Determination of homocysteine, penicillamine, and their symmetrical and mixed disulfides by liquid chromatography with electrochemical detection

A method for the determination of D-penicillamine, homocysteine, homocystine, penicillamine-homocysteine mixed disulfide, and penicillamine disulfide in human plasma and urine is described. The method involves separation of the various thiols and disulfides by high-performance liquid chromatography with detection by a dual Hg/Au amalgam electrochemical detector. D-Penicillamine and homocysteine are detected at the downstream electrode; the disulfides are first reduced to thiols at the upstream electrode and then the thiols are detected at the downstream electrode. Hydrodynamic voltammograms were measured for the various thiols and disulfides to determine optimum settings for the electrochemical detector, and the effect of mobile phase parameters on retention times was studied to optimize the separation. A convenient method for the preparation of calibration solutions of penicillamine-homocysteine mixed disulfide by thiol/disulfide exchange with standardization of the solution by H NMR spectroscopy is described. Detection limits are below the concentrations of homocystine and penicillamine-homocysteine mixed disulfide reported to be present in the plasma and urine, respectively, of homocystinuric patients under treatment with D-penicillamine.     

Antimicrobial garlic-derived diallyl polysulfanes: Interactions with biological thiols in Bacillus subtilis

BackgroundDiallylpolysulfanes are the key constituents of garlic oils, known to exhibit broad spectrum anticancer and antimicrobial activity. Studies in vitro, and in mammalian cells, have shown they react, via thiol-polysulfane exchange, with their major low molecular weight thiol, glutathione. However, there are no detailed reports of diallylpolysulfane effects on other common thiol metabolites (cysteine and coenzyme A) or major thiol cofactors (e.g. bacillithiol) that many Gram positive bacteria produce instead of glutathione.MethodsDiallylpolysulfanes were individually purified then screened for antimicrobial activity against Bacillus subtilis. Their impact on thiol metabolites (bacillithiol, cysteine, coenzyme A, protein thiols allyl thiols//persulfides) in B. subtilis cultures were analysed, by HPLC.ResultsDiallylpolysulfane bioactivity increased with increasing chain length up to diallyltetrasulfane, but then plateaued. Within two minutes of treating B. subtilis with diallyltrisulfane or diallyltetrasulfane intracellular bacillithiol levels decreased by ~90%. Cysteine and CoA were also affected but to a lesser degree. This was accompanied by the accumulation of allyl thiol and allyl persulfide. A significant level of protein-S-allylation was also detected.ConclusionsIn addition to the major low molecular weight thiol, diallylpolysulfanes can also have an impact on other thiol metabolites and protein thiols.General significanceThis study shows the rapid parallel impact of polysulfanes on different biological thiols inside Bacillus subtilis alongside the concomitant generation of allyl thiols and persulfides.     

Selective extraction of quercetrin in vegetable drugs and urine by off-line coupling of boronic acid affinity chromatography and high-performance liquid chromatography

Quercetrin, quercetin and chlorogenic acid were measured in urine or in drugs by combination of boronic acid affinity chromatography and HPLC. Simple reversed-phase HPLC with UV detection was used to determine quercetrin in five different Solidago virgaurea drugs. For determination of quercetrin in human urine immobilized boronic acid was applied for sample pretreatment. This procedure leads to a determination limit of 0.01 μg/ml with a recovery rate of 95.3%. The first results using this method for quercetrin pharmacokinetics are presented.     

Separation of urinary thiols as tributyltinmercaptides and determination using capillary isotachophoresis

The essential steps in the assay included electrolytic reduction of disulphides, neutralization, extraction of thiols with 0.1 M tributyltin hydroxide in octane, stripping of the extract with 2% acetic acid, fixing the washed-out amino thiols to a cation exchanger, elution with 2 M hydrochloric acid, oxidation with bromine and evaporation. The remaining octane extract was decomposed by dodecanethiol, the mercapto acids were washed out, oxidized with bromine and evaporated. Both residues were dissolved in water and analysed using capillary isotachophoresis at pH 3.0. Cysteamine was extracted from reduced urine at ca. pH 10, decomposed by dodecanethiol and re-extracted to boric acid followed by determination as a cation. The presence of the following thiols in urine has been confirmed: mercaptoacetic acid, 3-mercaptolactic acid, 2-mercaptopropionic acid, acetylcysteine, mercaptoethanol, cysteine, homocysteine and an un-identified amino thiol. Cysteamine and 3-mercaptopropionic acid could not be detected. Captopril, homocysteine and cysteine were determined quantitatively.     

