药物间置换相互作用的毛细管电泳-迎头分析法研究

将18-甲基炔诺酮加到人血清白蛋白-酮洛芬平衡溶液中,室温孵育达平衡后应用毛细管电泳-迎头分析法研究了18-甲基炔诺酮和酮洛芬在人血清白蛋白分子上的置换相互作用。一大体积样品虹吸进样至未涂层毛细管(65 cm×50 μm i.d.,有效长度35 cm),进样时间80 s,毛细管两端高度差11 cm,工作电压10 kV,运行缓冲液为67 mmol/L磷酸盐缓冲液(pH 7.4),游离酮洛芬浓度由前沿峰的高度直接测定。当酮洛芬样品溶液中酮洛芬的总浓度分别为100 μmol/L和200 μmol/L时,随着18-甲基炔诺酮加入量的增加(18-甲基炔诺酮的浓度由0增至200 μmol/L),游离酮洛芬的浓度分别从22.4 增至26.4 μmol/L和从82.1增至106.2 μmol/L。由上面的结果可推论:高浓度的18-甲基炔诺酮可将酮洛芬从它的第二类结合位点上置换出来。     

酮洛芬与蛋白结合作用的高效液相色谱-迎头分析法研究及其与高效毛细管电泳-迎头分析法的比较

采用高效液相色谱-迎头分析法(HPLC-FA),以67 mmol/L (pH 7.4, I=0.17 mol/L) 的等渗磷酸盐缓冲液为流动相,Pinkerton GFF　Ⅱ-S5-80内表面反相柱(150 mm×4.6 mm i.d., 5 μm)为固定相,254 nm下检测,研究了酮洛芬与人血清白蛋白(HSA)的结合作用,通过非线性回归参数估算求得酮洛芬与HSA的结合参数。与高效毛细管电泳-迎头分析法(HPCE-FA)相比,HPLC-FA法具有高灵敏度的优势,但进样量较大,分析时间较长。HPLC-F     

Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry

Dextromethorphan was extracted from human plasma samples (100 μL) using MonoTip C₁₈ tips, which are packed with C₁₈-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200 μL of distilled water and 50 μL of 1 mol/L glycine–sodium hydroxide buffer (pH 10). The mixture was extracted to the C₁₈ phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C₁₈ phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.5 μm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320 ng/mL of plasma, and the limit of detection was 1.25 ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug.     

Improved rapid assay of plasma uric acid by short-end injection capillary zone electrophoresis

A rapid and simple short-end injection capillary zone electrophoresis method was developed for the quantification of plasma uric acid. The separation was performed in an uncoated fused-silica capillary (50 μm ID, 60 cm total length, 10.2 cm effective length) by using as a background electrolyte a 75 mmol/L glycylglycine solution titrated with NaOH 5 mol/L to pH 9.0, a voltage of 28 kV, a cartridge temperature of 15 °C, and direct UV detection at 292 nm. Under optimized conditions, uric acid was determinate in little more than 1 min (1.076 minutes). In order to verify the accuracy of the analysis, urate levels were measured in 543 apparently healthy volunteers by the new assay and our previous method, and the obtained data were compared by Passing–Bablock regression, Bland–Altman test, and a new regression-based approach, which showed a good agreement between two methods.     

Determination of ibuprofen and tetrazepam in human urine by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatography method (MEKC) is proposed for the determination of ibuprofen and tetrazepam in human urine samples over a concentration range of therapeutic interest. A fused silica capillary (60 cm × 75 μm) is used. Ibuprofen and tetrazepam are detected via UV detection at 220 and 228 nm, respectively. Separation is performed at 25 °C and at a separation voltage of 30 kV, with 15 mM borate buffer (pH 10.2) containing 40 mM sodium dodecylsulfate as the electrolyte solution. Under these conditions the analytes were separated in <11 min. Sulfamethazine is used as an internal standard. Prior to determination, the samples are purified and enriched by means of an extraction–preconcentration step with a preconditioned C₁₈ cartridge and by eluting the compounds with methanol. Good linearity, accuracy, precision, robustness and solution stability were achieved for the technique. Detection limits of 200 μg L⁻¹ for ibuprofen and 300 μg L⁻¹ for tetrazepam were obtained. These analytes were then determined in real urine using the technique.     

