Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells

This paper, for the first time, reported the development of a simple, rapid, and reliable method for the separation and sensitive determination of four thiol compounds including homocysteine, cysteine, glutathione, and N‐acetylcysteine based on glass MCE with fluorescence detection using a highly reactive fluorogenic probe, 1,3,5,7‐tetramethyl‐8‐phenyl‐(2‐maleimide)‐difluoroboradiaza‐s‐indacene (TMPAB‐o‐M), as the labeling reagent. TMPAB‐o‐M reacted selectively with thiols to produce highly fluorescent derivatives and the highest derivatization efficiency was achieved within 6 min in physiological conditions. After the optimization of separation conditions, a baseline separation of the four thiol compounds was achieved with the detection limits ranging from 2 nM for glutathione to 4 nM for cysteine (S/N = 3) and RSDs (n = 5) in the range of 3.2–3.8%. The proposed method was significantly sensitive compared to those using electrochemical or even LIF detection in MCE‐based setup reported previously, and applied to the determination of intracellular thiols in macrophage RAW264.7 cells.     

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%.     

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%.     

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%.     

Miniaturized capillary electrophoresis system with a carbon nanotube microelectrode for rapid separation and detection of thiols

Multi-walled carbon nanotube (CNT) was mixed with epoxy to fabricate microdisc electrode used as a detector for a specially designed miniaturized capillary electrophoresis (CE)-amperometric detection system for the separation and detection of several bioactive thiols. The end-channel CNT amperometric detector offers favourable signal-to-noise characteristics at a relatively low potential (0.8 V) for detecting thiol compounds. Factors influencing the separation and detection processes were examined and optimized. Four thiols (homocysteine, cysteine, glutathione, and N-acetylcysteine) have been separated within 130 s at a separation voltage of 2000 V using a 20 mM phosphate running buffer (pH 7.8). Highly linear response is obtained for homocysteine, cysteine, glutathione, and N-acetylcysteine over the range of 5-50 μM with detection limits of 0.75, 0.8, 2.9, and 3.3 μM, respectively. Good stability and reproducibility (R.S.D. < 5%) are obtained reflecting the minimal adsorption of thiols at the CNT electrode surface. The new microchip protocol should find a wide range of bioanalytical applications involving assays of thiol compounds.     

Reversed‐phase liquid chromatography method for the determination of total plasma thiols after derivatization with 1‐benzyl‐2‐chloropyridinium bromide

A high‐performance liquid chromatography method was developed for simultaneous detection and quantitation of total cysteine, glutathione, homocysteine and cysteinylglycine in human plasma. The two key steps in the analysis are reduction of disulfides and treatment with 1‐benzyl‐2‐chloropyridinium bromide, which rapidly and quantitatively reacts with thiol groups to form stable S‐pyridinium derivatives with intense UV absorption. The derivatives are well separated on a Zorbax SB C₁₈ column using reversed‐phase high‐performance liquid chromatography and monitored at 315 nm. The calibration graphs were linear over concentration ranges covering most experimental and clinical cases with a regression coefficients better than 0.999. The detection and quantitation limits for all analytes were 0.2 and 0.5 µmol/L, respectively. The recoveries were 99.25–101.68%. The intra‐ and interassay imprecisions were 0.88–4.24 and 1.68–5.14%, respectively. The method was applied for plasma samples donated by apparently healthy volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

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.     

A validated HPLC‐fluorescence method with a semi‐micro column for routine determination of homocysteine,cysteine and cysteamine,and the relation between the thiol derivatives in normal human plasma

A semi‐micro column HPLC‐fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium‐7‐fluorobenzo‐2‐oxa‐1,3‐diazole‐4‐sulfonate (SBD‐F) were isocratically separated within 12 min on a semi‐micro ODS column (Daisopak‐SP‐120‐5‐ODS‐BP) with a mixture of 25 mm acetate buffer (pH 2.00) and CH₃CN as a mobile phase. The purity and similarity of SBD‐thiols by a multi‐wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal‐to‐noise ratio of 3 were 0.16, 0.47 and 0.03 µm , respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572–1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and −0.35, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of lipoic acid in human urine by capillary zone electrophoresis

Fast, simple, and accurate CE method enabling determination of lipoic acid (LA) in human urine has been developed and validated. LA is a disulfide‐containing natural compound absorbed from the organism's diet. Due to powerful antioxidant activity, LA has been used for prevention and treatment of various diseases and disorders, e.g. cardiovascular diseases, neurodegenerative disorders, and cancer. The proposed analytical procedure consists of liquid–liquid sample extraction, reduction of LA with tris(2‐carboxyethyl)phosphine, derivatization with 1‐benzyl‐2‐chloropyridinium bromide (BCPB) followed by field amplified sample injection stacking, capillary zone electrophoresis separation, and ultraviolet‐absorbance detection of LA‐BCPB derivative at 322 nm. Effective baseline electrophoretic separation was achieved within 6 min under the separation voltage of 20 kV (∼80 μA) using a standard fused‐silica capillary (effective length 51.5 cm, 75 μm id) and BGE consisted of 0.05 mol/L borate buffer adjusted to pH 9. The experimentally determined limit of detection for LA in urine was 1.2 μmol/L. The calibration curve obtained for LA in urine showed linearity in the range 2.5–80 μmol/L, with R ² 0.9998. The relative standard deviation of the points of the calibration curve was lower than 10%. The analytical procedure was successfully applied to analysis of real urine samples from seven healthy volunteers who received single 100 mg dose of LA.     

