N-methyl-D-glucamine improves the laser-induced fluorescence capillary electrophoresis performance in the total plasma thiols measurement

We describe an ultrarapid capillary electrophoresis with laser-induced fluorescence (CE-LIF) method for total plasma thiols measurement. Reduced thiols by 10% tri-n-butylphosphine (TBP) were derivatized in 10 min at room temperature with 5-iodoacetamidofluorescein (5-IAF) as fluorescent reagent. We show that CE-LIF allows a baseline separation of total plasma cysteinylglycine, homocysteine, cysteine, and glutathione in less than 5 min when N-methyl-D-glucamine in run buffer was added. CE was compared with high-performance liquid chromatography (HPLC) with fluorescence detection. The Bland-Altman test and Passing-Bablok regression demonstrates that the results obtained by CE-LIF and by HPLC are highly comparable. The simplified procedure of sample preparation, the short incubation and fast separation times, the high specificity, sensitivity and reproducibility, and the lower cost of analysis suggest that our proposed method can be considered valuable for the automation analysis in a routine laboratory.     

Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescence-capillary electrophoresis

We have recently described a new method to determine physiological thiols, in which the quantification of plasma homocysteine, cysteine, cysteinylglycine, glutathione, and glutamylcysteine was achieved after derivatization with 5-iodoacetamidofluorescein. Samples were separated and measured by capillary electrophoresis with laser-induced fluorescence in an uncoated fused-silica capillary, using a phosphate/borate run buffer and the organic base N-Methyl-D-glucamine as effective electrolyte addictive to obtain a baseline peak separation. In this paper, we propose an improvement of our method useful for the analysis of the intracellular thiols in different cultured cells. In particular, we studied run buffer and injection conditions in order to increase the sensitivity of the assay and we found that, by incrementing two times the injected volume and using the water plug before the sample injection, the sensitivity of our previous method was increased by about ten times. To maintain a good resolution between peaks, particularly between homocysteine and the internal standard d-penicillamine, we lengthened the run time by incrementing the concentration of the electrolyte buffer and the organic base d-glucamine and by decreasing the cartridge temperature from 40 to 25 degrees C. After these changes in electrophoretical parameters, cellular thiols were baseline-resolved in less than 14 min instead of 9 min as in our previous method, but the limit of quantification is increased from 50 to 1 nmol/L. This new procedure allows also to measure the intracellular thiols commonly found at low concentration, such as cysteinylglycine, glutamylcysteine, and homocysteine. The new analytical method performance was assessed by measuring the intracellular thiols in three different cell lines, i.e., HUVEC, ECV304, and R1 stem cells.     

Distribution of low-density lipoprotein-bound low-molecular-weight thiols: a new analytical approach

We have recently demonstrated that low-density lipoprotein (LDL) apoprotein is able to bind the most concentrated plasma thiols such as cysteine, cysteinylglycine, and homocysteine by disulfide linkage. However, the LIF CE assay employed to measure linked thiols was not sensitive enough to verify whether low concentrated plasma thiols as glutathione and glutamylcysteine are also linked to apoprotein. By modifying sample treatment and electrophoretic parameters we set up a new method with an LOQ of about 1.5 nmol/L, by which we demonstrate that LDL apoprotein binds all physiological plasma thiols. The increased sensitivity was obtained by drying released apoB thiols after reduction treatment, dissolving them directly in a low volume of derivatization buffer and decreasing the dilution factor of derivatized sample before CE injection. Moreover, by increasing the concentration of the electrolyte buffer, we improved the selectivity of peaks, in particular between glutathione (GSH) and the impurity peak derived from unreacted 5-iodoacetamidofluorescein, which in the previous electrophoretic conditions were overlapped. The method optimization, reached by searching the best combination between sample matrix and CE run buffer, is fully described. Given the potential pathologic significance of protein thiolation, the proposed method may be useful to understand the mechanisms and the balances that regulate the interaction between thiols and -SH free groups of proteins.     

Thiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detection

Thiols and in particular glutathione (GSH) play a central role in human metabolism, including the detoxification of xenobiotics, cell homeostasis, radioprotection, and antioxidant defence. Here, a new method is provided for the measurement of reduced and total forms of thiols in red blood cells. In order to minimize oxidation of reduced thiols, a water erythrocyte lysis (15 min at 4 degrees C) was performed followed by a protein precipitation step with acetonitrile. The supernatant was rapidly derivatized with 5-iodoacetoamidefluorescein that trapped thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 57 cm x 75 microm ID capillary by using 5 mmol/L sodium phosphate, 4 mmol/L boric acid as electrolyte solution with 75 mmol/L N-methyl-D-glucamine at pH 11.0. Under these conditions, cysteinylglycine (CysGly), cysteine (Cys), glutathione, and gamma-glutamylcysteine (GluCys) were baseline-resolved in approximately 4 min. Precision tests showed a good repeatability of our method both for migration times (coefficient of variation CV < 0.8%) and areas (CV < 3.3%). Furthermore, a good reproducibility of intrassay and interassay tests was obtained (CV < 5% and CV < 8%, respectively). The method was employed to investigate the effect of acidic precipitation on intracellular thiol concentration. Our data suggest that sample acidification causes a modification of the measured redox thiol status due to the development of a pro-oxidant environment; moreover, the thiol redox status of red blood cells was evaluated in 22 healthy volunteers.     

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.     

Albumin‐bound low molecular weight thiols analysis in plasma and carotid plaques by CE

We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS‐PAGE, thiols were freed from protein with tri‐n‐butylphosphine and successively derivatized with 5‐iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 μg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.     

Plasma total homocysteine and other thiols analyzed by capillary electrophoresis/laser-induced fluorescence detection: comparison with two other methods

We present a new analytical method for thiol quantification in plasma, based on the use of capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze 6-iodoacetamidofluorescein derivatives. Quantitative results of homocysteine, glutathione, cysteinylglycine, and cystationine are presented. A comparison of the quantitation of homocysteine in plasma, using high performance liquid chromatography/fluorescence detection and fluorescence polarization immunoassay is proposed. The results indicate that these techniques for plasma total homocysteine (tHcy) determination can be used interchangeably. The major advantage of CE-LIF is that it can quantitate the thiols in one run while keeping the price of consumables reasonable.     

Determination of homocysteine and other low-molecular-weight amino thiols in blood plasma

A current approach to determining low-molecular-weight amino thiols (cysteine, homocysteine, and glutathione) in model samples and blood plasma is considered. Procedures for determining 2–100 μM homocysteine in blood plasma with the use of microcolumn chromatography and capillary electrophoresis were developed. Photometric and fluorimetric detection techniques were used to identify amino thiols. Monobromobimane and 5-iodoacetamidofluorescein were used as labels. Mass spectrometry was used to confirm the structures of test amino thiol derivatives.     

Determination of thiols by capillary micellar electrokinetic chromatography with laser induced fluorescence detection using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as labeling reagent

A sensitive and effective micellar electrokinetic capillary chromatography with laser‐induced fluorescence detection approach was described for the determination of low molecular‐mass thiols using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as the labeling reagent. After precolumn derivatization, baseline separation of six thiol compounds including cysteine, glutathione, N‐acetylcysteine, homocysteine, 6‐mercaptopurine, and penicillamine were achieved within 18 min. The optimal running buffer was composed of mixtures involving 25 mM sodium dodecyl sulfate, 25% (v/v) acetonitrile and 15 mM sodium phosphate buffer, pH 7.5. The detection limits (S/N = 3) were found as low as 40 pM under argon ion laser‐induced fluorescence detector (λ_ex/λ_em = 488/520 nm), which were much better than the reported approaches. The accuracy and specificity of this assay for real samples were assured by a standard addition method. The proposed method has been applied to the analysis of thiols both in human plasma and plum flower samples with recoveries of 92.0–109.4%.     

