Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C₁₈ cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l⁻¹ and detection limits were between 1.1 and 2.4 mg l⁻¹.     

Determination of piracetam in human plasma by capillary electrophoresis

A capillary electrophoresis (CE) procedure has been developed for the determination of piracetam in human plasma. Analyses were performed on an uncoated silica capillary using borax buffer modified with the addition of α-cyclodextrin. The detection was UV, operated at 200 nm. The detection limit of the authentic samples was 1 μg/ml. The calibration curve was linear over a range of 4 to 24 μg/ml (r=0.997). Inter-assay R.S.D. was below 9.3%. The described method has been successfully applied to the quantitative determination of piracetam in human plasma and should be useful for clinical and bioavailability investigations.     

Determination of ciprofloxacin,enrofloxacin and flumequine in pig plasma samples by capillary isotachophoresis--capillary zone electrophoresis

Quinolones are a group of synthetic antibiotics that are widely used in veterinary medicine. Their residues may remain in tissues, milk, etc. intended for human consumption. The European Union fixes the maximum residue limits (MRLs) of veterinary medicinal products in foodstuffs of animal origin. Analytical methods are therefore needed to determine them in biological samples. In this study, we describe capillary isotachophoresis-capillary zone electrophoresis (ITP-CZE) to analyze three quinolones, enrofloxacin (ENR), ciprofloxacin (CPR) and flumequine (FLU), in pig plasma samples. We used solid-phase extraction with Oasis HLB cartridges as a sample pretreatment clean-up step. Capillary zone electrophoresis (CZE) requires low amounts of sample and is not as sensitive as one would wish. ITP-CZE is an easy way to increase the sample loadability and sensitivity. With this system sensitivity increases 40-fold. The detection limits for CPR, ENR and FLU were 70, 85 and 50 microg l(-1), respectively, which were lower than their MRLs in different kinds of samples. This method is simple and sensitive, and is therefore an alternative tool to the existing HPLC methods for analyzing the residuals of these quinolones in biological samples.     

Determination of idarubicin and idarubicinol in plasma by capillary electrophoresis

A rapid and sensitive capillary electrophoretic method for the determination of idarubicin and its metabolite idarubicinol in plasma has been developed and validated. Plasma is extracted by liquid-liquid extraction using chloroform. Idarubicin, idarubicinol and the internal standard daunorubicin can be separated in less than 5 min using a phosphate buffer of pH 5 with 70% acetonitrile. Laser-induced fluorescence detection with an Ar ion laser operated at 488 nm provides a sensitive and selective detection method without interferences from biological fluids. The small sample volume of 100 μl is of particular advantage for studies in pediatric oncology. The reproducibility of the method has been shown to be sufficient for drug monitoring or pharmacokinetic studies. The limit of quantification for idarubicin in plasma is 0.5 ng/ml.     

Determination of microcystins in environmental samples using capillary electrophoresis

The applicability of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) methods for the simultaneous determination of two frequently occurring microcystins (MCs-LR and -YR) and a new variant (MC-YA) in crude extracts of Hungarian bloom samples and cyanobacterial cultures was studied. It was found that the comparison of the results obtained by both CZE and MEKC measurements (due to the differences in their separation mechanisms) for the same sample can guarantee the reliability of the quantitative results. In our work environmental samples like lake waters, water bloom samples, cyanobacterial isolates were analysed. The three microcystins could be directly determined in water bloom samples collected from Hungarian lakes and laboratory culture samples of cyanobacteria.     

Determination of microcystins in environmental samples using capillary electrophoresis

The applicability of capillary zone electrophoreses (CZE) and micellar electrokinetic chromatography (MEKC) methods for the simultaneous determination of two frequently occurring microcystins (MCs-LR and -YR) and a new variant (MC-YA) in crude extracts of Hungarian bloom samples and cyanobacterial cultures was studied. It was found that the comparison of the results obtained by both CZE and MEKC measurements (due to the differences in their separation mechanisms) for the same sample can guarantee the reliability of the quantitative results. In our work environmental samples like lake waters, water bloom samples, cyanobacterial isolates were analysed. The three microcystins could be directly determined in water bloom samples collected from Hungarian lakes and laboratory culture samples of cyanobacteria.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.     

Determination of cimetidine in human plasma by free capillary zone electrophoresis

The separation of cimetidine from the metabolites cimetidine amide and cimetidine sulfoxide, endogenous creatinine and the internal standard ranitidine was achieved by capillary electrophoresis in less than 5 min. All compounds were well separated from cimetidine, including possible plasma ingredients, as the UV spectra of cimetidine standard and cimetidine from the plasma extract match. Plasma levels of cimetidine were determined in the range 250–3000 ng/ml in plasma and higher concentrations were determined by dilution of the sample with blank plasma.     

