Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis–laser induced fluorescence

Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. To determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser-induced fluorescence detection has been developed. Pretreatment of cell-lysate samples is very simple—precipitation of protein with acetonitrile in 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L⁻¹, pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5–500 ng mL⁻¹ and the LOD was 0.5 ng mL⁻¹. The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.     

Nature of the transient species formed during pulse radiolysis of thioacetamide in aqueous solutions

Thioacetamide (TA) is an organic compound having thioamide group similar to that in thiourea derivatives. Its reactions with e_aq⁻, H-atom and OH radicals were studied using the pulse radiolysis technique at various pHs and the kinetic and spectral characteristics of the transient species were determined. The initial adduct formed by the reaction of TA with OH radicals at pH 7 does not absorb light in the 300–600 nm region but reacts with the parent compound to give a transient species with an absorption maximum around 400 nm. At pH 0, the reaction of OH radicals with TA directly gives a similar transient species with absorption maximum at 400 nm. Transient species formed by H-atom reaction with TA and pH 0 has no absorption in the 300–600 nm region but at higher acidity a new transient species is formed which has absorption maximum at 400 nm. This transient absorption observed in the case of both OH and H atom reaction with TA is ascribed to the formation of a resonance stabilized radical similar to that obtained in the case of thiourea derivatives. The species produced by electron reaction viz. electron adduct was found to be a strong reductant and could reduce MV²⁺ with a high rate constant. H₂S was produced as a stable product in the reaction of e_aq⁻ and its G-value was determined to be about 0.8.     

Voltammetric Determination of Folic Acid with a Multi-Walled Carbon Nanotube-Modified Gold Electrode

The voltammetric behavior of folic acid (FA) at a multi-walled carbon nanotube (MWNT) modified gold electrode has been investigated by cyclic voltammetry, chronoamperometry and chronocoulometry. The modified electrode exhibits a good promoting effect on the electrochemical reaction of FA. FA can generate a well-defined anodic peak at around 0.83 V (vs. SCE) in 0.1 M H₃PO₄–NaH₂PO₄ buffer solution of pH 2.5. The peak results from a 2-electron transfer of FA, and the standard potential of FA is estimated to be 0.79 V (vs. SCE). The parameters affecting the response of FA, such as solution pH, accumulation time, accumulation potential, and amount of MWNTs are optimized for the determination of FA. Under the optimum conditions, the peak current changes linearly with FA concentration in the range from 2.0 × 10⁻⁸ M to 1.0 × 10⁻⁶ M. This method has been applied to the determination of FA in drug tablets, and the recovery is 93.9–96.9%. In addition, the influence of some coexistent species is examined. When a Nafion layer is introduced on the gold electrode before deposition of MWNTs, the resulting composite electrode can give better response to FA. At the same time, the interference by some foreign species is suppressed to some extent.     

Preparation and characterization of AuPt alloy nanoparticle–multi-walled carbon nanotube–ionic liquid composite film for electrocatalytic oxidation of cysteine

Gold–platinum (AuPt) alloy particles were fabricated directly on multi-walled carbon nanotubes (MWNT)–ionic liquid (i.e., trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide, [P_6,6,6,14][NTf₂]) composite coated glassy carbon electrode (GCE) by electrodeposition method. Scanning electron microscope image showed that they were well-dispersed nanocluster consisting of smaller nanoparticles, and their size was about 70 nm. X-ray diffraction experiment showed that they were single-phase alloy nanomaterial, and the calculated composition was consisting with that obtained by energy dispersive X-ray spectroscopy. The resulting modified electrode (i.e., AuPt–MWNT–[P_6,6,6,14][NTf₂]/GCE) presented high catalytic activity for the electrochemical oxidation of cysteine. The peak potential of cysteine shifted to 0.42 V (versus saturated calomel electrode) in 0.1 M H₂SO₄ and the peak current increased greatly in comparison with that on the corresponding Pt (or Au)–MWNT–[P_6,6,6,14][NTf₂]/GCE. Under the optimized conditions, the oxidation current of cysteine at 0.45 V was linear to its concentration in the range of 5.0 × 10⁻⁷ ∼ 4.0 × 10⁻⁵ M with a sensitivity of 43.8 mA M⁻¹.     

