Liquid chromatographic assessment of total and protein-bound homocysteine in human plasma

Homocysteine present in human blood plasma is derivatized with thiol selective ultraviolet labelling reagent, 2-chloro-1-methylpyridinium iodide, and separated from other plasma thiol derivatives by high-performance liquid chromatography (HPLC) with detection at 312 nm. The separation is carried out isocratically on LiChrospher RP-18 column using mobile phase consisting of pH 2.5 0.04 M trichloroacetic acid buffer and methanol in the ratio 9:1 (v/v) pumped at 0.5 ml min⁻¹ at 40°C. The homocysteine S-pyridinium derivative elutes at 6.5 min. To determine total and protein-bound homocysteine it is necessary to cleave disulphide bounds by the use of tri-n-butylphosphine in order to form free sulfhydryl group. The method provides quantitative information on total and protein-bound homocysteine based on assays with derivatization after reduction of whole plasma, and derivatization after reduction of acid precipitated proteins. The calibration graph is linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol sensitivity and is reproducible; intra- and inter-day, relative standard deviation range from 1.79 to 5.09% and from 2.80 to 5.60%, respectively. The method is applied to the determination of total and protein-bound homocysteine in the plasma of healthy individuals.     

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.     

毛细管电泳-激光诱导荧光检测法测定卡托普利和N-乙酰-L-半胱氨酸

建立了一种高效、快速的毛细管电泳分离-激光诱导荧光(CE-LIF)检测卡托普利(CAP)和N-乙酰-L-半胱氨酸(NAC)的分析方法。采用1,3,5,7-四甲基-8-溴甲基-二氟二硼-二吡咯甲川(TMMB-Br)为柱前衍生试剂,在50 mmol/L磷酸盐缓冲溶液(pH=8.5)中,40℃下进行衍生反应10min。以荧光素为内标,25mmol/L硼酸盐缓冲溶液(pH=9.5)为电泳背景电解质,14min内达到基线分离。CAP和NAC的检出限分别为0.65nmol/L、0.76nmol/L。将该方法应用于人体尿液和血清中这两种物质的测定,回收率在97.0%~105.7%之间。     

碳糊电极阳极吸附伏安法测定卡托普利

在0.40 mol/L的HAc-NaAc(pH=4.8)缓冲溶液中,卡托普利(Captopril,CPT)在碳糊电极(CPE)上有一灵敏的吸附氧化峰,峰电位为0.23 V(vs.SCE)。该氧化峰的二阶导数峰电流与卡托普利的浓度在2.0×10-8～1.0×10-6mol/L(富集90 s)范围内呈良好的线性关系,相关系数为0.9966,检出限为8.0×10-9mol/L,并成功应用于卡托普利片含量的测定。探讨了卡托普利在碳糊电极上的伏安性质和电极反应机理。     

Liquid chromatographic method for the simultaneous determination of imipenem and sulbactam in mouse plasma

The first analytical method is developed and validated for the simultaneous determination of imipenem and sulbactam in mouse plasma. Sample treatment is based on plasma stabilization with 4-(2-hydroxyethyl)piperazine-ethanesulfonic acid (HEPES) (0.5 mol/L; pH 7.0)-water-ethylene glycol (2:1:1, v/v/v), precipitation of plasma proteins with acetonitrile, centrifugation, evaporation, and reconstitution with borate buffer. Analytical determination is carried out by high-performance liquid chromatography with diode-array detection. Chromatographic separation is achieved within 11 min on a C(18) column by gradient elution with borate buffer (0.1 mol/L, pH 7.2) and methanol. Imipenem and sulbactam are monitored at 295 and 230 nm, respectively. The overall interday accuracy is in the range of 95% to 100% and from 98% and 101% for imipenem and sulbactam, respectively. Interday precision is below 8% and 4% for imipenem and sulbactam, respectively. Limits of quantitation of imipenem and sulbactam are 0.05 and 1.0 microg/mL, respectively. The mean extraction recoveries are 94.5% and 94.2% for imipenem and sulbactam, respectively. The described method allows an accurate, simple, and rapid identification and quantitation of imipenem and sulbactam in mouse plasma. This method is applied to the analysis of imipenem and sulbactam in mouse plasma after drug administration.     

