained using PLS regression (R² = 0.95 and RSMEP = 1.43% DW) compared to SVR (R² = 0.95; RMSEP = 1.56% DW). The RMSEP for both nitrogen and starch is below the reported seasonal flux for these analytes in Vitis vinifera. Nitrogen and starch concentrations in grapevine tissues can thus be accurately determined using ATR–FT-IR, providing a rapid method for monitoring vine reserve status under commercial grape production.     

Ion chromatography for rapid and sensitive determination of fluoride in milk after headspace single-drop microextraction with in situ generation of volatile hydrogen fluoride

In this article, we report a new method that involves headspace single-drop microextraction and ion chromatography for the preconcentration and determination of fluoride. The method lies in the in situ hydrogen fluoride generation and subsequent sequestration into an alkaline microdrop (15 μL) exposed to the headspace above the stirred aqueous sample. The NaF formed in the drop was then determined by ion chromatography. The influences of some crucial single-drop microextraction parameters such as the extraction temperature, extraction time, sample stirring speed, sulphuric acid concentration and ionic strength of the sample, on extraction efficiency were investigated. In the optimal condition, an enrichment factor of 97 was achieved in 15 min. The calibration working range was from 10 μg L⁻¹ to 2000 μg L⁻¹ (R² = 0.998), and the limit of detection (signal to noise ratio of 3) was 3.8 μg L⁻¹ of fluoride. Finally, the proposed method was successfully applied to the determination of fluoride in different milk samples. The recoveries of fluoride (at spiked concentrations of 200 μg L⁻¹ and 600 μg L⁻¹ into milk) in real samples ranged from 96.9% to 107.7%. Intra-day precision (N = 3) in terms of peak area, expressed as relative standard deviation, was found to be within the range of 0.24-1.02%.     

Selective spectrofluorimetric determination of glutathione in clinical and biological samples using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene

This work reports the development of a selective, sensitive and rapid spectrofluorimetric method for the determination of reduced glutathione (GSH) in the presence of relatively high levels of cysteine (Cys) in clinical and biological samples using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M). The fluorescence from TMPAB-o-M is strongly quenched by its maleimide moiety, but after reaction with thiol, the fluorescence is restored with a 350-fold intensity increase (fluorescence quantum yield from 0.002 to 0.73). In H₃Cit-Na₂HPO₄ buffer (pH 7.40), the derivatization is completed in just 5 min under 37 °C. The linear range is 0.005-0.2 μmol L⁻¹, with detection limit of 1.1 × 10⁻¹⁰ mol L⁻¹ (signal-to-noise ratio = 3). Almost all amino acids, including Cys, impose no interference even if present at relatively high concentrations (amino acids:GSH = 100:1, Cys:GSH = 1:1, molar ratio, C_GSH = 3 × 10⁻⁷ mol L⁻¹). The sample can be used directly without further treatment after the protein is removed. The developed method is precise with a relative standard deviation (R.S.D.) lower than 5.0% (n = 6) and has been applied to the determination of GSH in human blood and pig’s liver with recoveries between 94.4 and 105.6%.     

Transition-metal complexes of phenoxy-imine ligands modified with pendant imidazolium salts: Synthesis, characterisation and testing as ethylene polymerisation catalysts

