Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE‐based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4‐dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, ε‐aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs–Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.     

Determination of ethyl glucuronide in human serum by hyphenation of capillary isotachophoresis and zone electrophoresis

The determination of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in human serum by hyphenation of capillary ITP (CITP) and CZE is reported. For CITP step, 1 x 10(-2) M hydrochloric acid adjusted with epsilon-aminocaproic acid (EACA) to pH 4.4 was used as the leading electrolyte, and 1 x 10(-2) M nicotinic acid with EACA, pH 4.4, was used as the terminating electrolyte (TE). All electrolytes contained 0.2% hydroxypropylcellulose to suppress electroosmosis. In CITP, EtG was separated from fast serum macrocomponents chloride, phosphate, lactate, and acetate. Zones of microcomponents including EtG that migrated between acetate and nicotinate were forwarded to the second capillary filled with a BGE identical with the TE. Conductivity detection was used in the CITP step. Sensitive detection in the CZE step was performed using indirect spectrophotometric detection at 254 nm. The assay is based on a 1:5 dilution of serum with deionized water and has a concentration LOD for EtG in diluted sample of 9.8 x 10(-9) M. The method was used for the determination of EtG in sera of volunteers consuming alcohol.     

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.     

Determination of idarubicin in human urine by capillary zone electrophoresis with amperometric detection

A simple, reliable and reproducible method, based on capillary zone electrophoresis with amperometric detection, has been developed for the determination of idarubicin in human urine. A carbon disk electrode was used as working electrode. The optimal conditions of separation and detection were pH 5.6 phosphate buffer (0.20 mol/L), 22 kV for the separation voltage and 1.00 V (vs. Ag/AgCl, 3 mol/L KCl) for the detection potential. The linear range was from 4.0 x 10(-7) to 2.0 x 10(-5) mol/L with a regression coefficient of 0.9986, and the detection limit was 8.0 x 10(-8) mol/L. The method was directly applied to the determination of idarubicin in spiked human urine without any other sample pretreatment except filtration, and the assay results were satisfactory.     

Determination of idarubicin in human urine by capillary zone electrophoresis with amperometric detection

A simple, reliable and reproducible method, based on capillary zone electrophoresis with amperometric detection, has been developed for the determination of idarubicin in human urine. A carbon disk electrode was used as working electrode. The optimal conditions of separation and detection were pH 5.6 phosphate buffer ¶(0.20 mol/L), 22 kV for the separation voltage and 1.00 V (vs. Ag/AgCl, 3 mol/L KCl) for the detection potential. The linear range was from 4.0 × 10^–7 to 2.0 × 10^–5 mol/L with a regression coefficient of 0.9986, and the detection limit was 8.0 × 10^–8 mol/L. The method was directly applied to the determination of idarubicin in spiked human urine without any other sample pretreatment except filtration, and the assay results were satisfactory.     

Determination of creatinine in human serum by short-end injection capillary zone electrophoresis

A new ultra-rapid free-solution capillary zone electrophoresis method to measure serum creatinine is presented. Procedural parameters such as injection mode, concentration and pH of phosphate running buffer and acidic deproteinization of serum samples were investigated. Short-end injection permits a decrease of the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, so reducing the migration distance. Thus, when a capillary with an effective length of 10.2 cm and a 40 mmol/L sodium phosphate buffer pH 2.35 was used, the obtained migration time of the creatinine peak was the shortest never described before, about 1.1 min. These conditions give a good reproducibility of the migration times (coefficient of variation, CV% < 0.5) and the peak areas (CV% < 2.8). Intra- and interassay CV were 3.06 and 6.26%, respectively, and analytical recovery was 99.4%. We compared our proposed method to Jaffé colorimetric assay, by measuring serum creatinine in 128 normal subjects. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test. Creatinine concentration in healthy subjects was also used to investigate on its relationships with plasma thiols levels.     

Determination of uric acid in human urine by capillary zone electrophoresis with indirect laser-induced fluorescence detection

A CZE with indirect LIF detection method was used for the determination of uric acid (UA) in human urine. UA and its coexisting analytes (i.e. hypoxanthine, xanthine and ascorbic acid) could be well separated within 4.5?min at a voltage of 25?kV with 25°C cartridge temperature in a running buffer composed of 5?mM sodium borate, 10% methanol (v/v) and 0.3?μM fluorescein sodium (apparent pH of the final mixed hydro-organic solution of sodium borate, methanol and fluorescein is 9.5). Under the optimum condition, the method has good linearity relationships (correlation coefficients: 0.9973-0.9987) with ranges of 25-500, 25-350, 25-250 and 25-300?μg/mL for hypoxanthine, ascorbic acid, xanthine and UA, respectively. The detection limits for the analytes were in the range of 0.29-0.90?μg/mL. The intra-day RSD values for migration times and peak areas were less than 0.43 and 3.27%, respectively. This method was applied to the quantitation of UA in human urine with recoveries in the range of 93.1-107.3%.     