Measurement of cyst(e)amine in physiological samples by high performance liquid chromatography

Two methods for measurement of cyst(e)amine in physiological samples are described. One method involves reduction of disulfides present in the sample with tributylphosphine, reversed phase chromatography of thiols, and electrochemical detection of cysteamine and other thiols. The other method involves reduction of disulfides with dithiothreitol, derivatization of thiols with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, separation of these derivatives by reversed phase chromatography, and fluorometric detection of the thiol adducts. The endogenous concentration of cysteamine in rat liver was estimated to be less than 2.5 nmol/g. Cysteamine is produced in tissues postmortem; rapid sampling/freezing of tissues and rapid inactivation of enzymes during tissue preparation are essential for accurate measurement of endogenous cysteamine concentrations.     

Simple and rapid determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene

A rapid and simple background-free high-performance liquid chromatographic (HPLC) approach has been developed for simultaneously determining free thiol compounds including coenzyme A (CoA), cysteine (Cys), glutathione (GSH) and N-acetyl-cysteine (NAC) in biological samples by using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene (TMPAB-o-M) as fluorogenic reagent. After derivatization under physiological conditions within 6 min, baseline separation was finished in just 6 min using isocratic elution with reversed-phase HPLC and fluorescence detection. Excellent linearity was observed for all analytes over their concentration ranges of 1-500 nM and detection limits ranging 0.13 nM for CoA to 0.25 nM for Cys (S/N=3) were achieved. The utility of the proposed method has been validated by measuring thiol compounds mentioned above in tissue, fluid and cell samples. The results indicated that this approach was well suited for high-throughput quantitative determination of thiols and study of the physiological role of them.     

para-sulfobenzoyloxybromobimane: a new membrane-impermeable reagent useful for the analysis of thiols and their export from cells.

para-Sulfonylbenzoyloxybromobimane (sBBr) was shown to be similar to the fluorescent labeling agent monobromobimane (mBBr) in reacting rapidly and selectively with thiols to produce stable derivatives which are readily separated by HPLC. Chromatography of the sBBr derivative provides a useful means of confirming the identification of an unknown thiol based upon the chromatography of its mBBr derivative and can be useful for quantitative determination of polycationic thiols for which chromatography of the mBBr derivative is unsatisfactory. Unlike mBBr, which readily penetrates cells, sBBr was found not to be taken up by cells. These characteristics allow sBBr to be used, in conjunction with mBBr, to quantify the export of thiols from cells, as illustrated for GSH and the radioprotective drug WR1065, from V79 cells. Simultaneous determination of GSH and glutathione disulfides in cell culture medium could be achieved by labeling of thiols with sBBr followed by reduction of disulfides with dithiothreitol, labeling of the resulting thiols with mBBr, and HPLC analysis for both glutathione derivatives.     

Determination of cellular thiols and glutathione-related enzyme activities: versatility of high-performance liquid chromatography–spectrofluorimetric detection

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, γ-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and γ-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.     

A flow cytometric assay for intracellular nonprotein thiols using mercury orange

The level of nonprotein thiols was assayed in individual mammalian cells using flow cytometry. Previous determinations of glutathione (GSH, the most abundant nonprotein thiol in most cells) by flow cytometry were based on UV laser excitation of fluorochromes. Because of several shortcomings of UV excitation, an assay for GSH using visible light is of interest. Selective staining of nonprotein thiols with mercury orange (a mercurial compound that binds stoichiometrically to sulfhydryl groups) was obtained by restricting the staining time. By using various drugs that affect GSH levels and overall thiol levels in cells, it was shown that GSH is the primary thiol group being stained. Thus a quick, specific technique using mercury orange has been developed for the flow cytometric determination of nonprotein thiols and preferentially for GSH in individual mammalian cells.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

Determination of amphetamine in human urine by dansyl derivatization and high-performance liquid chromatography with fluorescence detection

A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated. The stability of amphetamine-dansyl chloride in different storage conditions was examined. The detection limit and linearity associated with this assay are discussed.     

Sensitive high-performance liquid chromatographic method for the determination of 2-phenylethylamine in human urine

A new sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of 2-phenylethylamine (PEA) in human urine. The analytical procedure involved a simple extraction of the analyte from urine, followed by precolumn derivatisation of the sample with o-phthalaldehyde. The HPLC separation was performed under isocratic conditions using an Erbasil S C₁₈ (250 × 4.0 mm I.D., particle size 3 μm) reversed-phase column. The limit of quantification was 0.5 ng of PEA/ml of urine. The method showed good linearity, accuracy and precision data in the concentration range 0.5–200 ng/ml of urine. The method was successfully applied to the determination of PEA urinary excretion in Parkinsonian patients after oral administration of the monoamine oxidase B (MAO-B) inhibitor, selegiline.     

Reaction of thiols with 7-methylbenzopentathiepin

Polysulfides typically react readily with thiols, thus, reactions of endogenous cellular thiols with the polysulfide linkage in naturally-occuring pentathiepin cytotoxins are likely to be an important aspect of their biological chemistry. Here, it is reported that the reaction of thiols with the pentathiepin ring system initially produces a complex mixture of polysulfides that further decomposes in the presence of excess thiol to yield the corresponding 1,2-benzenedithiol with concomitant production of H(2)S and dimerized thiol. In this reaction, a single molecule of the pentathiepin consumes approximately six equivalents of thiol. The reaction of thiols with the pentathiepin ring system is faster than the analogous reaction involving typical di- and trisulfides.     