High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 μg/ml with a 50-μl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 μl of the samples with Bond Elut C₁₈ cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7–9.9 (n=4) and 1.7–7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8–8.1% (n=3) and 2.9–4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.     

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length)×50 μm I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 μg AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5–100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 μg AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.     

Determination of duloxetine in human plasma by capillary electrophoresis with laser-induced fluorescence detection

A method based on capillary electrophoresis has been developed for the analysis of the novel antidepressant drug duloxetine in human plasma. The method makes use of laser-induced fluorescence detection after derivatisation of the analyte with 5-(4,6-dichlorotriazinyl)aminofluorescein at pH 11. A single step liquid/liquid extraction procedure with a mixture of hexane/2-propanol allows the sample clean-up with extraction yields always ≥84% and interference removal. The electrophoretic separation is achieved using uncoated fused silica capillaries (60.0 cm effective length, 75.0 cm total length, 50 μm internal diameter) and a background electrolyte composed of borate buffer (40 mM, pH 10.3), tetrabutylammonium bromide (10 mM), and acetone (10%, v/v). The applied voltage is 20 kV; the samples are injected by pressure (50 mbar × 8 s). The method has been fully validated in terms of linearity range (2.5–150 ng mL^?1), LOD and LOQ (1.0 and 2.5 ng mL^?1, respectively), precision (R.S.D. < 6.7%) and accuracy (recovery >78%). Application to samples obtained from patients under treatment with duloxetine gave good results. The method represents the first application of capillary electrophoresis to the analysis of duloxetine in human plasma.     

Determination of metformin in plasma by capillary electrophoresis using field-amplified sample stacking technique

A capillary electrophoresis method was described for the determination of metformin in human plasma based on the extraction of the ion-pair with bromothymol blue into chloroform. Phenformin was used as internal standard. Field-amplified sample stacking injection was employed with an electrokinetic injection voltage of 10 kV for 10 s. The running buffer was 0.1 M phosphate buffer (pH 2.5), running voltage was 20 kV and the UV absorbance detection was set at 195 nm. The limit of quantitation was 0.25 μg/ml. Linearity range of calibration curve was 0.25 to 3.5 μg/ml. Recoveries for three levels (0.25, 1 and 2 μg/ml) were 80.24%, 67.44% and 58.97% (n=5 for each level), respectively. The intra-day precisions for the three levels were 11.9%, 3.09% and 4.33% and the inter-day precisions were 12.4%, 4.57% and 4.94%, respectively. The concentrations of metformin hydrochloride in human plasma of eight volunteers were measured after orally administrating metformin enteric-capsule and tablet.     

Determination of opiates in urine by capillary electrophoresis

A method for the separation of a mixture of opiates comprising pholcodine, 6-monoacetylmorphine, morphine, heroin, codeine and dihydrocodeine by capillary electrophoresis using a running buffer of 100 mM disodium hydrogenphosphate at pH 6 is described. The characteristics of an analytical method based on this separation for the determination of these drugs following extraction from urine and using levallorphan as internal standard are reported. Detection limits in the region of 10 ng cm⁻³ are achieved when using electrokinetic injection. A comparison is made of the sensitivity and reproducibility of electrokinetic and hydrodynamic injection for these drugs. Data are presented to show the results obtained when the proposed method is applied to urine spiked with all the above opiates and also to urine from a subject following consumption of dihydrocodeine and pholcodine. The concentrations found are compared with those obtained by LC.     

Determination of josamycin in rat plasma by capillary electrophoresis coupled with post-column electrochemiluminescence detection

A novel determination method for josamycin (JOS) based on capillary electrophoresis-electrochemiluminescence detection has been described. In this study, platinum disk electrode (300 microm in diameter) was used as a working electrode and the conditions affecting separation and detection were investigated in detail. Under optimal condition: 40 cm separation capillary (75 microm i.d.); 1.25 V applied potential on the Pt disc of the ECL detector cell; 5 mM Ru(bpy)3(2+) and 50mM phosphate buffer (pH 7.5) in the detection cell; 12 kV separation voltage; 8s injection time; 10 kV injection voltage and 15 mM running buffer (pH 7.5), calibration curve was linear over the range from 10 ng/mL to 5.0 microg/mL with a detection limit of 3.1 ng/mL at a signal-to-noise ratio of 3. The method can be successfully applied for the determination of josamycin in rat plasma in 6 min and the extraction recoveries with spiked plasma samples were over 92%.     