场放大样品堆积毛细管区带电泳检测尿液中苯丙胺类毒品

建立了毛细管区带电泳(CZE)中场放大样品堆积(FASS)技术分析尿液中苯丙胺类毒品的方法。采用体积分数30%甲醇的100 mmol/L磷酸盐(pH 3)为分离缓冲液,利用缓冲体系与样品溶液体系电导率的差异,在毛细管中浓缩样品组分,对苯丙胺、甲基苯丙胺、3,4-(亚甲二氧基)苯丙胺(MDA)、3,4-(亚甲二氧基)甲基苯丙胺(MDMA)4种毒品进行了分离和定量测定,与常规毛细管区带电泳比较,检测灵敏度提高约2000倍。采用利多卡因为内标,对添加上述4种毒品的尿液进行提取和测定,分析的相对标准偏差在15%范围之内,可检测到的上述毒品质量浓度为0.002μg/mL,相对回收率在70%~120%内。该方法可用于生物检材中苯丙胺类毒品的检测。     

毛细管区带电泳法测定粉针剂中头孢拉定的含量

用毛细管区带电泳法测定头孢拉定的含量 ,未涂层毛细管柱 (75 μm×48.5cm ,有效长度 40cm) ,电压 2 8kV ,检测波长 2 3 0nm ,温度 2 0℃ ,进样 5×1 0 3Pa× 3s。运行缓冲液为 2 5mmol/L硼砂缓冲液。方法的线性范围 3 1 .2 2μg/mL～ 749.2 8μg/mL ,检测限为 1 .1 7μg/mL。     

Determination of ribavirin in human serum and plasma by capillary electrophoresis

The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 microL sample and reconstitution of the dried extract into 100 microL of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 microg/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-alpha-2b for a hepatitis C virus infection.     

Determination of norfloxacin in human urine by capillary electrophoresis with electrochemiluminescence detection

A fast and sensitive approach that can be used to detect norfloxacin in human urine using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection of is described. The separation column was a 75-μm i.d. capillary. The running buffer was 15 mmol L⁻¹ sodium phosphate (pH 8.2). The solution in the detection cell was 50 mmol L⁻¹ sodium phosphate (pH 8.0) and 5 mmol L⁻¹ The ECL intensity varied linearly with norfloxacin concentration from 0.05 to 10 μmol L⁻¹. The detection limit (S/N=3) was 0.0048 μmol L⁻¹, and the relative standard deviations of the ECL intensity and the migration time for eleven consecutive injections of 1.0 μmol L⁻¹ norfloxacin (n=11) were 2.6% and 0.8%, respectively. The method was successfully applied to the determination of norfloxacin spiked in human urine without sample pretreatment. The recoveries were 92.7–97.9%.      

Determination of aluminum in serum by capillary zone electrophoresis with laser-induced fluorescence detection

Summary A novel method of separating and detecting trace aluminum by capillary zone electrophoresis is described. Aluminum is reacted with lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzen-1-sulphonic acid] so that the complex can be selectively and sensitively detected by a laser-induced fluorescence detector after capillary electrophoretic separation. Using the proposed method, limits of detection in the sub parts per billion range are achieved. The technique is applied to the determination of aluminum in human serum.     

Analysis of 5-methyltetrahydrofolate in human blood, serum and urine by on-line coupling of capillary isotachophoresis and zone electrophoresis

The predominant circulating folate coenzyme in plasma/serum, 5‐methyltetrahydrofolate (5‐MTHF) was determined in human blood, serum and urine using a method based on the hyphenation of capillary ITP and zone electrophoresis. Measurements were done with a commercially available instrument for capillary isotachophoresis equipped with a column‐switching system. The choice of electrolytes was limited by the instability of 5‐MTHF and volatility of electrolytes for the potential coupling of the instrumentation with MS detector. To get an insight into the separability of individual sample components in an isotachophoretic analysis, we constructed zone existence diagrams for isotachophoretic electrolyte systems having a leading electrolyte composed of acetate and ammonium of pH 4.5 and 7.0, hydrocarbonate and ammonium, pH 7.8, chloride and ammonium, pH 5.6, and chloride and creatinine, pH 5.0, with hydroxide ion as the terminator. For isotachophoretic preseparation, the non‐volatile leading electrolyte with good buffering capacity composed of 1×10⁻² M HCl and 2.5×10⁻² M creatinine, pH 5.0, and terminating electrolyte composed of 1×10⁻² M MES was selected as the most suitable. The optimum BGE for CZE analysis from the standpoint of analyte stability, separability and volatility for MS coupling was 1×10⁻² M acetate with 3.5×10⁻² M ammonium, pH 4.5. Using this combination of electrolytes, LODs reached with optical detection at 220 nm were 1.6×10⁻⁷ M in human blood, 1.1×10⁻⁷ M in human serum and 4.7×10⁻⁶ M in human urine. Estimated content of 5‐MTHF in blood and serum samples of women following oral daily administration of 0.8 mg of folic acid was 1.2×10⁻⁵ and 5.8×10⁻⁶ M, respectively.     

