Milligram amount determination of thiol groups with ferricyanide. Use of 2,6-dichlorophenolindophenol as titrant

Summary Thiols are reacted at pH 7 with an excess of ferricyanide. The excess of reagent is determined by adding a measured excess of ascorbic acid followed by back titration of ascorbic acid with 2,6-dichlorophenolindophenol. A correction can be made for ascorbic acid, if present with thiols. Tested with dilute solutions of cysteine, glutathione, mercaptoacetic acid and some other water-soluble thiols gave results which are accurate to 0.1%. Glycine, cystine, methionine, etc. do not interfere.

---

Zusammenfassung Thiole reagieren bei pH 7 mit überschüssigem Cyanoferrat (III). Der Überschuß läßt sich durch Zusatz einer gemessenen Menge Ascorbinsäure und deren Rücktitration mit 2,6-Dichlorphenolindophenol bestimmen. Ist neben Thiol bereits Ascorbinsäure zugegen, so ist eine entsprechende Korrektur anzubringen. Die Anwendung auf Lösungen von Cystein, Glutathion, Mercaptoessigsäure und einigen anderen wasserlöslichen Thiolen erbrachte auf 0,1% genaue Ergebnisse. Glycin, Cystin, Methionin usw. stören nicht.

     

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.     

Reaction of gallium and indium with 4-(2-Thiazolylazo) resorcinol

Gallium is shown to react with TAR to form 1:1 and 1:2 Ga:TAR species, elucidation of the nature of which is complicated by hydrolysis. The species GaR(+) and GaHR(OH)(+), where H(2)R is the free reagent, occur at pH 2.7-4.8 for solutions that are equimolar or contain excess of metal ion; GaHR(2+) occurs at pH 2.13-2.86 with excess of metal ion. A 1:2 complex, probably GaR(2)(-), exists in 30% aqueous ethanol medium at pH approximately 5 if excess of ligand is present. The utility of the reaction for analysis is severely limited by the large number of interfering elements. Indium reacts similarly, forming a In RH(2+) complex which is converted into the 1:2 complex by excess of reagent at pH 4.6.     

Facile and sensitive method for the determination of mesna in plasma by high-performance liquid chromatography with ultraviolet detection

The use of 2-chloro-1-methylquinolinium tetrafluoroborate, an ultraviolet tagging reagent, for the ion-pair, reversed-phase high-performance liquid chromatography of mesna in human plasma is reported. In order to achieve this objective optimization of the two-step procedure, derivatization and separation of mesna S-quinolinium derivative from that of other thiols present in plasma and internal standard, was investigated. The derivatization was optimized in terms of pH, reagent excess and time of the reaction, and the mobile phase in terms of ion-pairing reagent concentration, pH, organic modifier content and temperature. Baseline separation was achieved on an analytical Waters Nova-Pak C18 (150x3.9 mm, 5 microm) column with a mobile phase consisting of pH 2.3 0.05 M trichloroacetic acid-acetonitrile (89:11, v/v) pumped at 1.2 ml/min. The peak height ratios of the mesna derivative to that of the internal standard (thiomalic acid) varied linearly with the concentration of the analyte added to normal plasma with a correlation coefficient of 0.9997. The lower limits of detection and quantitation were 40 pmol/ml (0.8 pmol on-column) and 160 pmol/ml (3.2 pmol on-column), respectively. The intra-run imprecision and inaccuracy were from 1.3 to 2.4 and from 1.3 to 2.0%, respectively.     

A CE‐LIF method based on long wavelength fluorescence labeling for the analysis of thiols in human urine

A rapid and robust CE method using a long wavelength fluorescent reagent 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(2‐maleimide)difluoroboradiaza‐s‐indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N‐acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4–105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.     

Existence of low-molecular-weight thiols in Caenorhabditis elegans demonstrated by HPLC-fluorescene detection utilizing 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide

A highly sensitive and simple method using HPLC-fluorescence detection with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a fluorogenic reagent demonstrated the existence of the low-molecular-weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD-Cl at 40 degrees C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 +/- 15 nm with excitation at 400 +/- 15 nm. The identification of the thiols was made by HPLC-electrospray ionization mass spectrometry (LC-MS) following isolation of the derivatives using HPLC-fluorescence detection. Low-molecular-weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.     

A continuous two-phase reaction system coupled on-line with capillary chromatography for the determination of polar solutes in water

Gas chromatographic procedures are described for the determination of carboxylic acids and chlorinated anilines in water samples. Propionic acid and 2,6-difluorobenzoic acid in aqueous solution have been simultaneously alkylated and extracted by means of a continuous two-phase reaction system, and then quantitated by on-line coupled capillary gas chromatography; tetrahexyl-ammonium hydrogen sulfate was used as phase transfer catalyst and pentafluorobenzyl bromide as reagent. A factorial design approach was used to optimize on-line derivatization of aqueous propionic acid with regard to pH and concentration of phase transfer catalyst. Alkylation and extraction, under optimized conditions, followed by quantitation of the pentafluorobenzyl ester by flame ionization detection furnished a linear calibration for concentrations between 0.1 and 10 μg/ml. The relative standard deviation was 9–15 %. The continuous two-phase reaction system was also used to determine (chlorinated) anilines present in water at concentrations of 0.1–1 μg/ml; pentafluorobenzoyl chloride was used as reagent and analysis was performed by capillary gas chromatography with flame ionization or electron capture detection. The on-line acylation of p-chloroaniline was optimized with regard to pH, reagent concentration, and reaction time. The on-line reaction system worked satisfactorily for both applications, although excess reagent caused some problems with the chromatography.     

