Ellman's Reagent Research Articles

Antioxidants selenomethionine and D-pantethine decrease the negative side effects of doxorubicin in NL/Ly lymphoma-bearing mice.

AimTo investigate the potential tissue-protective effects of antioxidants selenomethionine and D-pantethine applied together with doxorubicin (Dx) on NK/Ly lymphoma-bearing mice. The impact of this chemotherapy scheme on animal survival, blood cell profile, hepatotoxicity, glutathione level, and activity of glutathione-converting enzymes in the liver was compared with the action of Dx applied alone.MethodsThe hematological profile of animals was studied by the analysis of blood smears under light microscopy. Hepatotoxicity of studied drugs was evaluated measuring the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, De Ritis ratio, and coenzyme A fractions by McDougal assay. Glutathione level in animal tissues was measured with Ellman reagent, and the activity of glutathione reductase, transferase, and peroxidase was measured using standard biochemical assays.ResultsD-pantethine (500 mg/kg) and, to a lower extent, selenomethionine (600 µg/kg) partially reduced the negative side effects (leukocytopenia and erythropenia) of Dx (5 mg/kg) in NK/Ly lymphoma bearing animals on the 14th day of their treatment. This increased animal survival time from 47-48 to 60+ days and improved the quality of their life. This ability of D-pantethine and selenomethionine was realized via hepatoprotective and immunomodulating activities. D-pantethine also restored the levels of acid-soluble and free CoA in the liver of tumor-bearing animals, while selenomethionine caused the recovery of glutathione peroxidase levels in the liver, which was significantly diminished under Dx treatment. Both compounds decreased glutathione level in the liver, which was considerably induced by Dx.ConclusionsAntioxidants selenomethionine and D-pantethine partially reversed the negative side effects of Dx in NK/Ly lymphoma-bearing mice and significantly increased the therapeutic efficiency of this drug in tumor treatment.

Quantification of a peptide standard using the intrinsic fluorescence of tyrosine.

Absolute quantification of peptides is typically achieved using amino acid analysis, elemental analysis or derivatisation chemistry. Impurities, if present, may be accounted for using analytical high-performance liquid chromatography (HPLC) with detection of the peptide bond ultraviolet (UV) absorbance. To do this, peak areas from a UV chromatogram are used to estimate percentage purity on a mass basis, and this purity value is used as a correction. However, because the approach assumes that UV absorbance is uniformly proportional to mass, the result may be only semi-quantitative. Here, an alternative approach involving HPLC with detection of intrinsic tyrosine fluorescence is described. The fluorescence properties of a 21-residue synthetic peptide corresponding to an S-carbamidomethylated tryptic fragment of human serum albumin were characterised, and a method involving quantification relative to a non-peptidic calibrant, N-acetyl-l-tyrosine ethyl ester, was established. The method was used to quantify the thiol form of the peptide, and the results were compared with a parallel analysis involving derivatisation of the same material with Ellman’s reagent. When differences in fluorescence response (analyte versus calibrant) were accounted for, the measurements obtained via the two methods were in good agreement. Contributions from peptidic impurities were also considered, and their influence on the validity of the conclusions was evaluated. Despite some ambiguities introduced by the impurities, and the identification of some other potential sources of error, the results demonstrate that use of Tyr fluorescence is a promising solution to the challenging problem of absolute peptide quantification.Graphical A low-molecular-weight tyrosine derivative was used as a generic fluorescence calibrant for the quantification of a 21-residue peptideElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9334-1) contains supplementary material, which is available to authorized users.

