Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis

High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved by adding the organic base N-methyl-D-glucamine to the run buffer. However, because the active fractions of homocysteine and cysteine responsible for vascular injuries are still unknown, research calls for a set up of methods able to analyze different forms of plasma thiols. In this paper, we present an improvement of our previous method that allows the measurement of different thiol forms. Total, reduced, and free thiols were measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulfosalicylic acid. After derivatization with 5-iodoacetamidofluorescein, samples were separated and measured by CE-LIF using a phosphate/borate buffer in the presence of 75 mmol/L N-methyl-D-glucamine. Oxidized thiols and protein bound thiols were calculated by difference, free minus reduced and total minus free form, respectively. Linearity, reproducibility, analytical recovery, and sensitivity were evaluated. The assay was used to measure the thiols redox status in 15 plasma samples from healthy volunteers.     

Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection

We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.     

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%.     

RNA analysis by MEKC with LIF detection

We have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5'-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using CE with LIF. BODIPY conjugates of the four common ribonucleoside-5'-monophosphates were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions. After digestion of RNA or oligoribonucleotides to 5'-monophosphates by nuclease P1 and fluorescence labeling BODIPY conjugates were detected and resolved by CE-LIF without further purification steps. Comparative CE-LIF analyses with DNA digested to deoxyribonucleoside-5'-monophosphates showed that the assay is equally efficient and sensitive for RNA analysis. Conditions to determine the modified ribonucleosides inosine, xanthosine, pseudouridine and 2'-O-methyladenosine were also established. The limits of detection were in the range of 80-200 pM. After calibrating the assay with oligoribonucleotides, pseudouridine was quantified in total RNA of Drosophila, human liver, human kidney and t-RNA of Saccharomyces cerevisiae. These studies demonstrate good potential of fluorescence labeling of ribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF to determine RNA composition with high accuracy and sensitivity.     

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.     

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.     

高效液相色谱法测定人血浆中总高半胱氨酸含量

建立了快速检测人血浆中总高半胱氨酸含量的高效液相色谱（HPLC）方法。采用反相HPLC分析前,以巯基特异性荧光试剂ABDF对血浆中巯基进行衍生．方法简便、灵敏、准确,无干扰峰影响。平均回收率为97．75％,日内和日间精密度分别为4．49％和9．79％。     

Determination of gabapentin in human plasma by capillary electrophoresis-laser induced fluorescence detection with and without solid-phase extraction

We have developed two methods for the quantitation of gabapentin in human plasma. They are based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) with and without solid-phase extraction (SPE) and the derivatizing reagent 5-(4,6-dichlorotriazinyl)amino fluoresencin. The conditions for derivatization, separation and extraction were investigated in detail, and the optimal labeling conditions include a temperature of 40?°C, a reaction time of 30?min, and the use of a borate buffer of pH 9.0 as the reaction medium. A borate buffer of pH 9.2 served as a background electrolyte for CE separations. The CE-LIF and SPE-CE-LIF methods have linear ranges of 5–200?nmol?L^?1 and 0.2–10?nmol?L^?1, respectively, and the limits of detection are 0.5 and 0.02?nmol?L^?1, respectively. The SPE-CE-LIF method was successfully applied to the determination of gabapentin in blood plasma samples.

Determination of D-penicillamine and tiopronin in human urine and serum by HPLC-FLD and CE-LIF with 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene

Two rapid and sensitive analytical methods are developed for the determination of D-penicillamine (D-PEN) and tiopronin (TP) through high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A boron-dipyrrolemethene (BODIPY) fluorescence labeling reagent, 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene (TMMB-Br) was successfully applied to derivatize these two thiol drugs. Fluorescein was used as the internal standard (IS) for the quantification of D-PEN and TP in CE-LIF. The derivatization and separation conditions were optimized carefully. Under the optimum conditions, the HPLC and CE separation of D-PEN and TP could be achieved within 12?min. The limits of detection were as low as 2.0 nmol/L for HPLC-FLD and 0.47 nmol/L for CE-LIF. The drugs in human urine and serum samples were determined successfully, and the recoveries were 95.0–06.7% and 95.2–104.3%, respectively.     

A new HPLC method for serum neopterin measurement and relationships with plasma thiols levels in healthy subjects

Neopterin, a pyrazinopyrimidine compound, serves as a marker of cellular immune system activation, and it can be used as a prognostic predictor for certain types of diseases. We propose a new simple HPLC method to measure serum neopterin with highly sensitive fluorimetric detection. After TCA serum protein precipitation, the supernatant was diluted five times, injected into a C18 reversed-phase column and eluted at a flow rate of 1.5 mL/min by an isocratic water-acetonitrile (99:1) mobile phase. The natural fluorescence of the molecule was detected at excitation wavelength 353 nm and emission 438 nm. In these conditions the neopterin retention time was about 4 min. Our proposed method was compared with a validated chromatographic separation, and the obtained data of the serum neopterin from 35 healthy volunteers were analysed by Passing-Bablok regression and Bland-Altman test. Neopterin measurement in healthy subjects was also employed to investigate on its potential relationships with plasma thiols levels.     