Reaction between 3-((1R,2R)-2-{[1-(3,5-di-tert-butyl-2-hydroxy-phenyl)-meth-(E)-ylidene]-amino}-cyclohexyl)-1-isopropyl-4-phenyl-3H-imidazol-1-ium bromide (1a) or the derivative 3-((1R,2R)-2-{[1-(2-hydroxy-5-nitro-phenyl)-meth-(E)-ylidene]-amino}-cyclohexyl)-1-isopropyl-4-phenyl-3H-imidazol-1-ium bromide (1b) and metal halides MCl_x.yTHF (M = Zr, x = 4, y = 2; M = V, x = y = 3; M = Cr, x = y = 3), in THF, at −78 °C gives the metal complexes of general formula [MCl_x(κ²-N,O-OC₆H₂R¹R²C(H)N-C₆H₁₀-Im)₂][Br]₂ (where M = Zr, x = 2, R¹ = R² = ^tBu, 2; M = Zr, x = 2, R¹ = H, R² = NO₂, 3; M = V, x = 1, R¹ = R² = ^tBu, 4; M = Cr, x = 1, R¹ = R² = ^tBu, 5; M = Fe, x = 0, R¹ = R² = ^tBu, 6; Im = 1-isopropyl-4-phenyl-3H-imidazol-1-ium-3-yl). ¹H and ¹³C NMR spectroscopy of 2 and 3 indicate κ²-N,O-ligand coordination via the phenoxy-imine moiety with pendant imidazolium salt that is corroborated by a single crystal structure of 6. Compounds 2, 3, 4 and 5 were tested as precatalysts for ethylene polymerisation in the presence of methylaluminoxane (MAO) cocatalyst, showing low activity. Selected polymer samples were characterised by GPC showing multimodal molecular weight distributions.     

Application of a modified enzyme-linked immunosorbent assay for 3-amino-2-oxazolidinone residue in aquatic animals

Furazolidone has been banned from use in food animals because of its carcinogenicity and mutagenicity, but its continued misuse is widespread in aquacultures. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in aquatic products. In this regard, we modified a simplified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to address this need. A good linearity was achieved over a concentration range of 0.05-12.15 μg L⁻¹, and the IC₅₀ value was 0.96 μg L⁻¹. The sample preparation was simple and effective included water bath treatments, acid hydrolysis combined with overnight derivatization of AOZ by benzaldehyde. The limit of detection and the limit of quantification were 0.15 and 0.3 μg kg⁻¹. The recoveries of AOZ in all tissues were between 78.0-95.3% at the levels of 0.3, 1.0, and 2.0 μg kg⁻¹. The inter-assay variability was less than 19.1%. The modified ic-ELISA was applied in quantification of AOZ elimination in carp. The results showed that AOZ was quite difficult to eliminate. Good correlations of the results obtained by ELISA and LC-MS/MS were observed in incurred carp muscle (r = 0.9923) and carp plasma (r = 0.9915) at the levels of 2.5-571.8 μg kg⁻¹ (μg L⁻¹). Better results were obtained by modified ic-ELISA when compared with commercial ELISA kit. Therefore, the present assay is considered a rapid, accurate, reliable, and inexpensive method for the detection of furazolidone-residues in the edible tissues of aquatic animals.     

High throughput method for the analysis of cetrizine hydrochloride in pharmaceutical formulations and in biological fluids using a tris(2,2'-bipyridyl)ruthenium(II)-peroxydisulphate chemiluminescence system in a two-chip device

A fast, economic and sensitive chemiluminescence (CL) method has been developed for the analysis of cetrizine hydrochloride (CET) in pharmaceutical formulations and in biological fluids. The CL method is based on the oxidation of tris(2,2′-bipyridyl)ruthenium(II) (Ru (bipy)₃²⁺) by peroxydisulphate in a two-chip device. Up to 180 samples can be analysed per hour, consuming only minute quantities of reagents. Three instrumental setups were tested to find the most economical, sensitive and high throughput setup. In the first setup, a continuous flow of sample and CL reagents was used, whereas in the second setup, a fixed volume (2 μL) of (Ru (bipy)₃²⁺) was introduced into a continuous infusion of peroxydisulphate and the sample. In the third design, a fixed volume of sample (2 μL) was injected while the CL reagents were continuously infused. Compared to the first setup, a 200% signal enhancement was observed in the third setup. Various parameters that influence the CL signal intensity, including pH, flow rates and reagent concentrations, were optimized. A linear response was observed over the range of 50 μg L⁻¹ to 6400 μg L⁻¹ (R² = 0.9959) with RSD values of 1.1% (n = 15) for 1000 μg L⁻¹. The detection limit was found to be 15 μg L⁻¹ (S/N = 3). The amount of consumed sample was only 2 μL, from which the detected amount of CET was found to be 6.5 × 10⁻¹⁴ mol. This procedure was successfully applied to the analysis of CET in pharmaceutical formulations and biological fluids.     