Species- and concentration-dependent differences of acetyl- and butyrylcholinesterase sensitivity to physostigmine and neostigmine

Previous and more recent studies show that cholinesterase inhibitors (ChE-Is) are an important possibility for therapeutic intervention in Alzheimer's Disease, sepsis and other inflammatory syndromes. ChE-Is maintain high levels of acetylcholine (ACh) determining beneficial effects on the disease process. Despite numerous efforts to identify the appropriate choice of agents and dose of ChE-Is, a common protocol regarding concentration- and species-dependent differences in inhibitory potency (IC 50) of clinical relevant ChE-Is is still not available.To evaluate the in vitro sensitivity of Acetyl- and Butyrylcholinesterase (AChE, BChE), we compared the concentration-response effects of physostigmine and neostigmine on cholinesterases in whole blood from rat and human. A spectrophotometrical test system based on in vitro Ellman's reagent has been used to determine the kinetic properties of clinical relevant ChE-Is.In vitro, the enzyme activity of human AChE and BChE was inhibited in a concentration-dependent manner until a residual activity of 4–6% for AChE and 20–30% for BChE (IC 50 human AChE: 0.117 ± 0.007 μM physostigmine, 0.062 ± 0.003 μM neostigmine; IC 50 human BChE: 0.373 ± 0.089 μM neostigmine; 0.059 ± 0.012 μM physostigmine). The inhibition curve of rat BChE in contrast showed no concentration-dependency for physostigmine and neostigmine (87% residual activity even at high inhibitor concentrations). Rat AChE was inhibited in a concentration-dependent manner until a residual activity of 53%.The results suggest that cholinesterases from human and rat show marked species- and inhibitor-dependent differences in sensitivity to physostigmine and neostigmine. Knowledge of such differences may be critical in assessing the possible therapeutic effects of ChE-Is in both species and may guide researchers in the optimal design of future experiments regarding the application of ChE-Is.

A thiol fluorescent probe reveals the intricate modulation of cysteine's reactivity by Cu(II)

In this study, Cu(II)-mediated differential alteration of cysteine (Cys) reactivity is reported by using a Cys-specific fluorescent probe. The probe could react with Cys to give out strong fluorescence. When Cys was preincubated with Cu(II), the fluorescence of the probe was decreased due to the inhibition of Cys's reactivity by Cu(II). Remarkably, experimental results reveal that the probe could detect Cu(II) at subnanomolar concentrations. In contrast, Cu(II) could only partially inhibit the reaction between Cys and Ellman's reagent (DTNB). Furthermore, selectivity experiments show that Cu(II) is a much more potent inhibitor for Cys compared to other metal ions. Cell imaging experiments also confirm the inhibitory effects of Cu(II) on Cys's reactivity in living cells. We envision that the probe could add a useful tool for sensitive and selective detection of Cu(II) for biomedical research.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

Colic remains a life-threatening condition in the horse. Ischaemia and reperfusion following correction of small intestinal strangulation may produce oxidative stress. The ability to withstand oxidative stress depends on antioxidant levels and may be linked to horse survival. To measure peripheral antioxidant levels in horses undergoing exploratory laparotomy with small intestinal strangulation. Case-control study. Blood and plasma were collected from horses undergoing exploratory laparotomy for small intestinal strangulation and stored at -80°C. Controls involved non-colic horses. Total plasma glutathione was measured spectrophotometrically at 412 nm using the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) reaction. Samples containing scavenger (to remove reduced glutathione, GSH) were used to measure oxidised glutathione (GSSG). Glutathione reductase (GR) activity (u/l) was measured as the rate of GSH production at 412 nm. Glutathione peroxidise (GPx) activity (u/l) was measured as the change in optical density (340 nm) following the consumption of NADPH after GSSG production. All assays were purchased from BioAssay Systems (Hayward, California). Clinical data including arterial blood gas analysis were collected on admission. Glutathione reductase activity in horses with strangulating small intestinal lesions was significantly reduced compared to control horses (12.2 ± 1.1 u/l vs. 15.9 ± 0.8 u/l, P = 0.03, n = 6) whereas GPx activity did not significantly differ between colic and control horses (155.7 ± 48.7 u/l vs. 167.3 ± 30.1 u/l, P = 0.84, n = 6). Total glutathione, reduced or oxidised glutathione did not differ significantly between control and colic horses. A positive correlation existed between GR activity and Ca(2+) (r = 0.93) and K(+) (r = 0.75) whereas a strong negative correlation was present between GR activity and HCO3 (-) (r = -0.92) and PaCO2 (r = -0.96). Reduced plasma glutathione reductase activity with small intestinal strangulation indicates oxidative stress and may be related to systemic electrolyte/bicarbonate abnormalities. Ethical animal research: Study approval No. VREC219a. Explicit owner informed consent for inclusion of animals in this study was not stated. Supported by the School of Veterinary Sciences, University of Liverpool. Competing interests: None declared.