Determination of gabapentin in serum by capillary electrophoresis

A rapid capillary electrophoresis method for the quantification of gabapentin, a new anticonvulsant drug, in serum was developed. The assay involves derivatization of gabapentin with fluorescamine to provide a chromophore for UV-fluorescence detection. The migration time is about 11 min. The assay was linear between 0 and 20 mg/l. No other therapeutic drugs or amino acids interfered with the gabapentin peak. The relative standard deviation is 2.4% at a mean 11 mg/l (n=17). The mean serum level for 52 patients on this drug was 5.2 mg/l with a range of 0–12 mg/l.     

Determination of enzymatic activity by capillary electrophoresis

Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through whole live world. Their accurate quantitation in biological samples is important in many fields of biochemistry, not only in routine biochemistry and in fundamental research, but also in clinical and pharmacological research and diagnosis. Since the direct measurement of enzymes by masses is impossible, they must be quantified by their catalytic activities. Many different methods have been applied for this purpose so far. Although photometric methods are undoubtedly the most frequently used, separation methods will further gain their position in this field. The article reviews different possibilities for the assay of enzymatic activity by means of capillary electrophoresis (CE). Both the off-line and on-line enzyme assays based on CE are discussed.     

Determination of iothalamate in rat urine,plasma, and tubular fluid by capillary electrophoresis

A method for the quantitative determination of iothalamate (IOT) in rat urine, plasma and tubular fluid by capillary zone electrophoresis (CE) has been developed and validated. Samples of urine and tubular fluids were diluted with water and samples of plasma were deproteinized with two volumes of acetonitrile containing the internal standard, p-aminobenzoic acid (PABA). A BioFocus 2000 system (Bio-Rad, Hercules, CA, USA) was used. The UV detector was set at 254 nm. The samples were loaded into uncoated fused-silica capillary (40 cm×50 μm) by pressure injection. A borate buffer [20 mM, pH 12 (pH adjusted with 1.0 M NaOH)] was used as the electrophoretic buffer. The typical analytical conditions were: voltage, 22 kV; injection, 9 psi×s; capillary and carousel temperatures were 20°C and 18°C respectively. The linear relationship was observed between time-corrected peak area of IOT in water and urine or the corrected peak area ratio of IOT to PABA in plasma and the nominal concentration of IOT with correlation coefficient greater than 0.999. The intra- and inter-day coefficients of variation (CV) were less than 8%. The concentration of IOT in plasma, urine and tubular fluid determined by CE can be used for estimation of whole kidney and single nephron clearances.     

尿样中三种蛋白质的毛细管电泳分离检测方法研究

目的:建立毛细管电泳分离测定人尿样中转铁蛋白、白蛋白和血红蛋白的新方法.方法:通过选择运行缓冲溶液种类及浓度、pH、表面活性荆种类及浓度、分离电压、进样时间对蛋白质分离效果的影响,优化了毛细管电泳法分离转铁蛋白、白蛋白和血红蛋白的条件.结果:利用此方法测定三种蛋白质的含量,浓度在0.01到1.00 g L-1范围内与峰电流呈良好的线性关系,检出限均为10-4 g L-1.结论:所建立的方法用于人尿样中转铁蛋白、白蛋白和血红蛋白的测定,结果满意.     

Quantification of penicillin G during labor and delivery by capillary electrophoresis

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3 cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.     

Determination of meningococcal polysaccharides by capillary zone electrophoresis

Meningococcal polysaccharides are medically important molecules and are the active components of vaccines against Neisseria meningiditis serogroups A, C, W135, and Y. This study demonstrates that free solution capillary zone electrophoresis (CZE) using simple phosphate/borate separation buffers is capable of separating intact, native polysaccharides from these four serogroups. Separation appeared to be robust with respect to variations in test conditions and behaved in expected ways with respect to changes in temperature, ionic strength, and addition of an organic modifier. Serogroups W135 and Y are composed of sialic acid residues alternating with either galactose or glucose, respectively. Separation of these serogroups could be achieved using phosphate buffer and was therefore not dependent on differential complexation with borate. Addition of sodium dodecyl sulfate to the separation buffer (i.e., MEKC) resulted in peak splitting for all four serogroups. Changes in polysaccharide size did not affect migration time for the size range examined, but serogroup C polysaccharide (a sialic acid homopolymer) was separable from sialic acid monosaccharide. CZE quantification of multiple lots of each of the four serogroups was compared to wet chemical determination by phosphorus or sialic acid measurement. Results from CZE determination showed good agreement with the wet chemical methods.