Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into uncoated fused silica capillary (40 cm, 50 microm I.D.), then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. It was unnecessary to have sample pretreatments. During method validation, calibration plots were linear (r>or=0.9982) over a range of 150-3,000 ng/mL for M and C, 250-5,000 n g/mL for MA, and 50-1,000 ng/mL for K. The limits of detection were 15 ng/mL for M and C, and 5 ng/mL for MA and K (S/N=3, sampling 500 s at 10 kV). Comparing with capillary zone electrophoresis, the results indicated that this stacking method could increase 6,000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS. This method was feasible for application to detect trace levels of abused drugs in forensic analysis.     

A simple micellar electrokinetic capillary chromatographic method for the quantitative analysis of organic expectorants

A simple and rapid micellar electrokinetic capillary chromatographic method is described for the separation and quantification of five expectorant drugs, including ambroxol (AMB), bromhexine (BRM), carbocysteine, guaiacol and guaifenesin. The drugs were separated in a mixed solution of phosphate buffer (35 mM; pH 3.90) and acetonitrile (75:25, v/v) with sodium dodecylsulfate (120 mM) as the micellar source, and the separated drugs were directly monitored with UV detector (200 nm). Several key parameters affecting the separation and analysis of the drugs were studied and optimized. Based on the corrected peak-area ratios of the drugs to an internal standard (7-hydroxy-4-methyl-coumarin) versus the concentration of the drugs, the established method is applicable to quantify AMB and BRM each over 20-150 microM, carbocysteine over 100-1500 microM, guaiacol and guaifenesin each over 10-150 microM. The detection limits (S/N=3; 0.5 psi, 5 s injection) of the method for the analytes are in the range of 3.0-5.0 microM (except that of carbocysteine at 30 microM). The precision (relative standard deviation) and accuracy (relative error) of the method for the intra-day (n=3) and inter-day (n=5) analyses of the analytes at three levels are all below 4%. The method is speedy with a run time of about 6 min for the analysis of the five analytes. Application of this method to the analysis of AMB and BRM in pharmaceutical preparations or AMB in the urine of a dosed subject proved simple and effective.     

On-line stacking capillary electrophoresis for analysis of methotrexate and its eight metabolites in whole blood

Therapeutic drug monitoring of methotrexate (MTX) and its metabolites is significant for the evaluation of treatment response of acute lymphoblastic leukemia and the prevention of adverse effects. Those eight metabolites including 7-hydroxymethotrexate, MTX-polyglutamate derivatives (MTX-(Glu)(n), n=2-7), and 2,4-diamino-N(10)-methylpteroic acid, especially the MTX-(Glu)(n), only exist in blood cells and also present antifolate effects. Therefore, whole blood analysis has importance in clinical therapy. This study combined solid-phase extraction and on-line stacking capillary electrophoresis to examine the levels of MTX and its eight metabolites in whole blood. A higher conductivity buffer (HCB) was used to accumulate large amount of samples into a narrow zone, which resulted in effective stacking and sharp peaks. A fused-silica capillary was filled with phosphate buffer (80 mM, pH 2.5) containing 15% v/v tetrahydrofuran and 100 mM sodium dodecyl sulfate. Then HCB (100 mM phosphate, pH 2.5; 1 psi, 60 s) was injected for accumulating large volume of analytes (1 psi, 200 s). Owing to the pH difference between sample zone and HCB, dynamic pH junction was generated for focusing. Meanwhile, sweeping made further stacking by using sodium dodecyl sulfate in phosphate buffer. The limits of detection (S/N=3) were 0.1 microM for MTX, 0.2 microM for 7-hydroxymethotrexate and 2,4-diamino-N(10)-methylpteroic acid, 0.3 microM for MTX-(Glu)(n=2--5), and 0.5 microM for MTX-(Glu)(n=6--7). During validation, this method showed good linearity (r> / =0.9914) and reproducibility (relative standard deviation and relative error, both less than 13%). The applications were performed to monitor blood MTX and its metabolites in acute lymphoblastic leukemia patients. The blood concentrations could provide some information related to therapeutic response and adverse effects.     