Quantification of PEG-maleimide ligands and coupling efficiencies on nanoparticles with Ellman's reagent.

The surface modification of nanometer- and micrometer-sized particles and planar substrates with polyethylene glycol (PEG) ligands of varying length is a very common strategy to tune the hydrophilicity and biocompatibility of such materials, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times. Nevertheless, simple methods for the quantification of PEG ligands are comparatively rare. Here, we present a new concept for the quantification of PEG ligands for maleimide-functionalized PEG molecules and the determination of PEG coupling efficiencies, exploiting the quantitative reaction of maleimide with l-cysteine, and the subsequent determination of the unreacted thiol with the photometric Ellman's test. This is shown for heterobifunctional PEG spacers of varying length and amino-functionalized polystyrene nanoparticles (PS NP) without and with differently charged encoding dyes. The reaction of l-cysteine with the Ellman's reagent was monitored photometrically and with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) to derive the reaction mechanism and to obtain the stoichiometry factor for l-cysteine quantification. Mass balances and quantification of l-cysteine via its sulfur concentration using elemental analysis and inductively coupled plasma mass spectrometry (ICP-MS) confirmed the accuracy and reliability of this approach that can be extended to other surface groups and ligands.

Spectrophotometric Determination of Affinities of α‐Factors for Their G Protein‐Coupled Receptors in Saccharomyces cerevisiae

A new and relatively simple spectrophotometric technique has been developed for the accurate determination of α‐factor pheromone affinities for Ste2p whole cell receptor in yeast a‐cells. We designed and tested nine detector peptides containing mono‐ (ε412 = 14 500) or tri‐cysteine residues (ε412 = 43 660). The free unbound detector was detected using Ellman's reagent at 412 nm. Saturation binding studies using Saccharomyces cerevisiae Y 7925 ( MATa ) at a concentration of 2.5 × 1011 cells/mL with the highest affinity detector 1, [Orn6]α‐factor‐[Cys]3, resulted in a dissociation constant ( K D ) of 1.67 × 10−7 and total binding sites per cell (B cell = 29 500 sites/cell) comparable with those obtained using radiolabeled binding assays. Competitive binding assay using five nonchromogenic α‐factor analogs allowed for the determination of each K D value. [Orn6,d‐Ala9]α‐factor showed the highest receptor affinity ( K D = 1.03 × 10−7 M), which was threefold higher than that of native α‐factor. This assay provides rapid and convenient results for determining the relative affinities of nonchromogenic α‐factors and eliminates the need for radioactive waste disposal.

In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae.

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI50 values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs.

Towards the rare earth functionalization of nano-clays with luminescent reporters for biophotonics

Completely water dispersible nanoclays can be equipped with optical functions for eventual uses in biophotonics. For this purpose, 3-mercaptopropylmethyldimethoxysilane is grafted as a linker to the rims of strongly anisotropic clay disks for the subsequent covalent attachement of a rare earth β-diketonate, co-coordinated with epoxy-functional phenanthroline (“Eu(ttfa)3epoxiphen”). Silane grafting yields of approximately 80% with respect to available rim-SiOH groups can be obtained as demonstrated by reliable determination of the grafted mercapto-groups with 5,5′-dithiol-bis-(2nitrobenzoic acid) (“Ellman's reagent”). The final linkage of Eu(ttfa)3epoxiphen to the rim mercapto-groups competes with mere polar adsorption on the laponite faces; however, the rim linkage can be confirmed via the complex's excitation and emission spectra and X-ray diffraction of the solids obtained, and corresponding absorption spectra.

Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk

The objective of this study was to determine the contribution of phospholipids from buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The aggregation mechanisms were characterised using analysis with Ellman's reagent, one-dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that phospholipids from buttermilk were involved in the formation of protein aggregates through the MFGM fragments at a low temperature, whereas phospholipids could interact directly with the proteins at a higher temperature (80 °C).

Thiolated hemicellulose as a versatile platform for one-pot click-type hydrogel synthesis.

A one-pot synthetic methodology for the thiolation of O-acetyl-galactoglucomannan (AcGGM) was developed to merge hemicellulose chemistry with "click" chemistry. This was realized by the AcGGM-mediated nucleophilic ring-opening of γ-thiobutyrolactone via the activation of the polysaccharide pendant hydroxyl groups. The incorporation of thiol functionalities onto the hemicellulose backbone was visualized by (1)H and (13)C NMR spectroscopy and was assessed by an Ellman's reagent assay of the thiol groups. The versatility of the thiolated AcGGM was elaborated and demonstrated by conducting several postmodification reactions together with hydrogel formation utilizing thiol-ene and thiol-Michael addition "click" reactions. The one-pot synthesis of thiolated AcGGM is a straightforward approach that can expand the applications of hemicelluloses derived from biomass by employing "click" chemistry.

Effects of Siroheme Occupancy in Escherichia Coli Assimilatory Sulfite Reductase Hemoprotein

Sulfur reduction is a fundamental biological process in most bacteria and plants. As a model system we study the Escherichia coli assimilatory sulfite reductase (aSiR), an ∼800kD complex that catalyzes the concerted six-electron reduction of sulfite (SO3−2) to biologically useful sulfide (S−2). The complex consists of alpha and beta subunits, the flavoprotein and hemoprotein respectively. The active site lies in the hemoprotein subunit (SiRHP) that contains a siroheme cofactor coupled to a 4Fe-4S cluster used to shuttle electrons to the bound substrate.To investigate the structural importance of siroheme in SiRHP, we removed the cofactor by both mutagenic and auxotrophic methods. Electrophoretic mobility shift assays, circular dichroism, and dynamic light scattering were used to characterize changes in SiRHP lacking siroheme (apo SIRHP). In addition, the occupancy of the 4Fe-4S cluster was spectroscopically probed using an Ellman's reagent test. Apo SiRHP was found to exist as a tetramer instead of a monomer and no longer contained the 4Fe-4S cluster. Additionally, the apo SiRHP tetramer formed by the absence of siroheme was unable to bind the flavoprotein subunit required for functional aSiR assembly. These results suggest a possible mechanism for removing non-functional SiRHP subunits from the assembly pool within the cell.

Kinetic and thermodynamic studies on the disulfide-bond reducing potential of hydrogen sulfide

The significance of persulfide species in hydrogen sulfide biology is increasingly recognized. However, the molecular mechanisms of their formation remain largely elusive. The obvious pathway of the reduction of biologically abundant disulfide moieties by sulfide was challenged on both thermodynamic and kinetic grounds. Using DTNB (5,5′-dithiobis-(2-nitrobenzoic acid), also known as Ellman's reagent) as a model disulfide we conducted a comprehensive kinetic study for its reaction with sulfide. The bimolecular reaction is relatively fast with a second-order rate constant of 889 ± 12 M−1s−1 at pH = 7.4. pH dependence of the rate law revealed that the reaction proceeds via the bisulfide anion species with an initial nucleophilic thiol-disulfide exchange reaction to give 5-thio-2-nitrobenzoic acid (TNB) and TNB-persulfide with a pH independent second-order rate constant of 1090 ± 12 M−1s−1. However, kinetic studies and stoichiometric analyses in a wide range of reactant ratios together with kinetic simulations revealed that it is a multistep process that proceeds via kinetically driven, practically irreversible reactions along the disulfide → persulfide → inorganic polysulfides axis. The kinetic model postulated here, which is fully consistent with the experimental data, suggests that the TNB-persulfide is further reduced by sulfide with a second-order rate constant in the range of 5 × 103 – 5 × 104 M−1s−1 at pH 7.4 and eventually yields inorganic polysulfides and TNB. The reactions of cystine and GSSG with sulfide were found to be significantly slower and to occur via more complicated reaction schemes. 1H NMR studies suggest that these reactions also generate Cys-persulfide and inorganic polysulfide species, but in contrast with DTNB, in consecutive equilibrium processes that are sensitive to changes in the reactant and product ratios. Collectively, our results demonstrate that the reaction of disulfides with sulfide is a highly system specific process from both thermodynamic and kinetic aspects, which together with the considerable steady-state concentrations of the reactants in biological systems signifies physiological relevance.