Cholinesterase inhibition based determination of pancuronium bromide in biological samples

Pancuronium bromide (PCBr) inhibition effect on enzyme cholinesterase from pooled human serum (Che, EC 3.1.1.8 acylcholine acylhydrolase) was used for development of a spectrophotometric kinetic method for PCBr determination in human serum and urine. Optimal conditions for the basic and inhibitor reactions were established: pH=7.7 and substrate concentration c(benzoylcholine chloride)=1.33 mmol/L. Kinetic parameters were also determined: Michaelis-Menten’s constant K_M=0.40 mmol/L, maximal reaction rate V_max=52.2 μmol/L min, inhibition constant K_i=0,56 μmol/L and IC₅₀=1.31 μmol/L. Linear dependence between the reaction rate and inhibitor concentration exists in PCBr concentration range 8.20–68.25 nmol/L, which corresponds to the real sample concentrations from 0.328 to 2.730 μmol/L. The method detection and quantification limits were 2.01 nmol/L and 6.67 nmol/L, respectively. Precision of the method was tested for three pancuronium concentrations (10.70, 29.35 and 51.25 nmol/L). Relative standard deviation (RSD) was in the range 0.15–7.45%. Accuracy was examined by standard addition method. Influence of the substances usually present in serum and urine on the reaction rate was tested. The developed method was applied for PCBr content determination in serum model samples, urine model samples and in urine taken during surgery. The method has good sensitivity, accuracy, precision and it is suitable for clinical practice.      

Stacking and quantitative analysis of lovastatin in urine samples by the transient moving chemical reaction boundary method in capillary electrophoresis

A simple, sensitive, and useful concentration method for lovastatin (Lvt) in urine has been developed based on the transient moving chemical reaction boundary method (tMCRBM) in capillary electrophoresis. The MCRB is formed with acidic sample buffer (Gly-HCl) and alkaline running buffer (Gly-NaOH). The following optimal conditions were determined for stacking and separation: electrophoretic buffer of 100 mM Gly- NaOH (pH 11.52), sample buffer of 20 mM Gly-HCl (pH 4.93), fused-silica capillary of 76 cm × 75-μm i.d (67 cm from detector), sample injection at 14 mbar for 3 min. A 21- to 26-fold increase in peak height was achieved for detection of Lvt in urine under the optimal conditions compared with normal capillary zone electrophoresis. By combining the sample pretreatment procedure with the stacking method, the sensitivity of Lvt in urine was increased by 105- to 130-fold. The limits of detection (LOD) and quantification (LOQ) for Lvt in urine were decreased to 8.8 ng/mL and 29.2 ng/mL, respectively. The intra-day and inter-day precision values (expressed as RSD) were 2.23–3.61% and 4.03–5.05%, respectively. The recoveries of the analyte at three concentration levels changed from 82.65 to 100.49%.     

Gaseous sample introduction for the determination of silicon by ICP-AES

The generation of volatile species of silicon as a means to introduce silicon into an inductively coupled plasma has been studied. It is based on the reaction between silicon and fluoride ions in sulfuric acid media and it was carried out using different flow injection mountings. The first mounting works with an injection of 100 μL concentrated sulfuric acid and 150 μL silicon standard solution in a continuous 0.05 mol L^–1 NaF solution flow. The method shows a linear response between the intensity of emission at 251.611 nm line and the silicon concentration from 0.1 to 200 μg mL^–1, with a reproducibility of 2% and a detection limit of 0.004 μg mL^–1. The second mounting produces the volatile species by the reaction between two opposed aerosol flows in a home-made nebulization chamber. This chamber has a Cross-Flow and a Meinhard nebulizer at either end. A linear response ranging from 0.1 to 1000 μg mL^–1 of silicon solution is obtained and the reproducibility rises to 8%.The detection limit reached is 0.02 μg mL^–1. The silicon content in real water samples was determined by applying both the above-mentioned methods and a third method for reference. Received: 6 November 1996 / Revised: 19 December 1996 / Accepted: 3 January 1997     

Determination of EDTA in used fixing solutions by capillary electrophoresis

A capillary electrophoretic method for the determination of EDTA has been developed. EDTA was converted to Ni(II)-EDTA prior to separation, separated from Fe(III)-EDTA, thiosulphate, bromide and polythionates using a fused silica capillary (57 cm × 75 μm I.D.) filled with a borate buffer (50 mmol L^–1; pH 8.5; applied voltage, 30 kV) and detected at 214 nm. The separation time is about 6 min. The detection limit achieved is 2 × 10^–6 mol L^–1 for EDTA. This method was applied for the determination of free EDTA in used fixing solutions. Received: 27 February 1998 / Revised: 28 April 1998 / Accepted: 20 May 1998     

Determination of EDTA in used fixing solutions by capillary electrophoresis

A capillary electrophoretic method for the determination of EDTA has been developed. EDTA was converted to Ni(II)-EDTA prior to separation, separated from Fe(III)-EDTA, thiosulphate, bromide and polythionates using a fused silica capillary (57 cm × 75 μm I.D.) filled with a borate buffer (50 mmol L^–1; pH 8.5; applied voltage, 30 kV) and detected at 214 nm. The separation time is about 6 min. The detection limit achieved is 2 × 10^–6 mol L^–1 for EDTA. This method was applied for the determination of free EDTA in used fixing solutions. Received: 27 February 1998 / Revised: 28 April 1998 / Accepted: 20 May 1998     

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R _s = 1.80 for naproxen to R _s = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R _s value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R _s = 0.89).     