Simultaneous Detection of Homocysteine and Cysteine in the Presence of Ascorbic Acid and Glutathione Using a Nanocarbon Modified Electrode

The simultaneous electrochemical detection of homocysteine and cysteine using an absorbed ortho‐quinone species, catechol, at the nanocarbon modified glassy carbon electrode was achieved via 1,4‐Michael addition reaction. The detection was done in the presence and the absence of each other as well as with both glutathione and ascorbic acid present in order to investigate the selectivity of homocysteine and cysteine. A determination of homocysteine sensitivity is (0.882±0.296) nA nM^?1 with a LOD of ca. 11 nM and cysteine sensitivity is (7.501±0.202) mA µM^?1 with a LOD of ca. 5.0 µM within a range of 0–0.1 mM.     

Studies on the Interaction of Some Biologically Significant Radical Scavengers on Metal‐Ion Catalyzed Phloroglucinol‐Based Chemical Oscillator

The response of the Ce(III)‐catalyzed phloroglucinol (1,3,5‐trihydroxybenzene)‐based Belousov Zhabotinsky system to the addition of various antioxidants (ascorbic acid, glutathione, inosine, N‐acetylcysteine) is monitored at 30°C under stirred batch conditions. This method is convenient and has good sensitivity for the determination of these antioxidants. The addition of these antioxidants to the BZ mixture influences the oscillatory parameter (number of oscillations) to an extent that depends on the concentration of the antioxidant. The experimental results have shown that the number of oscillations decrease on increasing the concentration of antioxidant. The calibration plots show a linear relationship (R² = 0.98 for ascorbic acid and glutathione and R² = 0.99 for inosine and N‐acetylcysteine) between the number of oscillations and the [antioxidant] over the concentration range of 0.0125‐0.5, 0.05‐0.2, 0.025‐0.1, and 0.1‐0.5 mol L^?1 with detection limits 6.9 × 10^?5, 2.762 × 10^?4, 1.381 × 10^?4, and 5.524 × 10^?4 mol L^?1 for ascorbic acid, glutathione, inosine, and N‐acetylcysteine, respectively. Some aspects of the mechanism of these antioxidants on the BZ system have been discussed.     

Quantification of Intracellular Thiols by HPLC-Fluorescence Detection

Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R² > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5–153 pmol/1 × 10⁶ cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.     

Detection of homocysteine at carbon nanotube paste electrodes

The use of a carbon-nanotube paste (CNTP) electrode provides an effective means for the determination of homocysteine. A decrease of ca. 120 mV in the overpotential for the oxidation of homocysteine compared to a traditional carbon paste electrode, is reported along with greatly enhanced signal-to-noise characteristics. The analytical parameters have been assessed with a linear range from 5 to 200 μM and a detection limit of 4.6 μM. Furthermore, the generic nature of this increased reactivity of the CNTP surface towards thiol moieties has been demonstrated with cysteine, glutathione and n-acetylcysteine, providing a greatly enhanced electrochemical response compared to the carbon paste electrode.     

Commercial Screen‐Printed Gold Electrodes for the Detection and Quantification of Aminothiols in Human Plasma by Liquid Chromatography with Electrochemical Detection

In order to show the possibilities of screen‐printed electrodes in HPLC detection, a method is presented for routine quantification of aminothiols in human plasma. It uses commercial gold screen‐printed electrodes and provides a rapid, simple and economical procedure with a disposable detector and without any additional electrode. By working at 98 : 2 (0.05 % trifluoroacetic acid: methanol) and 0.9 V, it allows us to determine cysteine (Cys), cysteinyl‐glycine (Cys‐Gly), and homocysteine (HCys) at μmol/L levels. The method was validated, and linearity range, detection and quantification limits, precision and accuracy were evaluated by external calibration and by using N‐acetylcysteine as internal standard, which exhibited better precision.     

Ultrafast capillary electrophoresis method for the simultaneous determination of ammonium and diphenhydramine in pharmaceutical samples

Ammonium and diphenhydramine are active ingredients commonly found in the same pharmaceutical preparations. We report, for the first time, a sub‐minute method for the simultaneous determination of ammonium and diphenhydramine. The method is based on capillary electrophoresis with capacitively coupled contactless conductivity detection. Both analytes can be quantified in a single run (∼80 injections/h) using 30 mmol/L 2‐(N‐morpholino)ethanesulfonic acid and 15 mmol/L lithium hydroxide (pH 6.0) as background electrolyte. The separation by capillary electrophoresis was achieved on a fused‐silica capillary (50 cm total length, 10 cm effective length, and 50 μm inside diameter). The limits of detection were 0.04 and 0.02 mmol/L for ammonium and diphenhydramine, respectively. The proposed method also provided adequate recovery values for spiked samples (100–106 and 97–104% for ammonium and diphenhydramine, respectively). The results obtained with the new capillary electrophoresis method were compared with those of the high‐performance liquid chromatography method for diphenhydramine and the Kjeldahl method for ammonium and no statistically significant differences were found (95% confidence level).     