Automated high performance liquid chromatography with on-line reduction of disulfides and chemiluminescence detection for determination of thiols and disulfides in biological fluids

In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4 nM and the linear range in a log–log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.     

5-(2-Aminoethyl)dithio-2-nitrobenzoate as a more base-stable alternative to Ellman's reagent

[reaction: see text] 5-(2-Aminoethyl)dithio-2-nitrobenzoate (ADNB) reacts with free thiols with kinetics similar to those of Ellman's reagent but has dramatically improved stability under alkaline conditions, making it an excellent alternative to Ellman's reagent for the quantitation of thiol contents and enzymatic assays under basic pH conditions.     

Titrimetric determination of thiols: Tetrathionate, iron(III), cystine and hexacyanoferrate(III) as thiol reagents

Four analytical reagents, tetrathionate, iron(III), cystine and hexacyanoferrate(III) have been tested with respect to their specificity for oxidation of thiols to disulphides. Of a number of thiols studied, most have a strong tendency to oxidize beyond the disulphide stage with several of the commonly employed reagents. Tetrathionate, cystine and hexacyanoferrate(III) function in phosphate buffer of pH 7, but iron(III) does not require rigid control of pH, although the solution should be only feebly acidic. The reagents were used in excess and the thiosulphate or cysteine formed in the reaction of thiols with tetrathionate or cystine respectively was determined. The residual iron(III) was measured by adding ascorbic acid or mercaptoacetic acid and titrating with 2,6-dichlorophenolindophenol or iodine monochloride respectively; surplus hexacyanoferrate(III) was back-titrated with ascorbic acid. All four reagents react selectively with thiols even in the presence of several possible interfering substances and afford results that are accurate and precise.     

Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection

The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H₃PO₄/CH₃CN (96:4) to 0.15 M H₃PO₄/CH₃CN (85:15) over 30 min followed by isocratic elution with 0.15 M H₃PO₄/CH₃CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g^-1) and cystine (0.62 μmol g^-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g^-1) was observed in liver, spleen, heart and testicle.     

Determination of sulphur functions with N-iodosuccinimide

Titrimetric determination of thioureas, thiols, xanthates and dithiocarbamates with N-iodosuccinimide (NIS) is described. The method for xanthate can be applied to carbon disulphide (converted into xanthate with potassium ethoxide). Acidic and non-aqueous solutions of the oxidizing agent are stable. The procedures are rapid and accurate to 0.1% with a precision of 0.2%. Hydrogen sulphide and thiocarbonyl compounds interfere. The behaviour of N-bromosuccinimide and NIS with thiols in aqueous medium is compared. It is shown that iodine is the oxidizing species in both cases. The limitations of iodine as a reagent for thiol determination are discussed. Cysteine, which cannot be determined with iodine, can be determined with NIS. The role of methanol in non-aqueous determination of thiols is discussed. Methanol accelerates the oxidation, which is otherwise slow in acetonitrile medium.     

Allyl tetrahydropyranyl ether: a versatile alcohol/thiol protecting reagent

Allyl tetrahydropyranyl ether (ATHPE) can be used as a versatile protecting reagent. In combination with NBS/I₂, O-allyl group can easily be replaced by hydroxyls (including tertiary-OH) or thiols, in the molecules comprising other reactive functional groups such as halogen, nitro, acetonide and alkene under mild reaction conditions (near neutral pH and ambient temperature).     

2,6-Dicarboxypyridinium chlorochromate: an efficient and selective reagent for the oxidation of thiols to disulfides and sulfides to sulfoxides

2,6-Dicarboxypyridinium chlorochromate (2,6-DCPCC) was found to be an efficient reagent for the conversion of thiols to disulfides and sulfides to sulfoxides under neutral and anhydrous conditions in good to excellent yields. Selective oxidation of thiols in the presence of sulfides at room temperature is also observed with this reagent.     