Determination of nitrobenzene by differential pulse voltammetry and its application in wastewater analysis

A new method for the determination of nitrobenzene (NB) by differential pulse voltammetry (DPV) based on an adsorptive stripping technique was developed. Cyclic voltammetry (CV) was used in a comparative investigation into the electrochemical reduction of NB at a glassy carbon electrode (GCE). With this electrode, the sharp peak of NB appeared at −0.71 V (vs. Ag/AgCl). The experimental parameters were optimized. Studies on the effect of pH on the peak height and peak potential were carried out over the pH range ca. 9.0–11.5 with sodium carbonate/sodium hydrogen carbonate buffer solution. A solution of pH 9.9 was chosen as analytical medium. Cathodic peak currents were found to be linearly related to the concentration of NB over the range ca. 12.3–1.23 × 10⁴ μg L⁻¹ with relative standard deviations of ca. 3.26–6.75%. The detection limit of NB in water was 5.42 μg L⁻¹. The interference of organic and inorganic species on the voltammetric response was also studied. The proposed method was applied to the determination of NB in wastewater samples with an average recovery of ca. 95.9–102.4%.     

Preparation and study of colloid-chemical and optical properties of the nanocrystals of zinc sulfide modified with amino acids

The effect of some amino acids: cysteine, methionine, glycine, lysine, and aspartic acid, on the formation of nanoparticles of zinc sulfide in aqueous solutions at pH 5.5–10.0 was investigated. A method of obtaining stable sols of ZnS particles of 2–4 nm size with narrow distribution of the particle size was developed. The investigated nanoparticles are shown to be sphalerite, the cubic modification of zinc sulfide. The ZnS sols modified with methionine and glycine show intense luminescence at 415–425 nm.     

Study of the pH function of a mechanically renewed graphite-quinhydrone indicator electrode

A composition of quinhydrone and spectral graphite powders and also an epoxy resin with polyethylene polyamine as a binder is studied for fabricating a solid indicator electrode, renewed by mechanically cutting a thin surface layer, intended for measurements of the pH of solutions. It is shown that after the renewal of the electrode surface, the dissolution of the components of quinhydrone occurs in the near-electrode layer and the electrode potential depends on the pH of the solution and the time of the contact of the electrode with the solution. The pH dependence of potentials (E _t ) at t = const in the pH range 2.0–7.0 is linear and close to the theoretical one. In the pH range 7.0–13.9, the results of measurements of E _t by the proposed carbon-quinhydrone electrode, in contrast to the traditional quinhydrone electrode, are well reproducible and linearly depend on pH. This opens up the possibility of using the electrode for the determination of E _t also in alkaline media.     

Determination of the ethylene oxide adduct S-(2-hydroxyethyl)cysteine by a fluorometric HPLC method in albumin and globin from human blood

A new method has been developed for the quantification of 2-hydroxyethylated cysteine resulting as adduct in blood proteins after human exposure to ethylene oxide, by reversed-phase HPLC with fluorometric detection. The specific adduct is analysed in albumin and in globin. After isolation of albumin and globin from blood, acid hydrolysis of the protein and precolumn derivatisation of the digest with 9-fluorenylmethoxycarbonylchloride, the levels of derivatised S-hydroxyethylcysteine are analysed by RP-HPLC and fluorescence detection, with a detection limit of 8 nmol/g protein. Background levels of S-hydroxyethylcysteine were quantified in both albumin and globin, under special consideration of the glutathione transferase GSTT1 and GSTM1 polymorphisms. GSTT1 polymorphism had a marked influence on the physiological background alkylation of cysteine. While S-hydroxyethylcysteine levels in “non-conjugators” were between 15 and 50 nmol/g albumin, “low conjugators” displayed levels between 8 and 21 nmol/g albumin, and “high conjugators” did not show levels above the detection limit. The human GSTM1 polymorphism had no apparent effect on background levels of blood protein 2-hydroxyethylation. Received: 16 February 1998 / Revised: 27 March 1998 / Accepted: 1 April 1998     

Determination of the ethylene oxide adduct S-(2-hydroxyethyl)cysteine by a fluorometric HPLC method in albumin and globin from human blood

A new method has been developed for the quantification of 2-hydroxyethylated cysteine resulting as adduct in blood proteins after human exposure to ethylene oxide, by reversed-phase HPLC with fluorometric detection. The specific adduct is analysed in albumin and in globin. After isolation of albumin and globin from blood, acid hydrolysis of the protein and precolumn derivatisation of the digest with 9-fluorenylmethoxycarbonylchloride, the levels of derivatised S-hydroxyethylcysteine are analysed by RP-HPLC and fluorescence detection, with a detection limit of 8 nmol/g protein. Background levels of S-hydroxyethylcysteine were quantified in both albumin and globin, under special consideration of the glutathione transferase GSTT1 and GSTM1 polymorphisms. GSTT1 polymorphism had a marked influence on the physiological background alkylation of cysteine. While S-hydroxyethylcysteine levels in “non-conjugators” were between 15 and 50 nmol/g albumin, “low conjugators” displayed levels between 8 and 21 nmol/g albumin, and “high conjugators” did not show levels above the detection limit. The human GSTM1 polymorphism had no apparent effect on background levels of blood protein 2-hydroxyethylation. Received: 16 February 1998 / Revised: 27 March 1998 / Accepted: 1 April 1998     