Electrochemiluminescence quenching as an indirect method for detection of dopamine and epinephrine with capillary electrophoresis

An electrochemiluminescence (ECL) inhibition method was developed as an indirect detection method for the determination of dopamine and epinephrine separated by capillary electrophoresis (CE). When the concentration of Ru(bpy)(3) (2+) was 50 muM diluted by 50 mM phosphate (pH 8.5) in the cell and 0.5 M tripropylamine (TPA) was added to the running buffer (10 mM phosphate, pH 9.0), an inhibition of ECL of the Ru(bpy)(3) (2+)/TPA system by the analytes was observed. Under the optimized conditions, the relative standard deviations of migration time and negative peak area were less than 1% and 3%, respectively, for 1 microM dopamine or 1 microM epinephrine (n = 10). Linear ranges of 0.1-10 microM for both analytes and the detection limits (signal-to-noise ratio S/N = 3) of 10 nM for dopamine and 30 nM for epinephrine were obtained.     

Micellar electrokinetic capillary chromatographic method for the quantitative analysis of uricosuric and antigout drugs in pharmaceutical preparations

A simple MEKC method is described for the separation and quantification of seven widely used uricosuric and antigout drugs, including allopurinol (AP), benzbromarone (BZB), colchicine (COL), orotic acid (OA), oxypurinol (OP), probenecid (PB), and sulfinpyrazone (SPZ). The drugs were separated in a BGE of borate buffer (45 mM; pH 9.00) with SDS (20 mM) as the micellar source and the separated drugs were directly monitored with a UV detector (214 nm). Several parameters affecting the separation and analysis of the drugs were studied. Based on the normalized peak-area ratios of the drugs to an internal standard versus the concentration of the drugs, the method is applicable to quantify BZB, COL, and SPZ (each 5-200 microM), AP, OA, OP, and PB (each 10-200 microM) with detection limits (S/N = 3, 0.5 psi, 5 s injection) in the range of 0.6-4.0 microM. The precision (RSD; n = 5) and accuracy (relative error; n = 5) of the method for intraday and interday analyses of the analytes at three levels (30, 120, and 180 microM) are below 4% (n = 3). The method was demonstrated to be suitable for the analysis of AP and COL in commercial tablets with speed and simplicity.     

Optimization of sample stacking for the simultaneous determination of nonsteroidal anti-inflammatory drugs with a wall-coated histidine capillary column

A wall-coated histidine capillary column was developed for the on-line preconcentration of nonsteroidal anti-inflammatory drugs (NSAIDs) in capillary electrochromatography (CEC). A wide variety of experimental parameters, such as the sample buffer, background electrolyte (BGE) composition, concentration, sample plug lengths, water plug, and the effect of organic modifiers were studied. The relationship between peak height and injection times for the NSAIDs by variation of sample and BGE buffer concentration was investigated. On addition of sodium chloride (0.3-0.6%) to the sample zone, the stacking efficiency was increased. With acetate buffer (100 mM, pH 5.0)/ethanol (20% v/v) as BGE and sample solution in acetate buffer (0.2 mM, pH 5.0)/ethanol (20% v/v)/NaCl (0.3% w/v), NSAIDs could be determined at low microM levels without sample matrix removal. The detection limit was 0.096 microM for indoprofen, 0.110 microM for ketoprofen, 0.012 microM for naproxen, 0.023 microM for ibuprofen, 0.110 microM for fenoprofen, 0.140 microM for flurbiprofen, and 0.120 microM for suprofen. The method could be successfully applied to the simultaneous determination of NSAIDs in urine. The recoveries were better than 82% for all the analytes. The present method enables simple manipulation with UV detection for the determination of NSAIDs at low concentration levels in complex matrix samples.     