The use of Chromazurol S in the presence of a mixture of cationic and non-ionic surfactants for the spectrophotometric determination of iron in cereals

Simple and sensitive methods for the spectrophotometric determination of iron(III) in food, based on the formation of coloured complexes of Fe(III) with Chromazurol S (CAS) in the presence of tetradecyltrimethylammonium bromide (TTA) or octadecyltrimethylammonium chloride (ODTA) and Triton X-100 (TX100), have been developed. Optimum pH and the concentrations of CAS, TTA, ODTA, and TX100 ensuring maximum absorbance have been determined. For the Fe-CAS-TTA-TX100 system the molar absorptivity is 1.12 × 10⁵ L/(mol cm) at 650 nm; for Fe-CAS-ODTA-TX100 it is 1.35 × 10⁵ L/(mol cm) at 659.5 nm. Beer’s law was obeyed for iron concentration in the range 0.08–0.56 μg/mL for the complex Fe-CAS-TTA-TX100 and 0.08–0.64 μg/mL for Fe-CAS-ODTA-TX100. The influence of several interfering ions has been discussed. The stoichiometry of the complexes was established by applying Job’s method. The more sensitive method, based on the Fe-CAS-ODTA-TX100 system, has been applied to the determination of iron in cereals. To evaluate the accuracy of the elaborated method, the determined content of Fe was compared to the declared value as well as to the result obtained by the reference ICP-OES method.     

Rapid electrophoretic monitoring of the anaesthetic ketamine and its metabolite norketamine in rat blood using a contactless conductivity detector to study the pharmacokinetics

A method of capillary electrophoresis with contactless conductivity detection has been developed for non‐enantioselective monitoring the anaesthetic ketamine and its main metabolite norketamine. The separation is performed in a 15 μm capillary with an overall length of 31.5 cm and length to detector of 18 cm; inner surface of the capillary is covered with a commercial coating solution to reduce the electroosmotic flow. In an optimised background electrolyte with composition 2 M acetic acid + 1% v/v coating solution under application of a high voltage of 30 kV, the migration time is 97.1 s for ketamine and 95.8 s for norketamine, with an electrophoretic resolution of 1.2. The attained detection limit was 83 ng/mL (0.3 μmol/L) for ketamine and 75 ng/mL (0.3 μmol/L) for norketamine; the number of theoretic plates for separation of an equimolar model mixture with a concentration of 2 μg/mL was 683 500 plates/m for ketamine and 695 400 plates/m for norketamine. Laboratory preparation of rat blood plasma is based on mixing 10 μL of plasma with 30 μL of acidified acetonitrile, followed by centrifugation. A pharmacokinetic study demonstrated an exponential decrease in the plasma concentration of ketamine after intravenous application and much slower kinetics for intraperitoneal application.     

Spectrophotometric determination of total inorganic arsenic with hexamethylene ammonium-hexamethylenedithiocarbamate in nonionic triton X-100 micellar media

We have developed a cost-effective and sensitive spectrophotometric method for the determination of arsenic at trace level using a new reagent, hexamethylene ammonium-hexamethylenedithiocarbamate (HMA-HMDTC). Here we show that arsenic reacts with HMA-HMDTC in acidic conditions to yield the As(HMDTC)₃ complex. We studied the Beer’s law at 256 nm, which showed linearity over the concentration range 0.2–1.0 μg/mL of arsenic. We have shown that molar absorptivity, Sandell’s sensitivity and the detection limit of the method are 6.06 × 10⁴ L/mol cm, 0.0012 μg/cm² and 0.060 μg/mL, respectively. We have applied this new method to the determination of arsenic in drinking water.     