A PVC-based cryptand C2B22 membrane potentiometric sensor for zinc(II)

A PVC membrane electrode for zinc ions based on cryptand C2_B22 as membrane carrier was prepared. The electrode exhibits a linear stable response over a wide concentration range (5.0 × 10^–2– 5.0 × 10^–5 mol/L) with a slope of 24 mV/ decade and a limit of detection of 3.98 × 10^–5 mol/L (2.6 μg/g). It has a fast response time of about 30 s and can be used for at least 4 months without any divergence in potential. The proposed sensor revealed good selectivities for Zn²⁺ over a wide variety of other metal ions and could be used in a pH range of 4–7. It was used as an indicator electrode in potentiometric titration of zinc ion. Received: 26 February 1998 / Revised: 25 May 1998 / Accepted: 28 May 1998     

Pulse radiolysis study of dithio-oxamide in aqueous solutions

The reactions of e⁻ _aq, H-atoms, OH radicals and some one electron oxidants and reductants were studied with dithio-oxamide (DTO) in aqueous solutions using pulse radiolysis technique. The transient species formed by the reaction of e⁻ _aq with DTO at pH 6.8 has an absorption band with λ _max at 380 nm and is reducing in nature. H-atom reaction with DTO at pH 6.8 also produced the same transient species. The semi-reduced species was found to be neutral indicating that the electron adduct gets protonated quickly. However at pH 1, the species produced by H-atom reaction had a different spectrum with λ _max at 360 and 520 nm. Reaction of acetone ketyl radicals and CO₂ ⁻ radicals with DTO at pH 6.8 gave transient spectra which were identical to that obtained by e⁻ _aq reaction. However at pH 1, the spectrum obtained by the reaction of acetone ketyl radicals with DTO was similar to that obtained by H-atom reaction at that pH. The transient species formed by OH radical reaction with DTO in the pH range 1–9.2 also has two absorption maxima at 360 and 520 nm. This spectrum was identical with the spectrum obtained by H-atom reaction at pH 1. This means that all these radicals viz. OH, H-atom and (CH₃)₂COH radicals react with DTO at pH 1 by H-abstraction mechanism. The transient species produced was found to be sensitive to the presence of oxygen. One-electron oxidizing radicals such as Br₂ ^−· and SO₄ ^−· radicals reacted with DTO at neutral pH to give the same species as produced by OH radical reaction having absorption maxima at 360 to 520 nm. At acidic pHs, only Br₂ ^−· and Cl₂ ^−· radicals were able to oxidize DTO to give the same species as produced by OH radical reaction. The semioxidized species is a resonance stabilized species with the electron delocalized over the-N-C-S bond. This species was found to be neutral and non-oxidizing in nature.     

Electrocatalytic activity of poly(3-methylthiophene) electrodes

Poly 3-methylthiophene (P3MT) modified electrodes have shown an improvement for detecting catecholamines when compared to classical ones. Past work with this polymer electrode suggested the possible presence of “active sites,” which are believed to be the polymer’s center of electrocatalytic activity. The interaction of 1,5-anthroquinone-disolfonic acid (1,5-AQDS) at the P3MT electrode showed a nonreversible behavior resulting in the blocking of “the active sites,” suggesting the specific electcatalytical activity of this polymer is limited to catechol and similar compounds. In order to improve catecholamine detection, two methods of electropolymerization for P3MT were compared under similar conditions; (1) a constant potential for a specific length of time, and (2) potential cycling. It was found that cycling provided a more sensitive CV, i.e. increased number of active sites. Under a controlled pH study (pH range 2–9), the polymer electrode maintained its superior performance, manifested as lower ΔE and higher i, toward catechol over the traditional electrodes. Two different supporting electrolytes were used, sulfate and phosphate, and it was found that in neutral or basic solutions containing phosphate, the oxidation and reduction potentials of catechol shifted to lower values. Solutions containing sulfate exhibited no shift in the oxidation potential at any pH value.     