Speciation and determination of thiols in biological samples using high performance liquid chromatography-inductively coupled plasma-mass spectrometry and high performance liquid chromatography-Orbitrap MS

An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quadrupole MS or Orbitrap MS was employed for the separation and detection of thiols. For the determination of total thiols, oxidized thiols were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be close to 100%. Reduced thiols were derivatized by p-hydroxymercuribenzoate (PHMB) and then separated on a C8 column. Optimization of the extraction, separation and detection steps of the HPLC-ICP-MS and HPLC-Orbitrap MS methods was carried out. Detection limits for cysteine, homocysteine, selenocysteine, glutathione, selenomethionine and cysteinyl-glycine were found to be 18, 34, 39, 12, 128 and 103 fmol, respectively, using HPLC-Orbitrap MS and 730, 1110, 440, 1110 and 580 fmol for cysteine, homocysteine, selenocysteine, glutathione, and cysteinyl-glycine using HPLC-ICP-MS. Contrary to expectation, the LODs and RSDs are higher for the HPLC-ICP-MS instrument, therefore HPLC-Orbitrap MS was used for the determination of thiols in yeast samples. Three different brands of baker's yeast and a selenized yeast were analyzed. The GSH and cysteine levels found in these samples ranged from 4.45 to 17.87 μmol g(-1) and 0.61 to 1.32 μmol g(-1), respectively.     

High-performance liquid chromatography-fluorometry for the determination of thiols in biological samples using N-[4-(6-dimethylamino-2-benzofuranyl) phenyl]-maleimide

The selective determination of thiols in biological samples was investigated by high-performance liquid chromatography using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide, which was found to give fluorescent products when treated with certain thiols. Six kinds of thiol (reduced glutathione, cysteine, N-acetylcysteine, cysteamine, homocysteine and coenzyme A) could be separated simultaneously within ca. 12 min and determined at final level of sensitivity. The method was successfully applied to the determination of thiols in rat tissues and plasma and in human normal serum.     

Determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7‐tetramethyl‐8‐bromomethyl‐difluoroboradiaza‐s‐indacene

Altered levels of thiols in biological fluids are considered to be an important indicator for several diseases. In this article, 1,3,5,7‐tetramethyl‐8‐bromomethyl‐difluoroboradiaza‐s‐indacene is proposed as a fluorescent derivatization reagent for the determination of thiols including glutathione, cysteine, N‐acetylcysteine, and homocysteine by HPLC. Under the optimized derivatization and separation conditions, a baseline separation of all the four derivatives has been achieved using isocratic elution on an RP C₈ column within 26 min. With fluorescence detection at 505 and 525 nm for the excitation and emission, respectively, the LODs (S/N = 3) are from 0.2 nM (glutathione) to 0.8 nM (cysteine). The feasibility of this method in real samples has been evaluated by the determination of thiols in human plasma from the healthy persons and hypertensive patients with recoveries of 92–105.3%.     

Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near‐infrared laser‐induced fluorescence detection using a new BODIPY‐based probe as labeling reagent

A CZE with near‐infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ‐glutamylcysteine, cysteinylglycine, and N‐acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(4'‐iodoacetamido)difluoroboradiaza‐s‐indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N‐acetylcysteine to 0.31 nmol/L for γ‐glutamylcysteine, which are better than or comparable to those reported with other derivatization‐based CE‐LIF methods. As the first trial of NIR CE‐LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5–104.3%.     

Analysis of urine for cysteine, cysteinylglycine, and homocysteine by high-performance liquid chromatography

A new analytical method is proposed for simultaneous determination, by liquid chromatography, of the three main urinary thiols–cysteine, cysteinylglycine, and homocysteine. To measure the total amount of these thiols urine is reduced with sodium borohydride, to convert disulfides to thiols which are then derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. Separation and quantitation of the 2-S-quinolinium thiol derivatives formed were achieved by high-performance liquid chromatography with detection at 355 nm. Validation showed the method enabled reliable simultaneous determination of these aminothiols in urine. The calibration graphs for each analyte, obtained by use of normal urine spiked with increasing amounts of cysteine, cysteinylglycine, and homocysteine, were linear (R ²≥0.997) over the range covering most practical situations. The recovery of the assay was 98–100% and sensitivity was 0.12–0.25 μmol L⁻¹. The method was applied to 91 different samples of normal urine to establish reference values for the aminothiols, normalized on creatinine.     

