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.     

Simultaneous determination of aminomethylbenzoic acid,cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205nm. The novel method described here uses a running buffer of aqueous 200mM NaH2PO4 (pH 7.05) containing 100mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.     

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2–100 μM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 μM for clopidogrel and 1.1 and 3.2 μM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD < 7%), intermediate precision (RSD < 7%) and accuracy (85–96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent K_m of 145 ± 30 μM and V_max of 0.4, 1.5 and 3.4 μM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained.     

On-line drug-metabolism system using microsomes encapsulated in a capillary by the sol-gel method and integrated into capillary electrophoresis

A novel microsome-encapsulation technique using the sol-gel method was developed for the on-line drug-metabolism analytical system integrated into capillary electrophoresis. This analytical system allows both the metabolism of drugs and the determination of the metabolites in a single capillary simultaneously. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane-based silica matrices within a capillary in a single step under mild conditions. The availability of this system was evaluated using UDP-glucuronyltransferase, which is one of the most important microsomal enzymes. 4-Nitrophenol and testosterone, which were metabolized by the different isoforms of UDP-glucuronyltransferase, were used as substrates. The resultant monolithic reactor showed enzymatic activity at the same level as that of the soluble form. The following separation of the unreacted substrates and metabolites in the same capillary also showed high selectivity. Furthermore, the sample amount required for one analysis decreased more than 3 orders of magnitude from conventional reaction schemes in free solution. This on-line system could largely simplify the laborious procedures which were needed in conventional analytical schemes.     

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.     

Quantification of carnosine-related peptides by microchip electrophoresis with chemiluminescence detection

A microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of carnosine-related peptides, including carnosine, homocarnosine, and anserine, in biological samples. A simple integrated MCE-CL system was built to perform the assays. The highly sensitive CL detection was achieved by means of the CL reaction between hydrogen peroxide and N-(4-aminobutyl)-N-ethylisoluminol-tagged peptides in the presence of adenine as a CL enhancer and Co²⁺ as a catalyst. Experimental conditions for analyte labeling, MCE separation, and CL detection were studied. MCE separation of the above-mentioned three peptides took less than 120 s. Detection limits (signal/noise ratio [S/N] = 3) of 3.0 × 10⁻⁸, 2.8 × 10⁻⁸, and 3.4 × 10⁻⁸ M were obtained for carnosine, anserine, and homocarnosine, respectively. The current MCE-CL method was applied for the determination of carnosine, anserine, and homocarnosine in human cerebrospinal fluid (CSF) and canine plasma. Homocarnosine was detected at the micromolar (μM) level in the CSF samples analyzed, whereas the levels of carnosine and anserine in these samples were below the detection limit of the assay. Interestingly, both carnosine and anserine were detected in the canine plasma samples, whereas homocarnosine was not.     

High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10 min and reverse-phase (RP)-HPLC in fluorescence in ∼21 min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.     

Modified method for the determination of capillary electrophoresis nitric oxide-correlated nitrate in tissue homogenates

A modified capillary electrophoretic method for the determination of nitric oxide correlated nitrate in several tissue homogenates is described in this study. The method was developed using a running buffer consisting of 200 mM lithium chloride and 10 mM borate buffer at pH 8.5, in a fused-silica column total 82 cm, effective 43 cm length and 75 μm I.D. The signal was measured at 214 nm and controlled current of 200 μA (equivalent to 12.7 kV) was applied in the reversed polarity direction. The sample was injected by vacuum pressure 50 ms (25 nl). In these conditions, bromide as internal standard and nitrate appeared at 7.2 and 8.9 min, respectively. Whole validation procedures were applied and satisfactory results were obtained. The nitrate levels of the tissue homogenates of control and -NAME applied (heart, brain, kidney, stomach, lung, testis and liver) were monitored by the present method and it was decided that the method is precise and accurate.     

Measurement of feruloyl/p-coumaroyl esterase by capillary zone electrophoresis

Ferulic andp-coumaric acid can be separated from their corresponding aliphatic methyl esters by capillary zone electrophoresis, which allows the convenient determination of feruloyl andp-coumaroyl esterase activities using synthetic esters as substrates. A feruloyl-containing sugar ester from wheat bran was also efficiently separated and used as substrate for the enzyme assays.Penicillium expansum was shown to produce feruloyl/p-coumaroyl esterase activity when grown on wheat bran in solid-state culture.The authors are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK; A.M. McKay is also affiliated with the Department of Food Science (Microbiology), The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, UK.     