Synthesis and Transformations of 5‐Chloro‐2,2′‐Dipyrrins and Their Boron Complexes, 8‐Chloro‐BODIPYs

Symmetric dipyrrylketones 1 a , b were synthesized in two steps from the corresponding α‐free pyrroles, by reaction with thiophosgene followed by oxidative hydrolysis under basic conditions. The dipyrrylketones produced the corresponding 5‐chloro‐dipyrrinium salts or 5‐ethoxy‐dipyrrins on reaction with phosgene or Meerwein’s salt, respectively. Boron complexation of the dipyrrins afforded the corresponding 8‐functionalized BODIPYs (borondipyrromethenes) in high yields. The 5‐chloro‐dipyrrinium salts reacted with methoxide or ethoxide ions to produce monopyrrole esters, presumably via a 5,5‐dialkoxy‐dipyrromethane intermediate. In contrast, 8‐chloro‐BODIPYs underwent a variety of nucleophilic substitutions of the chloro group in the presence of alkoxide ions, Grignard reagents, and thiols. In the presence of excess alkoxide or Grignard reagent, at room temperature or above, substitution at the boron center also occurred. The 8‐chloro‐BODIPY was a particularly useful reagent for the preparation of 8‐aryl‐, 8‐alkyl‐, and 8‐vinyl‐substituted BODIPYs in very high yields, using Pd⁰‐catalyzed Stille cross‐coupling reactions. The X‐ray structures of eleven BODIPYs and two pyrroles are presented, and the spectroscopic properties of the synthesized BODIPYs are discussed.     

Extraction and spectrophotometric determination of copper with thiobenzoylacetone (3-mercapto-1-phenylbut-2-en-1-one)

The copper(II)-thiobenzoylacetone complex, extracted into benzene, is used for photometric determination of copper at 410 nm after excess of reagent has been removed by scrubbing with a buffer at pH12. The extraction is quantitative over a broad pH range (3.5-9.5). The method is highly sensitive for the determination of copper.     

Argentimetric assay of captopril in bulk drug and in tablets

Three simple, selective and cost-effective procedures for the determination of captopril in bulk drug and in tablets are described. All the procedures make use of silver nitrate as a reagent and involve titrimetry and spectrophotometry as measurement techniques. In titrimetry (Method A), the aqueous solution of the drug is titrated directly with the standard silver nitrate solution to a potassium chromate end-point. In one spectrophotometric method (Method B), the sample solution is treated with excess of silver nitrate and a known amount of methyl orange and the increase in absorbance at 520 nm, caused by a decrease in pH due to release of nitric acid, is measured and related to drug concentration. The other spectrophotometric method (Method C) involves the addition of a measured excess of silver nitrate to the sample solution followed by the determination of residual silver ion by an ion-associate complex formation reaction involving eosin and 1,10-phenanthroline. The decrease in absorbance at 550 nm, which corresponds to Ag⁺ reacted with the drug, is measured and is found to be linearly related to drug concentration. All experimental variables involved in the methods were investigated and optimized. Stoichiometry of the reaction that forms the basis for titrimetry is found. Method A is applicable in the range of 1.0–20.0 mg of drug while methods B and C can be conveniently used in the concentration ranges of 2.5–50.0 and 0.25–4.0 μg ml^?1, respectively. Several optical characteristics such as molar absorptivity, Sandell sensitivity, limits of detection and quantification, and correlation coefficient were calculated. The methods were applied to the analysis of tablets containing captopril. Statistical treatment of the results indicates that the procedures are precise and accurate. The excipients used as additives in tablets did not interfere in the proposed procedures as revealed by the recovery studies.     

Spectrophotometric study of the chelate of palladium(II) with methylthymol blue

Methylthymol blue reacts very sensitively with palladium(II) to form two complexes. The 1:1 complex has λ_max at 530 nm in acidity range 0.02 to 0.05 M with respect to perchloric acid and 1:2 (metal reagent ratio) complex at 500 nm in pH range 6.8 to 7.5. Optimum conditions including the range for adherence to Beer's law, effect of pH, effect of excess reagent and sensitivity are reported for the photometric determination of palladium using methylthymol blue.     

High-load, ROMP-generated oligomeric bis-acid chlorides: design of soluble and insoluble nucleophile scavengers

[reaction: see text] An efficient strategy for scavenging a host of nucleophiles utilizing an oligomeric bis-acid chloride (OBAC), generated from the ROM polymerization of trans-bicyclo[2.2.1]hept-5-ene-2,3-dicarbonyl dichloride, is described. The reactivity and high load of the OBAC reagent is exploited in the scavenging of amines, alcohols, and thiols that are present in excess following a common benzoylation event. Following the scavenging event, these oligomers can be precipitated with EtOAc and filtered (SiO(2)), leaving benzoylated nucleophiles in excellent yield and purity.     

Reactive ligands in radical processes catalyzed by copper complexes

The effect of the reactivity of donor ligands on the catalytic properties of copper complexes in the oxidative dimerization of mercaptans is considered. Catalytic compositions containing metal complexes in an excess of organic reagent ligands, which can show pronounced reductive properties (aromatic amines) or, on the contrary, oxidative properties (dimethyl sulfoxide) toward substrates, exhibit the greatest activity. In the course of the oxidation of mercaptans catalyzed by copper complexes, redox reactions accompanied by not only a change in the oxidation state of the metal but also the direct interaction of a substrate with an organic donor occur. In the presence of aromatic amines, the coupled oxidation of thiols and amines occurs, whereas dimethyl sulfoxide participates in the reaction as an oxidizing agent.