Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.     

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 free and total 7-hydroxycoumarin in urine and serum by capillary electrophoresis

A new method for the rapid determination of 7-hydroxycoumarin, the predominant metabolite of coumarin in humans, was developed for analysis in urine and serum, based on separation by capillary electrophoresis, with UV detection at 210 nm. The linear detection range for 7-hydroxycoumarin was 0–50 μg/ml while the limit of quantitation was 1 μg/ml. An internal standard, 3-(α-acetonylbenzyl)-4-hydroxycoumarin, was utilised for the determination of free 7-hydroxycoumarin, but it was found not to be suitable in the analysis of total 7-hydroxycoumarin present. Urine from two volunteers, who had been administered coumarin, was analysed by both capillary electrophoresis and by HPLC. The results from the two methods were compared and contrasted. The CE method was found to decrease the analysis time in comparison to HPLC analysis, with results available after 1.5 min as compared to 12 min with HPLC. There was no statistical difference between the results determined by either method.     

Determination of salicylic acid in human serum with capillary zone electrophoresis

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n=12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.     

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.     

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.     

Determination of three major catecholamines in human urine by capillary zone electrophoresis with chemiluminescence detection

A sensitive simple method is presented for the determination of three major catecholamines in human urine by capillary electrophoresis (CE) with on-line chemiluminescence (CL) detection. This was also the first time that the luminol-Ag(III) complex CL system was used for CE detection. This method was based on the enhancing effect of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on the CL reaction between luminol and the Ag(III) complex in alkaline solution. The separations and determinations were performed with an electrophoretic buffer consisting of 20.0mM sodium borate and 1.0mM luminol. Under optimized conditions, the three catecholamines were baseline separated and detected in less than 8 min. Detection limits of 7.9 × 10(-8)M, 1.0 × 10(-7)M, and 6.9 × 10(-8)M were observed for EP, NE, and DA, respectively. Relative standard deviation (RSD) values for the peak height were 4.7% to 5.4% (n = 5). Our proposed method was applied to the determinations of the catecholamines in urine samples from 12 healthy individuals and 26 pheochromocytoma patients. Our results suggest that this method might be useful to monitor the catecholamine levels in routine screening and to diagnose pheochromocytoma.     

Determination of protein charge by capillary zone electrophoresis

The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a "charge ladder" approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis determination method with UV detection of grepafloxacin and clinafloxacin has been developed. The separation was performed in 35 mM borate-35 mM phosphate buffer solution (pH 8.6), containing 6% (v/v) of acetonitrile. Analyses were realised using fused-silica capillaries (57 cm length x 75 microm I.D.) and the operating conditions were: 15 kV applied voltage, 30 degrees C and detection at 279 nm. Piromidic acid was used as an internal standard. The linear concentration range of application was 1.0-120.0 microg ml(-1) for both compounds, with a detection limit of 0.2 microg ml(-1) for grepafloxacin and 0.3 microg ml(-1) for clinafloxacin. The analysis yielded good reproducibility (RSD between 3.37 and 1.74%). It was applied to the determination of grepafloxacin and clinafloxacin in human and rat urine samples. The method was validated using HPLC as a reference method. Recovery levels were between 94.5 and 103%.     

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.     

Analysis of nitrate and nitrite in water and urine by capillary zone electrophoresis

A capillary zone electrophoresis method for the separation and analysis of nitrate and nitrite in water and urine was developed. No interference in the electropherogram from other anions is observed by using a polyacrylamide-coated column with a modified phosphate buffer at pH 3 for the separation, and UV absorption at 214 nm for the detection. The method does not require sample pretreatment or the use of organic solvents. The limit of detection for each analyte (S/N = 3), using a 75 μm I.D. capillary, is 0.5 μg/ml. Urine samples require 40-fold dilution in order to maintain migration time reproducibility to within 1% relative standard deviation.     

Determination of malondialdehyde in rat brain by capillary zone electrophoresis

A method for determination of malondialdehyde with capillary electrophoresis using UV detection at 267 nm has been developed. The buffer system consisted of 10 mM borax and 0.5 mM CTAB at pH 9.3. Malondialdehyde migrated as the first peak in the electropherogram at 2.6 min. Limit of detection was 1.2 μM corresponding to 7.8 pg. Malondialdehyde was determined before and after stimulating lipid peroxidation with the addition of ferrous ammonium sulphate to homogenates of rat brain tissue. Proteins were precipitated by boiling and removed from the brain homogenates with centrifugation. No further pretreatment was made before injecting the homogenates on the CE system. Non-precipitated homogenates could also be analyzed, but this required washing of the capillary with 0.1 M NaOH before introduction of the next sample.     

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.     

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.     

Determination of midecamycin by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the determination of midecamycin using an end-column amperometric detection with a carbon fiber micro-disk bundle electrode at a constant potential of +1.15 V vs. saturated calomel electrode. The optimum conditions of separation and detection are 1.00x10(-3) mol l(-1) Na(2)HPO(4)-3.49x10(-4) mol l(-1) NaOH (pH 11.4) for the buffer solution, 20 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, respectively. The limit of detection is 5.0x10(-7) mol l(-1) or 0.41 fmol (S/N=3). The linear range of the calibration curve is 1.00x10(-6)-1.00x10(-3) mol l(-1). The relative standard deviation is 1.4% for the migration time and 4.9% for the electrophoretic peak current. The method could be applied to the determination of midecamycin in human urine. In this case, a separation voltage of 14 kV was used.     