Separation and quantitation of fructose-6-phosphate and fructose-1 ,6-diphosphate by LC-ESI-MS for the evaluation of fructose-1,6-biphosphatase activity

An LC-ESI-MS method was developed for the identification and quantification of fructose-1,6-biphosphate (F1,6BP) and fructose-6-phosphate (F6P), respectively the substrate and the product of the enzymatic reaction catalysed by fructose-1,6-bisphosphatase (F1,6BPase). F1,6BPase, expressed predominantly in liver and kidney, is one of the rate-limiting enzymes of hepatic gluconeogenesis and has become a target for the development of new drugs for type 2 diabetes. The two sugar phosphates were separated on a Phenomenex Luna NH2 column (150 mm x 2.0 mm id) using the following mobile phase: 5 mM triethylamine acetate buffer/ACN (80:20) v/v in a linear pH gradient (from pH = 9 to 10 in 15 min) at the flow rate of 0.3 mL/min. The detection was performed with an IT mass spectrometer in negative polarity (full scan 100-450 m/z) and in SIM mode on the generated anions at m/z = 339 (F1,6BP) and m/z = 259 (F6P). Under the optimised final conditions, the method was validated for accuracy, specificity, precision (inter- and intradays RSD comprised between 1.0 and 6.3% over the range of concentrations used), linearity (50-400 microM), LODs (0.44 microM) and LOQs (1.47 microM), and the method was applied to F6P determination in the F1,6BPase catalysed hydrolysis of F1,6BP.     

Screening of drug metabolism by CE

The use of CE for rapid assessment of metabolic stability of drugs with cytochrome P450 (CYP) enzymes, based on relative rates of reduced nicotinamide adenine dinucleotide phosphate (NADPH) consumption and nicotinamide adenine dinucleotide phosphate (NADP) production, was investigated. The separation conditions were as follows: capillary, 80.5 cm (75 microm id, 72 cm effective length for UV detection, 58 cm effective length for fluorescence detection); 25 mM sodium phosphate buffer (pH 8.8); 28 kV (80 microA) applied voltage; UV, 260 nm; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 25 degrees C. For UV detection, the incubation conditions were as follows: CYP3A4: 20 pmol/mL; NADPH: 1 mM; EDTA: 1 mM; concentration of the substrate: 5-10 times its reported literature K(m) value; temperature: 37 degrees C; incubation time: 15 min. For fluorescence detection, the concentrations were reduced to CYP3A4: 4 pmol/mL, NADPH: 20 microM, EDTA: 20 microM and substrate: 10 microM. Blank incubations were performed in the absence of substrate. Compared with the blank, significant differences were found for the consumption of NADPH and the production of NADP. The development of this assay system allows rapid assessment of metabolic stability relative to standard compounds, as well as potential identification of the major CYP involved in the metabolism. It would reduce the backlog of compounds that require LC/MS analysis, and thereby expedite the process of metabolic stability screening.     

Pulsed amperometric detection with poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips for the determination of EPA priority pollutants

A miniaturized analytical system for separation and detection of three EPA priority phenolic pollutants, based on a poly(dimethylsiloxane)-fabricated capillary electrophoresis microchip and pulsed amperometric detection is described. The approach offers a rapid (less than 2 min), simultaneous measurement of three phenolic pollutants: phenol, 4,6-dinitro-o-cresol and pentachlorophenol. The highly stable response (RSD = 6.1%) observed for repetitive injections (n > 100) reflects the effectiveness of Au working electrode cleaned by pulsed amperometric detection. The effect of solution conditions, separation potential and detection waveform were optimized for both the separation and detection of phenols. Under the optimum conditions (5.0 mM phosphate buffer pH = 12.4, detection potential: 0.7 V, separation potential: 1200 V, injection time: 10 s) the baseline separation of the three selected compounds was achieved. Limits of detection of 2.2 microM (2.8 fmol), 0.9 microM (1.1 fmol), and 1.3 microM (1.6 fmol) were achieved for phenol, 4,6-dinitro-o-cresol and pentachlorophenol, respectively. A local city water sample and two over-the-counter sore-throat medicines were analyzed in order to demonstrate the capabilities of the proposed technique to face real applications.     