Quest for a turnover mechanism in peptide-based enzyme mimics

Creation of synthetic structures with an enzyme-like mechanism and turnover remains a significant challenge. In this study, peptides containing a cysteine thiol and histidine imidazole group were designed to mimic the active site of the cysteine protease papain. Ellman's reagent trapping experiments showed that rapid acetyl group exchange exists between the thiol and imidazole groups. This exchange rate increased significantly in peptides with bulky R-groups (phenylalanine) between the cysteine and histidine. A reduction of the cysteine thiol pKa and NMR results further supports closer proximity of the thiol and imidazole groups in peptides with faster acetyl group exchange.

A novel and automated assay for thiol/disulphide homeostasis

ObjectivesTo develop a novel and automated assay determining plasma thiol/disulphide homeostasis, which consists of thiol–disulphide exchanges. Design and methodsNative thiol and total thiol concentrations were synchronously measured as a paired test. In the first vessel, the amount of native thiol groups was measured by a modified Ellman reagent. At the parallel run, first, dynamic disulphide bonds were reduced to free thiol groups by NaBH4. The unused reductant remnants were completely removed by formaldehyde. Thus, the total thiol amount could be accurately measured. Mercaptoethanol solutions were used as calibrators. The half value of the difference between total thiol and native thiol amounts gave the disulphide bond amount. ResultsNo separation step for the assay was needed. All processes were performed using an automated analyser within about 10min. Plasma disulphide levels were 17.29±5.32μmol/L, native thiol levels were 397±62μmol/L and disulphide/native thiol per cent ratios were 4.32±1.49 in healthy subjects. Plasma disulphide levels were higher in patients with degenerative diseases and lower in patients with proliferative diseases. ConclusionAn easy, inexpensive, practical, fully automated and also optionally manual spectrophotometric assay can be used to determine plasma dynamic thiol/disulphide homeostasis.

Preparation and characterization of functionalized cellulose nanocrystals

In this work, a series of functional nanocrystals (F-CNCs) was successfully produced by an efficient preparation method, combining acid hydrolysis and Fischer esterification with various organic acids. Functionalities such as ATRP initiators, double bonds, triple bonds, and thiols could be incorporated on CNCs. Surface modification was confirmed by FT-IR, XPS, and elemental analysis. Physical properties of F-CNCs were assessed by AFM, XRD and TGA. Moreover, ATRP initiator functionalized CNCs were utilized to graft poly(methyl methacrylate) via ATRP, thiol functionalized CNCs were reacted with Ellman's reagent to determine the thiol content and dye disperse red 13 was attached to alkyne functionalized CNCs to estimate the propiolate content. The herein presented method is a highly versatile and straightforward procedure for the preparation of F-CNCs which is believed to be a better alternative for the commonly utilized, extensive, multistep, and time consuming post functionalization methods.

Changes in protein thiols in response to salt stress in embryogenic suspension cultures of Dactylis glomerata L.