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.     

Simultaneous determination of isoniazid and p‐aminosalicylic acid by capillary electrophoresis using chemiluminescence detection

It was found that isoniazid (ISO) or p‐aminosalicylic acid (PAS) could enhance the chemiluminescence (CL) emission from Cu (II)‐luminol‐hydrogen peroxide system, and the increased chemiluminescence signals were proportional to their concentrations, respectively. Based on this phenomenon, a chemiluminescence method coupled to capillary electrophoresis (CE) was established for simultaneous determination of ISO and PAS. The CE conditions including running buffer and running voltage were investigated in detail. The effects of the pH of H₂O₂ solution and the concentrations of luminol, H₂O₂ and Cu (II) on the CL signal were also investigated carefully. Under the optimized conditions, the analysis could be accomplished within 10 min, with the limits of detection of 0.3 µg mL^–1 for ISO and 1.1 µg mL^–1 for PAS, corresponding to 7.2 and 26.4 pg per injection (24 nL), respectively. Finally, the method was validated by determining the two analytes in pharmaceutical preparation and spiked human serum samples. The results of pharmaceutical tablet analysis were in good agreement with the labeled amounts. The recoveries for ISO and PAS in human serum were in the range of 92–104% and 90–113%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

High-resolution glycoprotein analysis using capillary electrophoresis

The high-resolution separation achievable with capillary electrophoresis has been applied successfully to the analysis of glycoproteins. Inherent in the implementation of this technology for glycoprotein analysis is the use of specific buffer additives. Bifunctional cationic reagents, such as simple alkyl chains bearing terminal amino or quaternary ammonium groups, have been particularly useful for the analysis of ovalbumin, an excellent model glycoprotein. Although dynamic coating of the capillary wall and the subsequent decrease in protein-wall interactions is known to be key in the effectiveness of these additives, much remains to be learned regarding the mechanism through which they function.     

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.     

Analysis of single nucleotide incorporation reactions by capillary electrophoresis

Single nucleotide incorporation assays have been used to probe the kinetic parameters of many DNA and RNA polymerases. Traditionally, oligonucleotide primers are 5'-(32)P labeled using T4 kinase and annealed to a complementary template with a 5' overhang. To quantify the reaction kinetics, the products of the primer extension reactions are usually separated using denaturing polyacrylamide gel electrophoresis and quantified using a phosphorimager or other method to measure radioactivity. We have developed a method using a 5' fluorescently labeled oligonucleotide to examine the kinetics of single nucleotide incorporation catalyzed by recombinant human mitochondrial polymerase gamma (Pol gamma) holoenzyme. Using laser-induced fluorescence detection in the P/ACE MDQ instrument, primers 5' labeled with fluorescent probes such as 6-carboxyfluorescein can be rapidly separated and quantified. However, we also show that only select probes can be used, presumably due to unfavorable interactions between Pol gamma and certain 5' labels.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

Plasma creatinine and creatine quantification by capillary electrophoresis diode array detector

Traditional clinical assays for nonprotein nitrogen compounds, such as creatine and creatinine, have focused on the use of enzymes or chemical reactions that allow measurement of each analyte separately. Most of these assays are mainly directed to urine quantification, so that their applicability on plasma samples is frequently hard to perform. This work describes a simple free zone capillary electrophoresis method for the simultaneous measurement of creatinine and creatine in human plasma. The effect of analytical parameters such as concentration and pH of Tris-phosphate running buffer and cartridge temperature on resolution, migration times, peak areas, and efficiency was investigated. Good separation was achieved using a 60.2-cm x 75-microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer, pH 2.25, at 15 degrees C, in less than 8 min. We compared the present method to a validated capillary electrophoresis assay, by measuring plasma creatinine in 120 normal subjects. The obtained data were compared by the Passing-Bablok regression and the Bland-Altman test. Moreover the performance of the developed method was assessed by measuring creatine and creatinine in 16 volunteers prior to and after a moderate physical exercise.