Microstructure of the LaSrCuO<Subscript>4 − δ</Subscript>|Ce<Subscript>0.9</Subscript>Gd<Subscript>0.1</Subscript>O<Subscript>2 − δ</Subscript> interface and its reversibility with respect to oxygen

The conditions of formation of electrode/electrolyte interfaces LaSrCuO_{4 − δ}|Ce_0.9Gd_0.1O_{2 − δ} are optimized. It is shown that electrode layers formed by the screen printing method have better developed surfaces and are more uniform and strong as compared with thick film layers applied by a brush. Symmetric LaSrCuO_{4 − δ}|Ce_0.9Gd_0.1O_{2 − δ}|LaSrCuO_{4 − δ} cells with porous electrodes are studied by impedance spectroscopy and cyclic voltammetry in the temperature range of 773–1173 K at the oxygen partial pressure of (28–2.1) × 10⁴ Pa. The oxygen process is shown to be limited by the charge transfer across the electrode/electrolyte interface. The exchange currents are calculated in the temperature range of 773–1173 K to amount from 1 × 10⁻³ to 3.5 × 10⁻² A/cm², which points to the high reversibility of the electrode/electrolyte interface with respect to oxygen.     

Lead selective membrane electrode using cryptand(222) neutral carrier

The construction, performance characteristics and applications of a polymeric membrance electrode for lead(II) ion are reported. The electrode was prepared by incorporating cryptand(222) as the neutral carrier into a plasticized poly(vinyl chloride) membrane. The influence of membrane composition, pH and concentration of internal reference solution were investigated. The electrode exhibits a potentiometric response for Pb²⁺ ion over the concentration range 10^–1–10^–5 M with a detection limit of 5 × 10^–6 M Pb²⁺. It shows a relatively fast response time of about 30 s and can be used for about two months without any considerable divergence in potential. The proposed sensor revealed good selectivities for Pb²⁺ in the presence of several metal ions and could be used in the pH range of 2.5 to 7.5. It was used as an indicator electrode in the potentiometric titration of Pb²⁺ with EDTA. Received: 20 November 1998 / Revised: 26 March 1999 / Accepted: 31 March 1999     

Electrochemical synthesis of self-doped polyaniline and its use to the electrooxidation of ascorbic acid

Self-doped polyaniline (PAN) film on platinum electrode surface has been synthesized via electrochemical copolymerization of aniline with orthanilic acid (OAA). Fourier transform infrared, UV–Vis, and elemental analysis indicate the formation of the copolymer and that the copolymer has the structure of a head-to-tail coupling of aniline and OAA units. It was found that the internal doping of PAN with OAA can extend the electroactivity of PAN in neutral and even in alkaline media. The obtained self-doped PAN (PAN-OAA)-coated platinum electrode is shown to be a good surface for the electrooxidation of ascorbic acid (AA) in phosphate buffer solution of pH 7. The anode peak potential of AA shifts from 0.63 V at bare platinum electrode to 0.34 V at the PAN-OAA-modified platinum electrode with greatly enhanced current response. A linear calibration graph is obtained over the AA concentration range of 5–60 mM using cyclic voltammetry. Rotating disk electrode voltammetry and chronoamperometry have been employed to investigate the electrooxidation of AA. The PAN-OAA-modified platinum electrode shows good stability and reproducibility.     

Lead selective membrane electrode using cryptand(222) neutral carrier

The construction, performance characteristics and applications of a polymeric membrance electrode for lead(II) ion are reported. The electrode was prepared by incorporating cryptand(222) as the neutral carrier into a plasticized poly(vinyl chloride) membrane. The influence of membrane composition, pH and concentration of internal reference solution were investigated. The electrode exhibits a potentiometric response for Pb²⁺ ion over the concentration range 10^–1–10^–5 M with a detection limit of 5 × 10^–6 M Pb²⁺. It shows a relatively fast response time of about 30 s and can be used for about two months without any considerable divergence in potential. The proposed sensor revealed good selectivities for Pb²⁺ in the presence of several metal ions and could be used in the pH range of 2.5 to 7.5. It was used as an indicator electrode in the potentiometric titration of Pb²⁺ with EDTA. Received: 20 November 1998 / Revised: 26 March 1999 / Accepted: 31 March 1999     

Immobilization of flavine adenine dinucleotide onto nickel oxide nanostructures modified glassy carbon electrode: fabrication of highly sensitive persulfate sensor