Determination of L-ascorbic acid in Lycopersicon fruits by capillary zone electrophoresis

This study shows an improved method for the determination of L-ascorbic acid (l-AA) in fruits of Lycopersicon by capillary zone electrophoresis (CZE). Two backgrounds electrolytes (BGEs) have been tested: (i) 400 mM borate at pH 8.0 and 1 x 10(-2)% hexadimethrine bromide, for the separation of Eulycopersicon subgenus species; and (ii) as in BGE(i) but supplemented with 20% (v/v) acetonitrile, for the separation of species of the Eriopersicon subgenus. The present procedures were compared with two routine methods-enzymatic assay and potentiometric titration with 2,6-dichlorophenol-indophenol. While these routine methods presented some difficulties in quantifying l-AA in several Lycopersicon fruits, CZE was successfully applied in all the analyzed samples. The proposed CZE protocols give lower detection limits (<0.4 microg ml(-1)); are cheaper, quicker, and highly reproducible; and can be applied to analyze large series of samples (ca. 50 samples per day) which is utmost importance, not only in screening trials for internal quality and tomato breeding programs, but also in systematic and routine characterization of Lycopersicon fruits.     

Determination of urinary orotic acid and uracil by capillary zone electrophoresis

We describe a simple method for measuring orotic acid and uracil concentration in urine by capillary zone electrophoresis in 20 mM Na-borate buffer, pH 9.2. The method was applied for studying a patient with HHH (hyperornithinemia, hyperammonemia and homocitrullinuria) syndrome. A high value of uracil excretion was found during periods of relatively low orotic acid excretion and normal ammonemia. The orotic acid level in urine was increased by increasing protein intake.     

Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry

The need for urgent diagnoses has propelled the development of automated analyses that can be performed in a short time at reasonable cost. One such method is immunoaffinity capillary electrophoresis. This emerging hybrid technology employs two powerful techniques coupled on-line for the direct and rapid determination of analytes present in biological fluids. The first technique, immunoaffinity, is used for the selective extraction of a molecule present in a complex matrix, utilizing a microscale-format chamber affinity device. An analyte (affinity target) present in serum or urine is captured by an immobilized molecular recognition antibody molecule (affinity ligand) bound to a solid support constituent (glass beads or an appropriate porous structure) of a microchamber affinity device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated affinity target material after elution from the microchamber affinity device. In this work, immunoaffinity capillary electrophoresis was developed for the identification and characterization of a single constituent of a complex matrix. Immunoreactive gonadotropin-releasing hormone was determined in serum and urine specimens derived from a normal individual and from a patient suffering from benign prostatic hyperplasia. Furthermore, the on-line immuno-separation system was coupled in tandem to mass spectrometry to obtain molecular mass information of the affinity isolated and CE separated neuropeptide. This hybrid immuno-analytical technology is simple, rapid, selective and sensitive. In addition, an attempt was also made to characterize other urinary constituents by CE–MS that may lead to marker activity in the urine of the diseased subject. The hyphenation of analytical techniques has proved valuable in enhancing their individual features. The future of bioanalysis using miniaturized affinity systems is discussed in this paper.     

Determination of purity degree and counter-ion content in lecirelin by capillary zone electrophoresis and capillary isotachophoresis

Capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) were applied for the determination of peptide purity degree and counter-ion content in lecirelin, the synthetic analogue of luteinizing hormone-releasing hormone (LHRH). CZE analyses were carried out in acidic background electrolyte (100 mM H3PO4, 50 mM Tris, pH 2.25) in bare fused silica capillary using UV-absorption detection at 206 nm. CITP analyses were performed in the electrophoretic analyzer with column coupling, equipped with contactless conductivity detectors both in preseparation capillary and in analytical capillary, and with UV-absorption detector (220 and 254 nm) in analytical capillary. Determinations of peptide purity were carried out in cationic mode with leading electrolyte (LE), 10 mM KOH/AcOH, pH 4.5, and terminating electrolyte (TE), 10 mM beta-alanine (BALA)/AcOH, pH 4.4. Degree of peptide purity determined by both CZE and CITP was in the range 60.1-80.9% for crude preparations of lecirelin and in the range 96.4-99.9% for HPLC purified batches. Concentrations of contaminating counter-ions, the anions of trifluoromethanesulfonic acid (TFMSA), trifluoroacetic acid (TFA) and acetic acid (AcOH), were determined by CITP analyses in anionic mode with LE 10 mM HCl/His, pH 6.0, and TE 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), pH 4.0, by the calibration curve method. Mass percentages of the counterion contents in the analyzed lecirelin batches varied from zero to ca. 9% (TFMSA), 3% (TFA) and 11% (AcOH), respectively.