Preparative enantioseparation of propafenone by counter‐current chromatography using di‐n‐butyl l‐tartrate combined with boric acid as the chiral selector

This paper extends the research of the utilization of borate coordination complexes in chiral separation by counter‐current chromatography (CCC). Racemic propafenone was successfully enantioseparated by CCC with di‐n‐butyl l ‐tartrate combined with boric acid as the chiral selector. The two‐phase solvent system was composed of chloroform/ 0.05 mol/L acetate buffer pH 3.4 containing 0.10 mol/L boric acid (1:1, v/v), in which 0.10 mol/L di‐n‐butyl l ‐tartrate was added in the organic phase. The influence of factors in the enantioseparation of propafenone were investigated and optimized. A total of 92 mg of racemic propafenone was completely enantioseparated using high‐speed CCC in a single run, yielding 40–42 mg of (R)‐ and (S)‐propafenone enantiomers with an HPLC purity over 90–95%. The recovery for propafenone enantiomers from fractions of CCC was in the range of 85–90%.     

胶束电动毛细管色谱法测定硫酸多粘菌素E药物中的多粘菌素E1和E2

采用胶束电动毛细管色谱法(MECC)对硫酸多粘菌素E药物中的主要成分多粘菌素E1和E2进行了分离,并测定了多粘菌素E1、E2的含量。分别考察了电泳电压、表面活性剂种类、Brij-35(月桂醇聚氧乙烯醚)浓度、乙腈含量、磷酸盐缓冲液的pH值、氯化钠浓度等实验参数对实验结果的影响,从而确定了最佳的分离条件: 电泳电压为10 kV,运行缓冲液为含有30 mmol/L Brij-35、5%(体积分数)乙腈、0.167 mol/L氯化钠的磷酸二氢钠缓冲液(0.01 mol/L,pH 4.1)。在优化的实验条件下,E1和E2得到了较好的分离,分离度达到1.94。以多粘菌素E1为例,柱效和峰面积的日间及日内测定的相对标准偏差(RSD)均小于5%。E1和E2在硫酸多粘菌素E药物中的含量分别为67%和32%。该方法简便、快速、准确、重现性好。     

Direct tris(2,2'-bipyridyl)ruthenium (II) electrochemiluminescence detection of polyamines separated by capillary electrophoresis

Capillary electrophoresis (CE) with tris(2,2'-bipyridyl) ruthenium (II) (Ru(bpy)3(2+)) electrochemiluminescence (ECL) detection technique was developed for the analysis of four polyamines (putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)) analysis. The four polyamines contain different amine groups, which have different ECL activity. There are several parameters which influence the resolution and ECL peak intensities, including the buffer pH and concentrations, separation voltage, sample injection, electrode materials, and Ru(bpy)3(2+) concentrations. Polyamines are separated by capillary zone electrophoresis in an uncoated fused-silica capillary (50 cmx25 micro m (ID) filled with acidic phosphate buffer (200 mmol/L phosphate, pH 2.0) - 1mol/L phosphoric acid (9:1 v/v) and a separation voltage of 5 kV (25 micro A), with end-column Ru(bpy)3(2+) ECL detection. A 5 mmol/L Ru(bpy)3(2+) solution plus 200 mmol/L phosphate buffer (pH 11.0) is added into the reagent reservoir. The calibration curve is linear over a concentration range of two or three orders of magnitude for the polyamines. The analysis time is less than 25 min. Detection limits for Put and Cad are 1.9x10(-7) mol/L and 7.6x10(-9) mol/L for Spd and Spm, respectively. Intraday and interday relative standard deviations of ECL peak intensities are less than 8%. The main advantages of this CE-ECL detection technique for polyamines analysis presented herein are the omission of chemical derivatization of the analytes and the high selectivity.     