Cation-selective exhaustive injection and sweeping MEKC for direct analysis of methamphetamine and its metabolites in urine

Direct analysis of methamphetamine, amphetamine, and p-hydroxymethamphetamine in urine was achieved by cation-selective exhaustive injection and sweeping micellar EKC. A bare fused-silica capillary (40 cm, 50 microm id) was filled with phosphate buffer (80 mM, pH 3, containing 20% ACN). Then a high-conductivity buffer (100 mM phosphate, pH 3; 6.9 kPa for 2.5 min) was injected. Samples were loaded using electrokinetic injection (10 kV, 600 s) which created long zones of cationic analytes. To enhance sensitivity by sweeping, the stacking step was performed using a phosphate buffer (50 mM, pH 3, containing 20% ACN and 100 mM SDS) at -20 kV before separation by MEKC. This method was capable of detecting the analytes at ppb levels. The calibration plots were linear (r(2) >or= 0.9948) over a range of 100-5000 ng/mL for methamphetamine, and 100-2000 ng/mL for amphetamine and p-hydroxymethamphetamine. The LODs (S/N = 3) were 20 ng/mL for methamphetamine, and 15 ng/mL for amphetamine and p-hydroxymethamphetamine. The method was applied to analysis of 14 urine samples of addicts and is suitable for screening suspected samples for forensic purposes. The results showed good agreement with fluorescence polarization immunoassay and GC-MS.     

Analysis of ethambutol and methoxyphenamine by capillary electrophoresis with electrochemiluminescence detection

A capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection method for the analysis of ethambutol (EB) and methoxyphenamine (MP) has been investigated. Complete separation of EB and MP was achieved in 8 min using a background electrolyte of 20 mM sodium phosphate at pH 10.0 and a separation voltage of 9 kV. ECL detection was performed with an indium/tin oxide (ITO) working electrode biased at 1.4 V (versus a Pt wire reference) in a 200 mM sodium phosphate buffer (pH 8.0) containing 3.5 mM Ru(bpy)3(2+) (where bpy = 2,2'-bipyridyl). Linear correlation (r > or = 0.993) between ECL intensity and drug concentration was obtained in the range 2-50 ng/ml. The limits of detection (LODs) for EB and MP in water were 1.0 and 0.9 ng/ml, respectively. The relative standard deviation values on peak size (10 ng/ml level) and migration time for the two drugs were in the ranges 5-8 and 0.2-0.7% (n = 7), respectively. Applicability of the CE-ECL method to the analysis of human plasma spiked with EB and MP was examined. The LODs for EB and MP in plasma were 0.4 and 0.3 microg/ml, respectively.     

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。     

Simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids by capillary zone electrophoresis with direct UV detection

Co-electroosmotic capillary zone electrophoresis (CZE) with direct UV detection was developed for simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids. These solutes were separated using a 30 mM phosphate buffer containing 1.0 mM tetradecyltrimethylammonium bromide (TTAB) and 20% (v/v) acetonitrile at pH of 6.5 and directly detected by UV at 190 nm. Calibration curves were linear in the range 0.01-2.0 mM, depending of the solutes. The detection limits ranged from 1.0 to 8.0 microM and the relative standards deviations (n=5) in range from 1.9 to 3.6% for the peak area. The proposed method was used to determine inorganic anions and carboxylic and aromatic acids in soil and plant tissue extracts.     

A novel amperometric method for antioxidant activity determination using DPPH free radical

A new method for the determination of antioxidant activity based on the amperometric reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) at the glassy carbon electrode is proposed. All experiments were done in three-electrode electrochemical cell at 140 mV vs. Hg(2)Cl(2) | 3 M KCl using ethanolic solution (phi=40%) and 0.033 M KCl in 0.033 M phosphate buffer, pH=7.4. The linear range obtained for Trolox in 100 microM DPPH ethanol-water solution was up to 30 microM, with a limit of detection of 0.05 microM. The developed method was applied for the evaluation of antioxidant activity of some water or ethanol soluble pure compounds of antioxidants and of the samples of tea, wine and some other beverages. The good correlation of measurements (R(2)=0.9993) expressed as Trolox equivalent was obtained between the proposed amperometric method and classic spectroscopic method.     

Carbon felt-based bioelectrocatalytic flow detectors: highly sensitive amperometric determination of hydrogen peroxide using adsorbed peroxidase and thionine.