Analysis of micafungin in serum by capillary zone electrophoresis

A method is developed for measuring the concentration of micafungin, an echinocandin antifungal agent, in serum, using capillary zone electrophoresis. With a 0.1M borate buffer (pH 10.0) as the run buffer, detection is carried out at 200 nm. Pretreatment is performed by adding acetonitrile to serum (1:2) for deproteinization, allowing the supernatant fluid to be taken for measurement. The detection limit of the assay is 0.5 mg/L at a signal-to-noise ratio of 3.0. Coefficients of variation for intra- and inter-assay precision are 3.45-4.47% and 2.61-7.15%, respectively, at a nominal concentration of 5.0-25.0 mg/L. Their recovery rates are 92-110%. It is convenient for the monitoring of micafungin therapy in patients contracting clinically important deep mycoses.     

毛细管区带电泳法快速测定人血血清蛋白

人血血清蛋白电泳分析是临床上诊断多种疾病的常用生化指标 ,也是临床实验室检查的常规项目。目前采用的方法有醋酸纤维薄膜电泳和琼脂糖电泳 ,尤以前者为主。由于醋酸纤维薄膜电泳法操作繁琐 ,每一步骤均需手工完成 ,影响因素较多 ,初学者往往不易掌握 ,且测定的重复性较差。高效毛细管电泳是近几年来迅速发展起来的分离分析技术 [1,2 ]。用该技术分析人血清蛋白国内外虽有报道 ,但由于人血清中蛋白质组分甚为复杂 ,采用不同的分离方法和条件可出现不同数量的组分峰 ( 6、7个甚至 1 0个以上组分 ) ,与目前临床上常用的醋酸纤维薄膜电泳图谱…     

Determination of aristolochic acid by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis (CZE) method for the determination of aristolochic acid (AA) in dietary supplements and selected herbs is described. A clear separation of AA from other sample constituents was achieved within 5 minutes without any sample clean up. A mixture of 20 mM-morpholinethanesulphonic acid+10 mM-BisTrisPropane+0.2% hydroxyethylcelullose in 10% methanol serves as a background electrolyte. The linearity, accuracy, intra-assay and detection limit of the developed method are 200–6000 ng/mL, 95–103%, 3.5%, and 50 ng/ml, respectively. Ease of use, sufficient sensitivity and low running cost are the most important attributes of the CZE method. The proposed CZE method was compared with HPLC.     

Determination of fosfomycin in pus by capillary zone electrophoresis

A method is described for the determination of fosfomycin in pus by capillary zone electrophoresis with reversed electroosmotic flow, and indirect UV absorbance detection. Sample pre-treatment is limited to removal of proteins and cell debris by adding the double volume of methanol, followed by vortexing for few seconds, and centrifugation at 15,000 x g for 2 min. The supernatant is directly injected into the instrument. Fosfomycin is separated from sample constituents with a background electrolyte at pH 7.25 (25 mM benzoate buffer with 0.5 mM hexadecyltrimethylammonium bromide added, adjusted to pH with tris(hydroxymethyl)-aminomethane (TRIS)). Separation is carried out in a capillary with 50 microm I.D., 64.5 cm total length, 56.0 cm to the detector, at 25 degrees C with -25 kV voltage applied. Due to the low absorbance of the analyte, indirect UV detection was performed at 254 nm using a bubble cell capillary. Sample was injected by pressure (450 mbar s). Repeatability for fosfomycin in spiked pus (from 8 or 10 consecutive injections of three different series at concentrations of 100 microg/mL of the antibiotic) was between 2.4 and 8.2% relative standard deviation (RSD). Accuracy (expressed as recovery of fosfomycin determined by three independent analysis at 10, 100 and 300 microg/mL fosfomycin added to plain pus) was between 75 and 102%. Intermediate reproducibility (n = 9 at three different days) was between 2 and 12% RSD. Limit of detection and limit of quantitation were 4.5 and 15 microg/mL, respectively. The concentration of fosfomycin in pus of patients treated with the antibiotic ranged up to 240 microg/mL. The concentration of other anionic pus constituents identified beside chloride (acetate, succinate, lactate, phosphate) ranged between 20 and 7800 microg/mL.     

Determination of dyes in foodstuffs by capillary zone electrophoresis

A rapid method based on capillary zone electrophoresis coupled with photodiode-array detection has been developed to determine the dyes Tartrazine E-102, Sunset Yellow FCF E110, Amaranth E-123, New Coccine E-124, Patent Blue V calcium salt E-131 and Allura Red AC E-129 in foodstuffs. Separation was done by using a Bare CElect-FS75 CE column, using a 10 mM phosphate buffer at pH 11.0. Hydrodynamic injections at 0.5 p.s.i. for 4 s (21 nl of sample) and 20 kV separation voltage were used. The quantitation limits for the six dyes ranged from 3 to 6 microg/ml. A linear relationship between 3 to 95 microg/ml, with correlation coefficient better than 0.995 was obtained. This method has been applied to the determination of the studied dyes in beverages, jellies and syrups.     

Determination of S-nitrosoglutathione in erythrocytes by capillary zone electrophoresis

A method for separation and quantification of S-nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25 degrees C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S-nitrosoglutathione analyses were checked. The results evidenced that S-nitrosoglutathione is formed in erythrocytes treated with S-nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。