Determination of aristolochic acid in Chinese herbal medicine by capillary electrophoresis with laser-induced fluorescence detection

We have demonstrated the analysis of aristolochic acids (AAs) that are naturally occurring nephrotoxin and carcinogen by capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), reduction of the analytes by iron powder in 10.0 mM HCl is required prior to CE analysis. The reduced AAs (RAAs) exhibit fluorescence at 477 nm when excited at 405 nm using a solid-state blue laser. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS, the determination of AA-I and AA-II by CE-LIF has been achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The simple CE-LIF method has been validated by the analysis of 61 Chinese herbal samples. Prior to CE analysis, OAAs were extracted by using 5.0 mL MeOH, and then the extracts were subjected to centrifugation at 3,000 rpm for 5 min. After reduction, extraction, and centrifugation, the supernatants were collected and subjected to CE analysis. Of the 61 samples, 14 samples contain AA-I and AA-II, as well as 10 samples contain either AAI or AAII. The relative standard deviation (RSD) values of the migration times for AA-I and AA-II are less than 2.5% and 2.1% for three consecutive measurements of each sample. The RSD values for the peak heights corresponding to AA-I and AA-II in most samples are about 8.0% and 10.0%, respectively. The result shows that the present CE-LIF approach is sensitive, simple, efficient, and accurate for the determination of AAs in real samples.     

Determination of tryptamine derivatives in illicit synthetic drugs by capillary electrophoresis and ultraviolet laser-induced fluorescence detection

A method based on separation by capillary electrophoresis combined with UV-laser-induced fluorescence detection (Lambdaex = 266 nm) was developed for the determination of nine tryptamine derivatives of forensic interest and potential matrix constituents. The composition of the separation electrolyte was optimized with respect to the resolution of solutes of interest and to the sensitivity of fluorescence detection. Native alpha-cyclodextrin was employed as a complex forming modifier of the electrophoretic separation and fluorescence-enhancing agent. With the help of a stacking procedure, limits of detection of 0.1-6 microg/L for all analytes were obtained. The repeatability for the peak area (at a concentration of the analyte about 100 times the LOD) was less than 2.3% RSD. A second HPLC method was developed, and its analytical parameters were evaluated for an estimation of the accuracy of the CE-LIF method and for method comparison. The results of the determination of tryptamine derivatives in the samples of forensic interest obtained with the two independent methods are in good agreement.     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.     

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.     

Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum

A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiolspecific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory.     

Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection

N-acetyl-l-cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N-acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (lambda(ex) = 330 nm, lambda(em) = 376 nm) using N-(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25-5000 nm (r(2) > 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 microL injection volume.     

Subminute and sensitive determination of the neurotransmitter serotonin in urine by capillary electrophoresis with laser-induced fluorescence detection

In this work, a sub-minute and sensitive capillary electrophoresis with laser-induced fluorescence (CE-LIF) method was developed for the analysis and quantitation of the neurotransmitter 5-hydroxytryptamine (5-HT) or serotonin in urine. The method involves precolumn derivatization with fluorescein isothiocyanate isomer I (FITC) using an excitation light from an argon ion laser of 488 nm and a 520 nm band pass emission filter. Different variables that affect derivatization (pH, FITC concentration, reaction time and temperature) and separation (buffer concentration, pH, applied voltage and injection time) were studied. The linear dynamic range obtained was between 0 and 188 nM with a detection limit of 16 nm with a RSD between 2 and 9%. The applicability of the proposed method was demonstrated by analysis of 5-HT in human urine, establishing a concentration of 57 nM in control urine. The method was validated by standard-addition methodology.     

Development and validation of a high-performance liquid chromatographic method using fluorescence detection for the determination of vardenafil in small volumes of rat plasma and bile

A new, simple and sensitive high-performance liquid chromatography (HPLC) method with fluorescence detection was developed and validated for the determination of vardenafil in small volumes of rat plasma and bile. The absorbance and fluorescence characteristics of vardenafil were studied and factors that affect the HPLC resolution and fluorescence intensity were examined and optimized. Vardenafil and the internal standard cisapride were extracted using acetonitrile. The separation was achieved on a C18 column at 35 degrees C using acetonitrile-50 mM ammonium acetate aqueous solution (pH 6.8) (40:60) as mobile phase. At a flow rate of 1 ml/min, the total run time was 18 min. Fluorescence was measured with excitation and emission set at 280 and 470 nm, respectively. The calibration curves were linear from 10 to 1000 ng/ml and 0.2-100 microg/ml for plasma and bile samples, respectively. The intra- and inter-day imprecision did not exceed 10.8%, and the accuracy was within 9.6% deviation of the nominal concentration. The method was used successfully to investigate the disposition and biliary excretion of vardenafil in rats.     

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%.     

Analysis of a new H2 receptor antagonist, 3-amino-5-[3-[4-(1-piperidinoindanyloxy)]propylamino]-1-methyl-1 H-1,2,4-triazole, in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of a new H2 receptor antagonist, 3-amino-5-[3-[4-(piperidinoindanyloxy)]propylamino] -1-methyl-1H-1,2,4-triazole (I), in human plasma and urine was developed. The method employs liquid-liquid extraction of the analyte and an internal standard and chromatographic separation using an alkylphenyl-bonded HPLC column. The total time of chromatography was less than 10 min. Sensitivity was 10 ng/ml for the plasma analysis and 1 microgram/ml for the analysis of I from urine. The coefficients of variation, based on interpolated concentrations, were less than 10%. The method was used for more than 5000 samples during clinical pharmacokinetic studies.