A simple method was used to fabricate flavin adenine dinucleotide (FAD)/NiOx nanocomposite on the surface of glassy carbon (GC) electrode. Cyclic voltammetry technique was applied for deposition nickel oxide nanostructures onto GC surface. Owing to its high biocompatibility and large surface area of nickel oxide nanomaterials with immersing the GC/NiOx-modified electrode into FAD solution for a short period of time, 10–140 s, a stable thin layer of the FAD molecules immobilized onto electrode surface. The FAD/NiOx films exhibited a pair of well-defined, stable, and nearly reversible CV peaks at wide pH range (2–10). The formal potential of adsorbed FAD onto nickel oxide nanoparticles film, E ^o′ vs. Ag/AgCl reference electrode is −0.44 V in pH 7 buffer solutions was similar to dissolved FAD and changed linearly with a slope of 58.6 mV/pH in the pH range 2–10. The surface coverage and heterogeneous electron transfer rate constant (k _s ) of FAD immobilized on NiOx film glassy carbon electrode are 4.66 × 10⁻¹¹ mol cm⁻² and 63 ± 0.1 s⁻¹, indicating the high loading ability of the nickel oxide nanoparticles and great facilitation of the electron transfer between FAD and nickel oxide nanoparticles. FAD/NiOx nanocomposite-modified GC electrode shows excellent electrocatalytic activity toward S₂O₈²⁻ reduction at reduced overpotential. Furthermore, rotated modified electrode illustrates good analytical performance for amperometric detection of S₂O₈²⁻. Under optimized condition, the concentration calibration range, detection limit, and sensitivity were 3 μM–1.5 mM, 0.38 μM and 16.6 nA/μM, respectively.     

Construction and Analytical Applications of Plastic Membrane Electrodes for Oxytetracycline Hydrochloride

 Oxytetracycline hydrochloride-selective electrodes of both the coated wire and the conventional polymer membrane types based on oxytetracyclinium phosphotungstate and phosphomolybdate have been prepared. A Nernstian response is shown by these electrodes within 1.0×10⁻⁶–1.0×10⁻² M concentration ranges depending on the type of electrode. The response is unaffected by the change of pH over the range 4–11. The standard electrode potentials, E°, were determined at different temperatures and used to calculate the isothermal temperature coefficients of the electrodes. The electrodes show good selectivity to oxytetracycline hydrochloride with respect to many inorganic cations, sugars and amino-acids. Oxytetracycline hydrochloride is determined successfully in pure solutions and in pharmaceutical preparations using calibration by standard addition and potentiometric titration. A regeneration process for the exhausted electrodes has been developed. Received February 2, 2000. Revision April 7, 2000.     

Solid-contact ionophore-based electrode for determination of pH in acidic media

Solid-contact electrode for pH measurements in acidic media is described. The sensor membrane is made of polyvinyl chloride plasticized with bis(2-ethylhexyl)phthalate and contains neutral pH-selective ionophore hexabutyltriamidophosphate and potassium tetrakis-p-Cl-phenylborate cation exchanger. The transducer layer of the solid-contact electrode contains the same membrane composition and also carbon black and electron–ion-exchanger resin EI-21(a cation exchange resin containing fine dispersion of metal copper) for stabilization of the electrode potential. The electrode is suitable for measurements of pH in the range 0–6 and works also in hydrofluoric acid (HF) solutions up to 0.1 M HF. Chronopotentiometric measurements show diffusion-limited polarization at the interface between sensor membrane and transducer layer. The slope of the linearized polarization curve correlates with the long-term stability of the electrode potentials providing a tool for prediction of the long-term stability of solid-contact potentiometric sensors.     

Electrocatalytic reduction of hydrogen peroxide at Prussian blue modified electrodes: a RDE study

Electrocatalytic reduction of hydrogen peroxide at Prussian blue modified electrode has been studied with rotating disk electrode in pH 5.5 and 7.3 solutions. It has been shown that the electrocatalytic cathodic reduction obeys Koutecky–Levich relationship at electrode potentials ranging from 0.1 to −0.4 V vs. Ag/AgCl for low concentrations of peroxide not exceeding 0.3 mM. Within this potential window, the calculated kinetic cathodic current ranges within the limits of 2.15–6.09 and 1.00–3.60 mA cm⁻² mM⁻¹ for pH 5.5 and 7.3, respectively. For pH 5.5 and 7.3 solutions, a linear slope of the dependence of kinetic current on electrode potential of −10.8 and −2.89 mA cm⁻² mM⁻¹ V⁻¹, respectively, has been obtained. At a higher concentration of peroxide, exceeding 0.6 mM, deviations from Koutecky–Levich relationship have been observed. These deviations appear more expressed at higher potentials and higher solution pH. The results obtained have been interpreted within the frame of two-step reaction mechanism, including (1) dissociative adsorption of hydrogen peroxide with the formation of OH radicals and (2) one-electron reduction of these radicals to OH⁻ anions. At a higher concentration of peroxide, and especially at a higher pH, the second process becomes rate limiting.