The aim of the present study is to assess the rate of protein disulphide formation and the activity of NADPH-dependent thioredoxin and glutaredoxin systems, responsible for the reverse reduction of protein and mixed protein-glutathione disulphides, in embryogenic suspension cultures of Dactylis glomerata, subjected to salt stress. Two concentrations of NaCl previously established as enhancing (0.085 mol/L) and inhibiting (0.17 mol/L) somatic embryogenesis were used. The quantitative (by colour reaction with Ellman's reagent) and qualitative (by diagonal gel electrophoresis) analyses showed a significant increase in protein disulphide formation in salt-treated cultures compared to controls. The ratio of disulphides to free thiols is higher in 0.17 mol/L NaCl-treated cultures. The activity of the thioredoxin–thioredoxin reductase system has been increased accordingly in 0.085 mol/L NaCl-treated cultures but decreased at the higher salt concentration. The activity of glutaredoxins was also estimated, by using glutathionylated bovine serum albumin as substrate and following the decrease of NADPH absorbance at 340 nm in the presence of glutathione and glutathione reductase. Mild salt (0.085 mol/L NaCl) treated cultures again showed the highest activity compared to controls and 0.17 mol/L NaCl-treated cultures. Based on these observations it was suggested that salt treatment resulted in increased protein disulphide formation and thioredoxin and glutaredoxin systems are important regulators of this process, strongly involved in salt stress response. The highest activity at 0.085 mol/L NaCl may be also related to the regulatory mechanisms, involved in the potentiating of somatic embryogenesis at this salt concentration.

A chromogenic assay of substrate depletion by thiol dioxygenases

A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5′-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman’s reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.

A moderate protective effect of quercetin against γ-irradiation- and storage-induced oxidative damage in red blood cells for transfusion

Purpose: To investigate the extent of γ-irradiation-induced oxidative membrane damage and antioxidant activity of quercetin in long-term, cold stored (4°C) acid-citrate-dextrose- preserved human red blood cells (RBC).Materials and methods: The extracellular activity of lactate dehydrogenase (LDH) was measured to assess RBC membrane integrity. Lipid peroxidation and reduced glutathione (GSH) levels were quantified by thiobarbituric acid-reactive substances (TBARS) and Ellman's reagent, respectively.Results: During storage of non-irradiated RBC (up 21 days) the LDH activity in the supernatant increased with time. In contrast to a low dose of ionizing radiation (30 Gy), irradiation at higher, but still clinically relevant doses, of 40–50 Gy resulted in elevation of the post-storage extracellular LDH activity. Quercetin (2–50 μM) dissolved in dimethyl sulfoxide (DMSO) significantly increased the LDH release in the irradiated and non-irradiated RBC, reflecting an increase of RBC membrane permeability. In the presence of ethanol as a solvent quercetin protected RBC against storage-induced oxidative damage – it inhibited the LDH release, GSH depletion, and lipid peroxidation.Conclusion: The level of protection offered by quercetin against the radiation- and storage-induced oxidative damage to RBC does not seem to be sufficient to warrant its application as an additive for conservation purposes. The findings indicate that the solvent can modulate a response of RBC to water-insoluble antioxidants changing their properties from anti-oxidative to pro-oxidative.

Impurity profiling of carbocisteine by HPLC-CAD, qNMR and UV/vis spectroscopy

For the impurity profiling of the mucolytic and anti-inflammatory drug carbocisteine a high performance liquid chromatographic (HPLC) method using corona charged aerosol detection (CAD) was developed and fully validated following the ICH guideline Q2(R1). The response was linear (R2>0.995) over a small concentration range (0.05–0.25 or 0.10–0.60% respectively) and a detection limit of at least 0.03% was registered. The separation was achieved on a mixed mode column combining hydrophobic C18 and strong cation exchange retention mechanisms using a mass spectrometer compatible volatile mobile phase consisting of trifluoroacetic acid 10mM and acetonitrile 12% (V/V). Impurities, not assessable by HPLC-CAD such as the volatile chloroacetic acid and the unstable cysteine, were determined by quantitative NMR (qNMR) with maleic acid as internal standard and UV/vis spectroscopy after reaction with Ellman's reagent, respectively. Six batches of three different manufacturers were tested by means of those methods. The purity varied from below 99.0 to higher than 99.8 per cent. The major impurities of all batches were the starting material cystine and N,S-dicarboxymethylcysteine being a synthesis by-product.