Horseradish peroxidase (HRP) and thionine (TN) were co-adsorbed onto a porous carbon felt (CF), and the resulting HRP and TN-adsorbed CF (HRP-TN-CF) was successfully used as a working electrode unit of a novel bioelectrocatalytic flow detector for a highly sensitive amperometric determination of hydrogen peroxide (H(2)O(2)). Co-adsorbed TN was essential to enhance the cathodic peak current of H(2)O(2), and the current responses of the HRP-TN-CF-based detector were much larger than those of the HRP-CF-based detector (without TN). When air-saturated 0.1 M phosphate buffer (pH 7.0) was used as a carrier at a flow rate of 3.9 ml/min, cathodic peak currents of H(2)O(2) (sample injection volume, 200 microl) obtained at an applied potential of 0 V (vs. Ag/AgCl) increased linearly up to 50 microM with a detection limit of 0.1 microM. Repetitive 100 sample injection of 100 microM H(2)O(2) induced no serious current decrease, and RSD was 0.41 to 1.21% (n = 100). The HRP-TN-CF retained 42% of its original activity after 8 days of storage in 0.1 M phosphate buffer at 4 degrees C.     

Amino acid analysis using disposable copper nanoparticle plated electrodes

A disposable copper nanoparticle-plated screen-printed carbon electrode (designated as Cun-SPE(100-nm)) provides a new material for the determination of native amino acids. All 20 underivatized amino acids can be sensitively determined at 0.0 V vs. Ag/AgCl in pH 8 phosphate buffer solution. The precisely controlled copper nanoparticles can boost up the CuIIO/CuI2O redox signal on the working surface without any prior pretreatment procedure. The formation of a reversible 1:1 CuIIO-amino acid complex on the Cun-SPE(100-nm) was proposed to play a key role in the reaction mechanism. Stable detection responses were obtained for all amino acids by flow injection analysis with detection limits (S/N = 3) that lie in the range of 24 nM-2.7 microM. Selected amino acids from six representative chemical natures were separated by HPLC and detected at the Cun-SPE(100-nm) with promising results.     

Determination of arsenic species by capillary zone electrophoresis with large-volume field-amplified stacking injection.

Determination of arsenic species by large-volume field amplified stacking injection-capillary zone electrophoresis (LV-FASI-CZE) is reported in this paper. Whole column injection was employed. The optimum buffer pH for the separation of weak acids was discussed. It was found that the optimum buffer to analyze the stacked arsenate (As(V)), monomethylarsonate (MMA), and dimethylarsinate (DMA) was 25 mM phosphate at pH 6.5. However, the optimum buffer to analyze the concentrated arsenite (As(III)) was 20 mM phosphate - 10 mM borate at pH 9.28. The limits of detection of the method developed were 0.026 mg/L for As(III), 0.023 mg/L for As(V), 0.043 mg/L for MMA, and 0.018 mg/L for DMA. An enrichment factor of 34-100 for several arsenic species was obtained. In the end, this method was applied to determine the arsenic concentration in the environmental reference materials to show the usefulness of the method developed.     

毛细管电泳电致化学发光检测血浆中布比卡因

布比卡因是一种外科局部麻醉剂,使用过量会导致中枢神经系统和心脏血管系统中毒^[1],可引起心脏停博.高效液相色谱和毛细管电泳(CE)^[2]是该药常用的检测方法.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Micellar electrokinetic capillary chromatography of macrolide antibiotics. Separation of tylosin, erythromycin and their related substances.

The separation of tylosin by micellar electrokinetic capillary chromatography with a mixed micelle system is described. Good selectivity was obtained with sodium phosphate buffer (80 mM, pH 7.5) containing 20 mM sodium cholate and 7 mM cetyltrimethylammonium bromide (CTAB). This method permits tylosin to be separated from its closely related substances within 15 min. The influences of type of buffer, buffer pH, the concentrations of sodium cholate and CTAB were investigated. The robustness of the method was examined for tylosin by means of a full-fraction factorial design. Quantitative results are presented. Using a similar buffer system (80 mM sodium phosphate, pH 6.0, 20 mM sodium cholate and 5 mM CTAB), separation of erythromycin and its main related substances was also obtained. However, detection sensitivity and resolution are not sufficient for analysis of related substances in erythromycin commercial samples.