Ultrasensitive and Simple Method for Determination of N‐Acetyl‐L‐Cysteine in Drug Formulations Using a Diamond Sensor

A boron‐doped diamond (BDD) electrode coupled to flow injection analysis (FIA) was firstly developed for determination of N‐acetyl‐L ‐cysteine (NAC) in drug formulations. The effects of experimental parameters including pH, applied potential and scan rate on the response were investigated. FIA amperometry was applied as an automatic method for the quantitative detection of trace amounts of NAC. A wide linear range of 0.5–50 µmol/L and a low detection limit of 10 nmol/L were obtained. The results of amperometric determinations show a very good reproducibility, and the RSD for the measurement based on 10 measurements was <3.7 % and <4.1 % for intra‐ and inter‐day, respectively. The benefits of the proposed method are fast, simple, sensitive and no requirement of complicated operational steps.     

LC‐ESI‐MS/MS analysis and pharmacokinetics of heterophyllin B,a cyclic octapeptide from Pseudostellaria heterophylla in rat plasma

Heterophyllin B (HB) is a cyclic octapeptide isolated from Pseudostellaria heterophylla. HB is used as the quality control index for evaluating P. heterophylla in the Chinese Pharmacopoeia. A rapid and sensitive LC‐ESI‐MS/MS method was developed and validated for the analysis of HB in rat plasma. Sample preparation consisted of a solid‐phase extraction step for the removal of interference and preconcentration of the target analyte HB and the internal standard N‐acetylcysteine before chromatographic analysis by MS/MS detection. The separation of HB and N‐acetylcysteine was performed using a Hypersil GOLD^TM C₁₈ column and a mixture of methanol–water (60:40, v/v) containing 10 mmol/L ammonium formate and 0.1% formic acid as the mobile phase. The determination step was optimized in the selected reaction monitoring mode for the highly selective and sensitive quantitation of HB in rat plasma. Intra‐ and inter‐assay precision (as relative standard deviation) was ≤9.1%, and accuracy was between 92.6 and 102.7%. The validated method was successfully applied to quantify HB concentrations up to 7 h after tail intravenous injections of 2.08, 4.16 and 8.32 mg/kg HB in rats. The LC‐MS/MS method identified the relevant pharmacokinetic parameters of HB and its studied analog. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of glutathione and cysteine in yeasts by hydrophilic interaction liquid chromatography followed by on‐line postcolumn derivatization

In the present study, we report a new method for the determination of two primary thiols, cysteine (CYS) and glutathione (GSH), by hydrophilic interaction LC. The polar analytes are separated isocratically using a mobile phase consisting of 65% acetonitrile/35% ammonium acetate (15 mmol/L, pH 2.0) and are detected at 285 nm following on‐line postcolumn derivatization by the thiol‐selective reagent methyl propiolate. The main figures of merit included linearity in the range of 5–200 μmol/L and an LOD 0.6 μmol/L for both compounds. The absence of matrix effect allowed the determination of CYS and GSH in various yeast samples. GSH was present in most of the samples at levels ranging between 0.9 and 3.1 mg/g, whereas CYS was determined in only one sample at a significantly lower concentration. In terms of accuracy, the percent recoveries ranged between 91.2 and 105.6% for GSH, and 91.6 and 106.9% for CYS.     

Ion‐pair‐based hollow‐fiber liquid‐phase microextraction combined with high‐performance liquid chromatography for the simultaneous determination of urinary benzene,toluene, and styrene metabolites

In the current study, a novel technique for extraction and determination of trans,trans‐muconic acid, hippuric acid, and mandelic acid was developed by means of ion‐pair‐based hollow fiber liquid‐phase microextraction in the three‐phase mode. Important factors affecting the extraction efficiency of the method were investigated and optimized. These metabolites were extracted from 10 mL of the source phase into a supported liquid membrane containing 1‐octanol and 10% w/v of Aliquat 336 as the ionic carrier followed by high‐performance liquid chromatography analysis. The organic phase immobilized in the pores of a hollow fiber was back‐extracted into 24 μL of a solution containing 3.0 mol/L sodium chloride placed inside the lumen of the fiber. A very high preconcentration of 212‐ to 440‐fold, limit of detection of 0.1–7 μg/L, and relative recovery of 87–95% were obtained under the optimized conditions of this method. The relative standard deviation values for within‐day and between‐day precisions were calculated at 2.9–8.5 and 4.3–11.2%, respectively. The method was successfully applied to urine samples from volunteers at different work environments. The results demonstrated that the method can be used as a sensitive and effective technique for the determination of the metabolites in urine.