In-line preconcentration of oxidized and reduced glutathione in capillary zone electrophoresis using transient isotachophoresis under strong counter-electroosmotic flow

Transient isotachophoresis (tITP) can improve the sensitivity of capillary electrophoresis (CE). In general, it was carried out under the condition of suppressed electroosmotic flow (EOF). However, some special conditions, such as extreme low pH background electrolyte and coating were needed to achieve the requirements of suppressed EOF. In this work, an approach of tITP under the strong counter-EOF in open system (counter-EOF-tITP) is presented for the rapid and sensitive preconcentrating the reduced glutathione (GSH) and the oxidized glutathione (GSSG) without modifying the capillary and the commercial CE instrument. The parameters of the experimental system, such as the concentration of leading electrolyte, the injected amount of terminating electrolyte and the injected pressure of sample were investigated in detail to understand the mechanism of counter-EOF-tITP. The sensitivity enhancement factors were of 320 for GSH and 280 for GSSG. In addition, the detection limit of 23.4 and 18.0 μg L⁻¹ for GSH and GSSG was achieved, respectively. The method's applicability was demonstrated by determining GSH and GSSG in tomato and human serum.     

Study of ferrocenyl-substituted Co2(CO)6-bispropargylic alcohol complexes as substrates for the formation of chains and macrocycles

Treatment of [Fc-1-R¹-1′-R²] (R¹ = H, R² = CH(O); R¹ = H, R² = CMe(O); R¹ = R² = CMe(O)) with LiCCCH₂OLi (prepared in situ from HCCCH₂OH and n-BuLi) affords the ferrocenyl-substituted but-2-yne-1,4-diol compounds of general formula [Fc-1-R¹-1′-{CR(OH)CCCH₂OH}] (R¹ = R = H (1a); R¹ = H, R = Me (1b); R¹ = CMe(O), R = Me (1c)) in low to high yields, respectively (where Fc = Fe(η⁵-C₅H₄)₂). In the case of the reactions of [Fc-1-R¹-1′-R²] (R¹ = H, R² = CH(O); R¹ = R² = CMe(O)), the by-products [Fc-1-R¹-1′-{CR(OH)(CH₂)₃CH₃}] (R¹ = R = H (2a); R¹ = CMe(O), R = Me (2c)) along with minor quantities of [Fc-1,1′-{CMe(OH)(CH₂)₃CH₃}₂] (3) are also isolated; a hydrazide derivative of dehydrated 2c, [1-(CMeCHCH₂CH₂CH₃)-1′-(CMeNNH-2,4-(NO₂)₂C₆H₃)] (2c′), has been crystallographically characterised. Interaction of 1 with Co₂(CO)₈ smoothly generates the alkyne-bridged complexes [Fc-1-R¹-1′-{Co₂(CO)₆-μ-η²-CR(OH)CCCH₂OH}] (R¹ = R = H (4a); R¹ = H, R = Me(4b); R¹ = CMe(O), R = Me (4c)) in good yield. Reaction of 4a with PhSH, in the presence of catalytic quantities of HBF₄ · OEt₂, gives the mono- [Fc-1-H-1′-{Co₂(CO)₆-μ-η²-CH(SPh)CCCH₂OH}] (5) and bis-substituted [Fc-1-H-1′-{Co₂(CO)₆-μ-η²-CH(SPh)CCCH₂SPh}] (6) straight chain species, while with HS(CH₂)_nSH (n = 2,3) the eight- and nine-membered dithiomacrocylic complexes [Fc-1-H-1′-{cyclo-Co₂(CO)₆-μ-η²-CH(S(CH₂)_n-)CCCH₂S-}] [n = 2 (7a), n = 3 (7b)] are afforded. By contrast, during attempted macrocyclic formation using 4b and HSCH₂CH₂OCH₂CH₂SH dehydration occurs to give [Fc-1-H-1′-{Co₂(CO)₆-μ-η²-C(CH₂)CCCH₂OH}] (8). Single crystal X-ray diffraction studies have been reported on 2c′, 4b, 4c, 7b and 8.     