Oriented antibody immobilization for atrazine determination by a flow-through fluoroimmunosensor

An atrazine flow-through fluoroimmunosensor was developed, based on an oriented antibody covalently bound to Protein-A (Prot-A) immobilized on Controlled Pore Glass (CPG). Atrazine was detected “in-situ” by placing the immobilized antibody in the optical path of the flow cell. Immobilization of 30 μg of polyclonal anti-atrazine antibody on 0.5 g of Prot-A-CPG provided the highest sensitivity. The effect of several solvents on the covalently immobilized antibodies regeneration was evaluated, the optimum conditions being achieved by pumping 5% acetonitrile (pH = 3) at 0.15 mL/min for 100 s. The detection limit of the immunosensor was 0.7 μg/L and the reproducibility was 2% and 4% for 5 μg/L and 40 μg/L, respectively, in the optimum working concentration range (0.7–50 μg/L). This device allowed 12 samples per hour to be analyzed and had a life-time of 200 assays. Simazine and desisopropylatrazine (DIA) were not cross-reactive, desethylatrazine (DEA) has a cross-reactivity of 8% and propazine and prometryn of 44% and 27%, respectively. The immunosensor was applied to the determination of atrazine in tap and ground water samples spiked at the ?10 and 30 μg/L concentration level. Received: 30 April 1999 / Revised: 16 July 1999 / Accepted: 21 July 1999     

Analysis of fenvalerate in its formulations and environmental samples using spectrophotometry

Two rapid, simple, sensitive, and nonextractive spectrophotometric methods were described for the determination of fenvalerate (syntheitic pyrethroid) in its formulations, water and grain samples. The methods are based on the hydrolysis of fenvalerate with methanolic NaOH to form 3-phenoxybenzaldehyde. The resultant aldehyde group was condensed with 4-aminoantipyrine in the basic medium to form a red product having λ_max at 489 nm or condensed with4,4′-methylene-bis-m-nitroaniline to form a plae red product with an absorption maximum of 513 nm. Beer’s law was obeyed over the range 0.6–10 μg/mL (molar absorptivity 2.184 × 10⁴ L/mol cm) for 4-aminoantityrine and over the range of 1–12 μg/mL (molar absorptivity 4.162 × 10⁴ L/mol cm) for 4,4′-methylene-bis-m-nitroaniline. The formations of color derivatives with the reagents are instantaneous and stable for 40 and 32 h, respectively. The methods were rapid, simple, sensitive, and free from nontarget species. The proposed methods have been applied to the determination of fenvalerate in its formulations and environmental samples. The text was submitted by the authors in English.     

A Simple Liquid Chromatography Method for the Determination of Captopril in Urine

A simple and specific method for the determination of total captopril in human urine was developed. 2-Chloro-1-methylquinolinium tetrafluoroborate was used as a thiol precolumn derivatizing reagent after conversion of a disulfide forms to free captopril with tris(2-carboxyethyl)phosphine hydrochloride. The 2-S-quinolinium derivative of captopril was separated on a Zorbax SB C-18 column using reversed-phase ion-paring chromatography and monitored by spectrophotometric detector at 355 nm. The calibration curve for the derivatized captopril showed linearity in the range 0.1–200 μmol L⁻¹ of urine with a regression coefficient corresponding to 0.9999. The detection and quantitation limits were 0.05 and 0.1 μmol L⁻¹, respectively. The intra-day imprecision was from 0.01 to 10.58%. This method can be used for routine clinical monitoring of the thiol-drug. Omission of the reduction step gives result for concentration of the reduced form of captopril.     

Quantification of plasma homocitrulline using hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry

Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d₇-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice (n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids (p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples.     

Capillary electrophoresis study of human serum albumin binding to basic drugs

Summary The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.     

Study on the protein binding of ketoprofen using capillary electrophoresis frontal analysis compared with liquid chromatography frontal analysis

A method of capillary electrophoresis frontal analysis (CEFA) is developed for the first time to study the binding of ketoprofen to human serum albumin (HSA) and compared with high-performance liquid chromatography frontal analysis (LCFA). The separation is performed in an uncoated fused-silica capillary (60-cm x 75- micro m i.d., 50-cm effective length) with a phosphate buffer (pH 7.4, ionic strength of 0.17M) as the running buffer. The applied voltage is 13 kV and the detection is set at 254 nm. A trapezoidal peak of the unbound ketoprofen appears after HSA elution in the electropherogram. The plateau height of the peak is employed to determine the unbound concentration of ketoprofen in the HSA equilibrated sample solution. The CEFA method provides the advantages of small sample injection volume and rapidity and the disadvantage of low sensitivity compared with LCFA. CEFA is applicable to the binding parameter estimation of ketoprofen to the secondary binding site; an association constant (K(2)) of 0.24 x 10(6)M(-1) and the number for the binding site per molecule HSA of 2.54 is estimated. In contrast, LCFA measures parameters for both primary and secondary sites, which are 1.05 x 10(6)M(-1) and 0.94 for K(1) and n(1), respectively, and 0.12 x 10(6)M(-1) and 3.16 for K(2) and n(2), respectively. It is found that ketoprofen binds mainly at the primary site at a molecular ratio of ketoprofen versus HSA lower than 0.75, and the binding at the secondary site occurs at a higher ratio.