Determination of low molecular weight organic acids in soil,plants, and water by capillary zone electrophoresis

Determination of low molecular weight organic acids in soils and plants by capillary zone electrophoresis was accomplished using a phthalate buffer and indirect UV detection mode. The influence of some crucial parameters, such as pH, buffer concentration and surfactant were investigated. A good separation of seven organic acids was achieved within 5 min using an electrolyte containing 15 mmol L(-1) potassium hydrogen phthalate, 0.5 mmol L(-1) myristyltrimethylammonium bromide (MTAB), and 5% methanol (MeOH) (v/v) at pH 5.60, separation voltage -20 kV, and temperature 25 degrees C. The relative standard deviation (n=5) of the method was found to be in range 0.18-0.56% for migration time and 3.2-4.8% for peak area. The limit of detection ranged between 0.5 micro mol L(-1) to 6 micro mol L(-1) at a signal-to-noise ratio of 3. The recovery of standard organic acids added to real samples ranged from 87 to 119%. This method was simple, rapid and reproducible, and could be applied to the simultaneous determination of organic acids in environmental samples.     

Quantification of glutathione in plasma samples by HPLC using 4-fluoro-7-nitrobenzofurazan as a fluorescent labeling reagent

A rapid and highly sensitive high-performance liquid chromatograpy method with fluorescence detection has been developed for determination of glutathione (GSH) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized glutathione was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (77:23, v/v) at a flow rate of 1.0 mL/min with the column temperature 2°C. The eluted derivatives were fluorometrically detected at an excitation wavelength 470 nm and an emission wavelength 530 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1 μmol/L to 10.0 μmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra- and inter-day coefficient of variation being 6.3%, 6.9%, respectively. This method has been used to determine glutathione concentrations in plasma samples from healthy individuals.     

柱前衍生化高效液相色谱-荧光检测法测定桑叶中的1-脱氧野尻霉素

采用柱前衍生化高效液相色谱-荧光检测法测定了桑叶中的1-脱氧野尻霉素(DNJ)。用0.05 mol/L HCl提取桑叶中的DNJ,采用6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)试剂在pH 8.5硼酸盐缓冲液下对DNJ进行衍生化,以0.02 mol/L磷酸二氢钾缓冲液(pH 5.0)-乙腈(体积比为85∶15)为流动相,利用C18色谱柱(5 μm,250 mm×4.6 mm)分离,在激发波长为250 nm、发射波长为395 nm条件下进行荧光检测,DNJ的AQC衍生物与衍生化试剂的水解产物分离良好。方法的线性范围为0.5～25 mg/L,检出限为0.02 mg/L（S/N=3）。实验测得桑叶中DNJ含量为0.12%;回收率为96.1%～98.6%。     

Determination of tryptophan and kynurenine in human plasma by liquid chromatography-electrochemical detection with multi-wall carbon nanotube-modified glassy carbon electrode

A novel method was developed for the simultaneous determination of kynurenine and tryptophan by high‐performance liquid chromatography with electrochemical detection at multi‐wall carbon nanotube (MWCNT)‐modified glassy carbon electrode. The separation and detection conditions were optimized. The typical HPLC experiments were conducted by using a reversed‐phase ODS column with a mobile phase consisting of stock acetate buffer (pH 5)–methanol (4:1, v/v) using an isocratic elution at the flow rate of 1.0 mL/min. The obtained LODs for kynurenine and tryptophane were 0.5 and 0.4 µmol/L, respectively. The analytical method for human plasma samples was validated and confirmed by LC‐UV and LC‐MS. The recoveries were in the range of 84.8–110%, and the precision was lower than 5.9%. Copyright © 2010 John Wiley & Sons, Ltd.     

纳米纤维固相萃取-高效液相色谱法测定血浆中的5-羟色胺

基于纳米纤维的富集作用,建立了血浆中5-羟色胺(5-HT)的柱前衍生高效液相色谱-电化学检测(HPLC-ECD)分析方法.用10%(v/v)高氯酸溶液沉淀血浆蛋白,离心后取上清液,用0.1 mol/L 的四苯硼酸钠溶液调节pH值至8.5,加入衍生剂邻苯二甲醛溶液于30 ℃衍生4 min,经纳米纤维固相萃取柱净化富集后,...     