Determination of biologically active low-molecular-mass thiols in human blood. II. High-performance capillary electrophoresis with photometric detection

A new high-performance capillary electrophoresis assay for aminothiols in human blood, including homocysteine, a marker of several human metabolism disorders, has been developed. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fused-silica capillary (65 cm x 50 microm I.D.) at 30 degrees C. With the electric field of 250 V cm(-1), separation of homocysteine, glutathione and cysteine occurred at less than 10 min. Detection at 250 or 234 nm was used to confirm the monobimane-thiols peaks. The detection limit was approximately 5 nmol/ml for all labeled aminothiols. The proposed method for these compounds' analysis included simple sample preparation, high selectivity, good linearity (r2>0.999), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<5% for three times consequently injected sample); high reliability and the small volumes required for analysis made it suitable for clinical studies.     

Simultaneous determination of total homocysteine,cysteine, glutathione,and N‐acetylcysteine in brain homogenates by HPLC

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N‐acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2‐carboxyethyl) phosphine, pre‐column derivatization of free thiol groups with 2‐chloro‐1‐methylquinolinium tetrafluoroborate followed by ion‐pair reversed‐phase high‐performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10 min. Linearity was observed in the range of 10–300, 0.7–10, 2–30, and 3–20 μmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2 μmol/L homogenate for cysteine, homocysteine, glutathione, and N‐acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21–4.77, 1.53–14.35, 0.47–1.92, and 1.61–8.95% for cysteine, homocysteine, glutathione, and N‐acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.     

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.     

Qualitative and quantitative determination of biologically active low-molecular-mass thiols in human blood by reversed-phase high-performance liquid chromatography with photometry and fluorescence detection

The reversed-phase high-performance liquid chromatographic method employing photometry and fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine (tHcy), cysteine (Cys), and glutathione (GSH) in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with trichloroacetic acid, conjugation of the thiols with monobromobimane (mBrB). The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions) to obtain reliable quantitative results within the concentration range corresponding to normal and pathological levels of these thiols in human blood.     

Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry

For the first time a liquid chromatography method with high resolution mass spectrometric detection has been developed for the simultaneous determination all key metabolites of the sulfur pathway in yeast, including all thiolic (cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), γ-glutamyl-cysteine (Glu-Cys)) and non-thiolic compounds (methionine (Met), s-adenosyl-methionine (AdoMet), s-adenosyl-homocysteine (AdoHcy), and cystathionine (Cysta)). The developed assay also permits the speciation and selective determination of reduced, oxidized and protein bound fractions of all of the five thiols. Iodoacetic acid (IAA) was chosen as the derivatizing reagent. Thiols were extracted from sub-mg quantities of yeast using hot 75% ethanol. The detection limits were in the range of 1–12 nmol L⁻¹ for standard solution (high femotomole, absolute), except AdoMet (116 nmol L⁻¹), which was unstable. In freshly harvested yeast, most of the thiols were in the reduced forms and low levels of protein-bound GSH and Glu-Cys were found. In a selenium enriched yeast, the thiols were mainly in the oxidized forms, and a significant amount of protein-bound Cys, HCys, GSH, Cys-Gly and Glu-Cys were found. The method was also applied to the metabolic study of the adaptive response of Saccharomyces cerevisiae to hydrogen peroxide, cadmium, and arsenite, and the change in concentration of thiols in the sulfur pathway was monitored over a period of 4 h.     

A Native‐Chemical‐Ligation‐Based Turn‐on Fluorescent Probe for Selective Detection of Cysteine

Selective and quantitative detection of biological thiols such as cysteine, homocysteine, and glutathione is often necessary because abnormal levels of such thiols can cause some diseases. Here, we report that bis(pentafluorophenyl) 1,4‐benzenedicarbothionic acid diester can serve as a turn‐on fluorescent probe for selective detection of cysteine vis‐a‐vis homocysteine and glutathione. When cysteine was added to a mixture of the diester and a sodium phosphate buffer solution with THF (60 vol%), which is non‐fluorescent, the mixture became green‐fluorescent. In contrast, addition of homocysteine or glutathione did not make the mixture fluorescent. A native‐chemical‐ligation‐based mechanism is proposed.