Analysis of biological thiols: derivatization with monobromotrimethylammoniobimane and characterization by electrophoresis and chromatography

A new method for analysis of biological thiols based upon their conversion to fluorescent derivatives by reaction with monobromotrimethylammoniobimane (qBBr) is described. The derivatives are separated by chromatography and by electrophoresis on cellulose thinlayer chromatography plates. The use of two-dimensional mapping makes it possible to differentiate between a wide variety of biological thiols including N-acetylcysteine, CoA, cysteine, cysteinylglycine, cysteamine, ergothioneine, glutathione, γ-glutamylcysteine, homocysteine, mercaptopyrimidine, pantetheine, 4′-phosphopanetheine, thiosulfate, and thiouracil. For applications to biological samples thiols were isolated from crude extracts by binding to a mercuriagarose gel. Following removal from the gel with dithiothreitol, the thiols were derivatized with qBBr. The methods were tested by showing that glutathione is the major thiol in human red blood cells, that glutathione and ergothioneine are the major thiols in Neurospora crassa conidia, and that Bacillus cereus vegetative cells lack glutathione but contain cysteine, pantetheine, and an unidentified thiol in significant amounts.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.     

Recent advances in separation and detection methods for thiol compounds in biological samples

Biological aminothiols, such as cysteine, homocysteine, and glutathione, widely occur in animal tissues and fluids. The altered levels of the thiols (reduced forms) and their disulfides (oxidized forms) in physiological liquids have been linked to specific pathological conditions and closely associated with several human diseases. Therefore, it is well recognized that the determination of thiols and disufides is important in order to understand their physiological roles. The derivatization utilizing a suitable labeling reagent followed by chromatographic separation and detection is the most reliable means for sensitive and selective assays. Many reagents have typically been synthesized and successfully used for the determination of thiols and disulfides in biological specimens. The development of new reagents for highly sensitive detection is still continuing. This review describes the approaches for the separation assay of various thiol compounds, obtained through the analytical papers published in 2000–2008. The derivatization reagents are categorized with each type of chromophore and fluorophore and evaluated in terms of their reactivity, stability, detection wavelength, handling, sensitivity and selectivity. Application examples of the reagents for bioanalysis are also described in the text.     

Screening of Thiol Compounds: Depolarization-Induced Release of Glutathione and Cysteine from Rat Brain Slices

Superfusates from rat brain slices were screened for thiol compounds after derivatization with monobromobimane by reversed-phase HPLC. Only glutathione and cysteine were detected. The Ca(2+)-dependent release of these compounds from slices of different regions of rat brain was investigated, applying a highly sensitive and reproducible quantification method, based on reduction of superfusates with dithiothreitol, reaction of thiols with iodoacetic acid, precolumn derivatization with o-phthalaldehyde reagent solution, and analysis with reversed-phase HPLC. This methodology allowed determination of reduced and total thiols in aliquots of the same superfusates. Mostly reduced glutathione and cysteine were released upon K+ depolarization and the Ca2+ dependency suggests that they originate from a neuronal compartment. The GSH release was most prominent in the mesodiencephalon, cortex, hippocampus, and striatum and lowest in the pons-medulla and cerebellum. This underscores a physiologically significant role for glutathione in CNS neurotransmission.     

Discriminating formation of HNO from other reactive nitrogen oxide species

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GSONH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GSONH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO(-)),indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GSONH2. Detection of GSONH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.     

The determination of S-nitrosothiols in biological samples--procedures, problems and precautions

Despite the considerable number of published studies in the field of S-nitrosothiols (RSNO), the determination of these compounds in biological samples still represents an analytical challenge, due to several technical obstacles and often long sample preparation procedures. Other problems derive from the intrinsic lability of RSNO and the absence of certified reference material, analytically validated methods or suitable internal standards. Also, thiols and nitrites are usually present at high concentrations in biological matrices, and all precautions must be adopted in order to prevent artifactual formation of RSNO. Preanalytical steps (sampling, preservation and pre-treatment of samples) are particularly critical for the obtainment of reliable measurements. Three main mechanisms have been identified capable of compromising the assays: metal-catalyzed RSNO decomposition, reduction of the S-NO bond by thiols (transnitrosylation reactions) and enzymatic degradation of S-nitroso-glutathione (GSNO) by endogenous γ-glutamyltransferase (GGT) activity possibly present in the sample. If not adequately controlled, these factors likely contribute to the wide dispersion of values reported in the literature for RSNO and GSNO concentration in biological fluids, blood in the first place. The use of metal chelators, thiol reagents and GGT inhibitors appears therefore mandatory.