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Hydrogen sulfide (H 2 S) plays major functional and structural roles in diverse physiological functions and the pathogenesis of a variety of disorders in biological matrices. The significance of H 2 S has prompted the development of sensitive and selective methods to determine its concentration in biological samples. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation. MBB may prevent the oxidation of sulfide and the reaction of sulfide with several different species (such as superoxide radicals, hydrogen peroxide and peroxynitrite). An isomers of MBB, 3-(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1,2-a] pyrazole-1, 5-dione (MMB), is cheaper than MBB and its use in the analysis of H 2 S has not previously been reported. In the present study, we compared the derivatization reactions of hydrogen sulfide with MMB and MBB and developed a sensitive method to quantify H 2 S in blood. In our method, H 2 S was incubated in the dark with excess MMB in 0.1 M Tris-HCl buffer (pH 10.1) at 50 °C for 120 min. 50 μL aliquots of the derivatized product were analyzed using HPLC system with gradient elution of 0.1% ( v/v ) formic acid-acetonitrile. The limit of detection for the derivatized product was 0.03 nmol/mL. The derivatization reaction was suitable for detecting low concentrations of H 2 S. The derivate product is stable over time, permitting batch storage and analysis. [ABSTRACT FROM AUTHOR]

The organic sulfur compound 3-mercaptopropionic acid (3-MPA) is an important thiol intermediate in organic sulfur metabolism in natural environments. It is generated during degradation of sulfur-containing amino acids (e.g. methionine) and from demethylation of dimethylsulfoniopropionate (DMSP). This pathway is an alternative enzymatic process in the DMSP catabolism that routes sulfur away from the climatically-active dimethyl sulfide (DMS). 3-MPA detection and subsequent quantification in different matrices is difficult due to its extreme reactivity. We therefore developed a sensitive method for determination of 3-MPA based on pre-column derivatization with monobromobimane and analysis by high-performance liquid chromatography (HPLC) with fluorescence detection. This methodology was first tested with 3-MPA standards under low (0.005–0.2 μmol L −1 ) and high (1–25 μmol L −1 ) concentrations. For the optimization of the reaction, CHES and, alternatively, Tris–HCl buffers were evaluated in the derivatization step, with Tris–HCl showing more effective separation of thiol derivatives and a better 3-MPA peak shape. The detection limit was 4.3 nmol L −1 with a 10 μL sample injection, and mean recoveries of 3-MPA ranged from 97 to 105% in estuarine waters with different salinities (0.17 and 35.9 ppt). The linearity ( r > 0.99) and repeatability of detector response, with intra- and inter-day precision (% CV) of 2.68–7.01% and 4.86–12.5%, respectively, confirmed the reliability of the method. Previous 3-MPA analytical methods required immediate analysis due to unstable derivatives, but in this method we achieved high stability of the derivatized samples when stored at 4 °C, with only a 3–5% loss after more than one year of storage. This method was successfully applied to measure 3-MPA concentrations and rates of 3-MPA production in a variety of intertidal estuarine sediment slurries. Dissolved 3-MPA concentrations in these sediment slurries varied between 2 and 237 μmol L −1 and, 3-MPA net fluxes ranged in wet sediments between −3.6 ± 1.7 and 30 ± 5 μmol L −1 g −1 h −1 . Thus, the application of this optimized methodology showed an efficient performance for measuring 3-MPA in environmental samples, with a straightforward sample derivatization and a simple analysis of stable 3-MPA derivatives. [ABSTRACT FROM AUTHOR]

Abstract: Metal-binding thiols, involved in detoxification mechanisms in plant and other organism under heavy metal stress, are receiving more and more attentions, and various methods have been developed to determine related thiols such as cysteine (Cys), glutathione (GSH) and phytochelatins (PCs). In present study, an HPLC method was established for simultaneous determination of Cys GSH and PC2–6 after treatment with disulfide reductant of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and thiolyte reagent of monobromobimane (mBBr). The separation of thiol derivatives was performed on an Agilent Zorbax Eclipse XDB-C18 column (4.6mm×30mm, 1.8μm) with a linear gradient elution of 0.1% (v/v) trifluoroacetic acid (TFA)–acetonitrile (ACN) at 0.8mLmin−1. The temperature of the column was maintained at 25°C. The excitation and emission wavelengths were set at 380 and 470nm, respectively. The thiol derivatives were well separated in 19min, and the total analysis time was 30min. The established method was proved selective, specific and reproducible, and could be applicable to determine Cys, GSH and PC2–6 and to evaluate their roles in detoxification mechanisms in Cd-treated Lolium perenne L. under ambient and elevated carbon dioxide (CO2). It was found that the total SH contents and proportions of thiols in roots and shoots were dependent on Cd concentration, whereas the total SH contents decreased and the proportions of thiols altered without significance at elevated CO2 level. [Copyright &y& Elsevier]

Abstract: A simple, specific and sensitive derivatization with monobromobimane (mBrB) and the corresponding HPLC-fluorescence quantitation method for the analysis of bucillamine in human plasma was developed and validated. The analytical procedure involves a simple protein precipitation, pre-column fluorescence derivatization, and separation by reversed-phase high performance liquid chromatography (RP-HPLC). The calibration curve showed good linearity over a wide concentration range (50ng/mL to 10μg/mL) in human plasma (r 2 =0.9998). The lower limit of quantitation (LLOQ) was 50ng/mL. The average precision and accuracy at LLOQ were within 6.3% and 107.6%, respectively. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (300mg) of bucillamine to 20 healthy Korean volunteers. [Copyright &y& Elsevier]

Abstract: 2,3-Dimercaptosuccinic acid (DMSA) is an orally effective chelating agent for the treatment of heavy metal poisoning. The increasing therapeutic use of DMSA has stimulated the need for sensitive and selective methods for its determination in biological samples, as well as study on pharmacokinetics and tissue distribution. According to the previously reported method, an improved method was established for the determination of DMSA in mice blood and tissues, in which oxidized DMSA was reduced by the disulfide-reducing agent, dithiothreitol (DTT), and DMSA was converted to a highly fluorescent and stable derivative by reaction with monobromobimane (mBBr) in alkaline solution. Acetonitrile was used for deproteinization and dichloromethane was used for condensation and purification, which significantly shortened the amount of time used to process the sample. Meanwhile isocratical elution was performed and excellent separation of the DMSA derivative was obtained, this enabled a run finish within 20min. The limits of quantitation were 0.025μg/ml in brain and 0.1μg/ml in blood, lung, heart, intestine, liver, spleen and kidney, respectively. The calibration curves were linear in all samples (r 2 >0.992) with a range of 0.025–1.6μg/ml for brain homogenate and 0.1–6.4μg/ml for blood and homogenates of lung, heart, intestine, liver, spleen and kidney, respectively. Therefore, the method is simple, rapid and sensitive, and it could be applicable to the studies in an animal model to evaluate the distribution of DMSA in blood and tissues. [Copyright &y& Elsevier]