Search

(1) Background: Hydrogen sulfide (H2S) is a widely recognized gasotransmitter, with key roles in physiological and pathological processes. The accurate quantification of H2S and reactive sulfur species (RSS) may hold important implications for the diagnosis and prognosis of diseases. However, H2S species quantification in biological matrices is still a challenge. Among the sulfide detection methods, monobromobimane (MBB) derivatization coupled with reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most reported. However, it is characterized by a complex preparation and time-consuming process, which may alter the actual H2S level; moreover, a quantitative validation has still not been described. (2) Methods: We developed and validated an improved analytical protocol for the MBB RP-HPLC method. MBB concentration, temperature and sample handling were optimized, and the calibration method was validated using leave-one-out cross-validation and tested in a clinical setting. (3) Results: The method shows high sensitivity and allows the quantification of H2S species, with a limit of detection of 0.5 µM. Finally, it can be successfully applied in measurements of H2S levels in the serum of patients subjected to inhalation with vapors rich in H2S. (4) Conclusions: These data demonstrate that the proposed method is precise and reliable for measuring H2S species in biological matrices and can be used to provide key insights into the etiopathogenesis of several diseases and sulfur-based treatments. [ABSTRACT FROM AUTHOR]

Generally, volatile thiols are hard to be measured with electrospray-ionization-type LC-MS due to the volatility. Therefore, we here evaluated the pretreatment of their S -bimanyl derivatization by monobromobimane to enable the detection as nonvolatile derivative. Consequently, we successfully developed the convenient and efficient method through the quantitative analysis of 2-furanmethanethiol (volatile thiol odorant of coffee aroma) in coffee bean. [ABSTRACT FROM AUTHOR]

There are limited studies that report the physiological levels of H 2 S in the eye. The currently available UV/Vis methods lack the required sensitivity and precision. Hence, the purpose of this study was to develop and validate a sensitive and robust pre-column derivatization LC-MS/MS method to measure changes in H 2 S levels in tissues from isolated porcine eyes. H 2 S was derivatized and an LC-MS/MS method was developed to monitor the derivatized product, Sulfide-dibimane (Sdb) using a reverse phase Waters Acquity BEH C18 column (1.7 μm, 2.1 × 100 mm). H 2 S quantification was performed using multiple-ion reaction monitoring (MRM) in positive mode, with the transitions of m / z 415.0 → m / z 223.0 for Sdb and m / z 353.0 → m / z 285.0 for internal standard (griseofulvin). This method provided a suitable way to quantify H 2 S and was then successfully adapted to measure H 2 S levels in isolated porcine iris-ciliary body tissues previously treated in the presence or absence of varying concentrations of lipopolysaccharide (LPS, 5–100 ng/ml), a pro-inflammatory agent. Isolated iris-ciliary bodies (ICB) from porcine eyes were cut into quadrants of approximately 50 mg and homogenized using a 1:3 volume of homogenizing buffer. H 2 S in the supernatant was then derivatized with monobromobimane and quantified. [Display omitted] • Hydrogen sulfide (H 2 S) is an endogenous molecule involved in various physiological and pathological processes. • The currently available UV/Vis methods lack sensitivity and precision in quantifying concentrations of H 2 S in eye tissues. • We developed and validated an LC-MS/MS method through derivatization to quantify H 2 S concentrations in ocular samples. • Small and dynamic changes in H 2 S levels in iris-ciliary body samples from isolated porcine eyes using only 50 mg of tissue. • LC-MS/MS is a specific and sensitive method and opens opportunities to study the mechanisms of ocular diseases. [ABSTRACT FROM AUTHOR]

Biothiols are extremely powerful antioxidants that protect cells against the effects of oxidative stress. They are also considered relevant disease biomarkers, specifically risk factors for cardiovascular disease. In this paper, a new procedure for the simultaneous determination of human serum albumin and low-molecular-weight thiols in plasma is described. The method is based on the pre-column derivatization of analytes with a thiol-specific fluorescence labeling reagent, monobromobimane, followed by separation and quantification through reversed-phase high-performance liquid chromatography with fluorescence detection (excitation, 378 nm; emission, 492 nm). Prior to the derivatization step, the oxidized thiols are converted to their reduced forms by reductive cleavage with sodium borohydride. Linearity in the detector response for total thiols was observed in the following ranges: 1.76–30.0 mg mL−1 for human serum albumin, 0.29–5.0 nmol mL−1 for α-lipoic acid, 1.16–35 nmol mL−1 for glutathione, 9.83–450.0 nmol mL−1 for cysteine, 0.55–40.0 nmol mL−1 for homocysteine, 0.34–50.0 nmol mL−1 for N-acetyl-L-cysteine, and 1.45–45.0 nmol mL−1 for cysteinylglycine. Recovery values of 85.16–119.48% were recorded for all the analytes. The developed method is sensitive, repeatable, and linear within the expected ranges of total thiols. The devised procedure can be applied to plasma samples to monitor biochemical processes in various pathophysiological states.

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]

Abstract: The thiol-specific derivatization reagent monobromobimane (MBB) is applied – for the first time – under flow conditions. Sequential injection analysis allows the handling of precise volumes of the reagent in the micro-liter range. The effect of the main chemical and instrumental variables was investigated using captopril (CAP), N-acetylcysteine (NAC) and penicillamine (PEN) as representative pharmaceutically active thiols. Previously reported hydrolysis of MBB due to interaction with nucleophilic components of the buffers was avoided kinetically under flow conditions. The proposed analytical scheme is suitable for the fluorimetric determination of thiols at a sampling rate of 36h−1. [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]

Biothiols are extremely powerful antioxidants that protect cells against the effects of oxidative stress. They are also considered relevant disease biomarkers, specifically risk factors for cardiovascular disease. In this paper, a new procedure for the simultaneous determination of human serum albumin and low-molecular-weight thiols in plasma is described. The method is based on the pre-column derivatization of analytes with a thiol-specific fluorescence labeling reagent, monobromobimane, followed by separation and quantification through reversed-phase high-performance liquid chromatography with fluorescence detection (excitation, 378 nm; emission, 492 nm). Prior to the derivatization step, the oxidized thiols are converted to their reduced forms by reductive cleavage with sodium borohydride. Linearity in the detector response for total thiols was observed in the following ranges: 1.76–30.0 mg mL−1 for human serum albumin, 0.29–5.0 nmol mL−1 for α-lipoic acid, 1.16–35 nmol mL−1 for glutathione, 9.83–450.0 nmol mL−1 for cysteine, 0.55–40.0 nmol mL−1 for homocysteine, 0.34–50.0 nmol mL−1 for N-acetyl-L-cysteine, and 1.45–45.0 nmol mL−1 for cysteinylglycine. Recovery values of 85.16–119.48% were recorded for all the analytes. The developed method is sensitive, repeatable, and linear within the expected ranges of total thiols. The devised procedure can be applied to plasma samples to monitor biochemical processes in various pathophysiological states. [ABSTRACT FROM AUTHOR]