Vortex-assisted surfactant-enhanced-emulsification liquid-liquid microextraction for the determination of triazine herbicides in water samples by microemulsion electrokinetic chromatography

A novel method based on the combination of microemulsion electrokinetic chromatography (MEEKC) and vortex‐assisted surfactant‐enhanced‐emulsification liquid–liquid microextraction (VSLLME) was developed for the determination of five triazine herbicides (simazine, atrazine, ametryn, prometryn, and terbutryn) in water samples. The five triazine herbicides were baseline separated by using the microemulsion buffer containing a 10 mmol/L borate buffer at pH 9.5, 2.5% (w/v) SDS as surfactant, 0.8% (w/v) ethyl acetate as oil phase, and 6.0% (w/v) 1‐butanol as cosurfactant. The optimum extraction conditions of VSLLME were as follows: 100 μL chloroform was used as extraction solvent, 5.0 × 10^?5 mol/L Tween‐20 was chosen as the surfactant to enhance the emulsification, and the extraction process was carried out by vortex mixing for 3 min. Under these optimum experimental conditions, the calibration curve was linear in the range of 2.0–200.0 ng/mL, with the correlation coefficients (r²) varying from 0.9927 to 0.9958. The detection limits of the method varied from 0.41 to 0.62 ng/mL. The purposed method was applied to the determination of five triazine herbicides in real water samples, and the recoveries were between 80.6 and 107.3%.     

胶束电动毛细管色谱检测鱼肉中的七种生物胺

建立了一种利用胶束电动毛细管色谱同时检测鱼肉中组胺、腐胺、2-苯乙基胺、尸胺、色胺、亚精胺及精胺7种生物胺的方法。样品经6%过氯酸萃取后,由苯甲酰氯衍生化,以含0.06 mol/L脱氧胆酸钠的0.02 mol/L硼酸(pH 9.2)-甲醇(体积比为95∶5)混合液为电泳介质,电泳电压25 kV,温度25 ℃,检测波长214 nm,在12 min内实现了7种生物胺的完全分离。7种生物胺的浓度与其峰面积在一定的范围呈良好的线性关系,检出限除组胺为15 μg/g外,其余均为5 μg/g。迁移时间和峰面积的日内、日间相对标准偏差均小于5%。该法用于海鱼中7种胺类物质含量的测定,结果令人满意。     

Kinetics and mechanism of the addition of nucleophiles to alpha,beta-unsaturated thiol esters

Compounds containing the UV-absorbing chromophores p-methoxycinnamate, p-methoxycinnamide, or anthranilate and an alpha,beta- or alpha,beta,gamma,delta-unsaturated thiol ester (crotonyl or sorboyl) have been prepared. These compounds are subject to nucleophilic attack at the C=C conjugated to the thiol ester carbonyl group. The kinetics of the reactions of these thiol esters with N-acetyl-l-cysteine (NAC), N-acetylcysteamine, and N(2)-acetyl-L-lysine (NAL) have been studied, and the thiol addition products have been identified. The reaction rates increased at higher pH, and the reaction of NAC thiolate with a crotonyl thiol ester in 1:1 (v/v) acetonitrile/aqueous HEPES exhibited buffer catalysis as a result of protonation of the enolate intermediate. At the same concentration, NAC underwent approximately 300-fold more reaction than NAL with a crotonyl thiol ester at pH 9.8. Additionally, a crotonyl thiol ester was found to be 7.9 times more reactive than a sorboyl thiol ester toward NAC addition. These unsaturated thiol esters may serve as a means of covalently binding UVA and UVB sunscreens to the outer layer of skin to provide long-lasting protection.     