Headspace liquid-phase microextraction using ionic liquid as extractant for the preconcentration of dichlorodiphenyltrichloroethane and its metabolites at trace levels in water samples

A novel technique, high temperature headspace liquid-phase microextraction (HS-LPME) with room temperature ionic liquid (RTIL), 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄MIM][PF₆]) as extractant, was developed for the analysis of dichlorodiphenyltrichloroethane (p,p′-DDT and o,p′-DDT) and its metabolites including 4,4′-dichlorodiphenyldichloroethylene (p,p′-DDE) and 4,4′-dichlorodiphenyldichloroethane (p,p′-DDD) in water samples by high performance liquid chromatography with ultraviolet detection. The parameters such as salt content, sample pH and temperature, stirring rate, extraction time, microdrop volume, and sample volume, were found to have significant influence on the HS-LPME. The conditions optimized for extraction of target compounds were as follows: 35% NaCl (w/v), neutral pH condition, 70 °C, 800 rpm, 30 min, 10 μL [C₄MIM][PF₆], and 25 mL sample solutions. Under the optimized conditions, the linear range, detection limit (S/N = 3), and precision (R.S.D., n = 6) were 0.3-30 μg L⁻¹, 0.07 μg L⁻¹, and 8.0% for p,p′-DDD, 0.3-30 μg L⁻¹, 0.08 μg L⁻¹, and 7.1% for p,p′-DDT, 0.3-30 μg L⁻¹, 0.08 μg L⁻¹, and 7.2% for o,p′-DDT, and 0.2-30 μg L⁻¹, 0.05 μg L⁻¹, and 6.8% for p,p′-DDE, respectively. Water samples including tap water, well water, snow water, reservoir water, and wastewater were analyzed by the proposed procedure and the recoveries at 5 μg L⁻¹ spiked level were in the range of 86.8-102.6%.     

Evaluation of the toxicity of ZnO nanoparticles to Chlorella vulgaris by use of the chiral perturbation approach

The toxicity of ZnO nanoparticles (NPs) has been widely investigated because of their extensive use in consumer products. The mechanism of the toxicity of ZnO NPs to algae is unclear, however, and it is difficult to differentiate between particle-induced toxicity and the effect of dissolved Zn²⁺. In the work discussed in this paper we investigated particle-induced toxicity and the effects of dissolved Zn²⁺ by using the chiral perturbation approach with dichlorprop (DCPP) as chiral perturbation factor. The results indicated that intracellular zinc is important in the toxicity of ZnO NPs, and that ZnO NPs cause oxidative damage. According to dose–response curves for DCPP and the combination of ZnO NPs with (R)-DCPP or (S)-DCPP, the toxicity of DCPP was too low to perturb the toxicity of ZnO NPs, so DCPP was suitable for use as chiral perturbation factor. The different glutathione (GSH) content of algal cells exposed to (R)-DCPP or (S)-DCPP correlated well with different production of reactive oxygen species (ROS) after exposure to the two enantiomers. Treatment of algae with ZnO NPs and (R)-DCPP resulted in reduced levels of GSH and the glutathione/oxidized glutathione (GSH/GSSG) ratio in the cells compared with the control. Treatment of algae with ZnO NPs and (S)-DCPP, however, resulted in no significant changes in GSH and GSH/GSSG. Moreover, trends of variation of GSH and GSH/GSSG were different when algae were treated with ZnSO₄·7H₂O and the two enantiomers. Overall, the chiral perturbation approach revealed that NPs aggravated generation of ROS and that released Zn²⁺ and NPs both contribute to the toxicity of ZnO NPs.