Reuse of the xylanase enzyme in the biobleaching process of the sugarcane bagasse acetosolv pulp

In this work, pretreatment-enzymatic series of the bagasse-sugarcane pulp and alkaline extraction of enzyme treated pulp were carried out. In the pretreatment an enzyme dose was utilized and acetosolv pulp suspension of 3% (w/v) with different solvents (distilled water, 0.05 mol/L acetate buffer pH 5.5 and 0.05 mol/L phosphate buffer pH 7.25) stirred at 85 rpm for 2 or 4 h. The enzymes used were pulpzyme and cartazyme, both commercial. The accompaniment of the enzymatic activity was carried out through measurement in initial and finish of each enzymatic pretreatment. The xylanase-treated pulps and xylanase-alkaline-extracted pulps were analyzed regarding kappa number and viscosity. Pulpzyme recovery was better in phosphate buffered medium (84, 46, and 23% for first, second, and third enzymatic treatment, respectively) although in aqueous medium reached only 2% for every treatments. However, the improvement of pulp properties was evidenced only in aqueous medium for pulpzyme. Cartazyme recovery was similar for both solvents (water and acetate buffer), reaching values around 19% for first enzymatic treatment and 9% for second one. Nevertheless, the pulp properties increased only in acetate buffered medium.     

Quantification of polyamines in human erythrocytes using a new near-infrared cyanine 1-(epsilon-succinimidyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium with CE-LIF detection

A novel near-infrared (NIR) cyanine 1-(epsilon-succinimidyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium (MeCy5-OSu) has been developed in our laboratory. Simultaneous determination of MeCy5-OSu-derivatized polyamines spermine (Spm), spermidine (Spd), cadaverine (Cad), and putrescine (Put) based on the separation by CE combined with diode LIF detection has been accomplished. The highest derivatization efficiency was achieved in 0.2 mol/L borate buffer (pH 8.8) for 20 min at 25 degrees C. Polyamine derivatives were separated within 14 min in the phosphate running buffer (pH 3) containing 50 mmol/L phosphoric acid, 40 mmol/L SDS, and 35% methanol v/v. Linearity of response was obtained in the range of 10-200 nmol/L. The detection limits (S/N = 3) for Spm, Spd, Cad, and Put were 0.8, 1, 3, and 2 nmol/L, respectively. The proposed method has been successfully applied to the analysis of polyamines in erythrocytes of two healthy persons and one cancer patient. Average recoveries for erythrocyte samples were 93.6-106% and coefficients of variation ranged from 1.8 to 5.4%. The analysis of polyamines in erythrocytes can be used for studying the relationship between their changes and the carcinogenesis process involved in erythrocytes.     

Simultaneous separation and determination of seven chelating agents using high‐performance liquid chromatography based on statistics design

We describe an optimization approach to determine simultaneously occurring chelating agents (glycine, malonic acid, citric acid, glycolic acid, lactic acid, DL‐malic acid, and ethylenediaminetetraacetic acid) in an electroplating effluent using high‐performance liquid chromatography. With chromatography signal area and overall resolution considered as responses, detection conditions were optimized via multiple functions combined with response surface methodology and Plackett–Burman design. Optimized detection conditions were as follows: 15 mmol/L ammonium phosphate buffer (pH 2.5), a 94:6 v/v ratio of ammonium phosphate buffer/acetonitrile, a column temperature of 23.3°C, and a mobile phase flow rate of 1 mL/min. The experimental values conformed to the predicted values and were repeatable (relative standard deviation < 6.4%) and linear (r² > 0.991) over concentration ranges of 1–100 µmol/L. Moreover, the quantification limit (signal‐to‐noise ratio = 10) and the detection limit (signal‐to‐noise ratio = 3) ranged from 0.03 to 0.15 µmol/L and from 0.01 to 0.04 µmol/L, respectively. These results indicate that high‐performance liquid chromatography coupled with statistical design may be a simple and rapid method for simultaneously determining multiple chelating agents in electroplating wastewater effectively.     

Chiral separation of ephedrine isomers by capillary electrophoresis using bovine serum albumin as a buffer additive

A method utilizing bovine serum albumin (BSA) as buffer additive for chiral separation by means of capillary electrophoresis is described. Parameters that affect chiral separation, such as buffer pH, buffer concentration, BSA concentration, and organic modifier, are investigated. Baseline resolution of ephedrine-pseudoephedrine and norephedrine-norpseudoephedrine isomers are achieved in an uncoated capillary with a 20 mmol/L phosphate buffer at pH 9.0 in the presence of 10 micromol/L BSA and 15% (v/v) 2-propanol at 25 degrees C. The developed method can be applied for the analysis of ephedra plant extracts that contain the four test drugs.