Development of an electrophoretic method based on nanostructured materials for HbA1c determination

Glycosylated hemoglobin (HbA1c) detection is performed routinely in hospitals as it is the most widespread confirmatory diagnosis of diabetes mellitus. Here we present a novel CE method for measuring HbA1c by introducing silica nanoparticles (NPs) modified with a boronic acid derivative (sugar loadings of 51 ± 2 μg/mg) as pseudo‐stationary phase. Before the sample injection, SiO₂NP─B(OH)₂ were introduced via pressure. Electrophoretic separation was explored through variation of the buffer pH and separation voltage, being the best separation, resolution and shorter separation time achieved with a 25 mM phosphate buffer pH 6.5. The calibration curve obtained was expressed as Area = 182.05%⁻¹ × HbA1c − 377.02; R² = 0.9826, using a UV/VIS absorbance detector at 415 nm (diode array). No interferences were observed from carbamylated or acetylated hemoglobin and the method shows a noteworthy stability. A paired t‐test was applied to compare the developed CE method with a commercial HbA1c test and no significant variations have been observed at a 90% significance level.     

Separation of fluorescent oligosaccharide derivatives by microcolumn techniques based on electrophoresis and liquid chromatography.

Various aldose oligosaccharides can be quantitatively derivatized into primary amines for subsequent reaction with fluorogenic reagents, such as 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde or 3-benzoyl-2-naphthaldehyde. Capillary electrophoresis (CE) and microcolumn liquid chromatography (LC), coupled with laser-induced fluorescence detection, were evaluated as a means of separating complex oligosaccharide mixtures. Whereas microcolumn LC and open-tubular CE appear confined in their utility to relatively small oligosaccharides, unprecedented results were obtained with polyacrylamide gel-filled capillaries on hydrolyzed malto-oligosaccharides and enzymatically degraded samples of chondroitin sulfate and hyaluronic acid.     

Direct monitoring of glycohemoglobin A1c in the blood samples of diabetic patients by capillary electrophoresis. Comparison with an immunoassay method

The capillary electrophoresis (CE) study was focused on quantifying glycohemoglobin A1c, HbA1c, in whole blood samples of patients suffering diabetes mellitus. The results showed that dynamic polyionic coating of the capillary made the method very reproducible. The precision evaluation, method comparison and bias estimation in CE were performed during 20 days with patient blood samples and with four control samples. The influence of the storage time and temperature on the glycohemoglobin levels were also tested. High resolution in CE could be used to show evidences of the ageing of the samples stored at -70 degrees C. The results showed that the ageing peak was not originated from HbA1c, because it did not affect the HbA1c level which was in balance with the results of fresh samples measured with immunoassay. The HbA1c % values of blood samples of 105 patients measured with the CE technique varied between 3.6 and 11.8 and they were approximately 2-3% lower than measured with immunoassay. The correlation (R2) of CE results with immunoassay and HPLC results were 0.962 and 0.781, respectively.     

Nanometer‐sized alumina packed microcolumn solid‐phase extraction combined with field‐amplified sample stacking‐capillary electrophoresis for the speciation analysis of inorganic selenium in environmental water samples

In this paper, a new method of nanometer‐sized alumina packed microcolumn SPE combined with field‐amplified sample stacking (FASS)–CE‐UV detection was developed for the speciation analysis of inorganic selenium in environmental water samples. Self‐synthesized nanometer‐sized alumina was packed in a microcolumn as the SPE adsorbent to retain Se(IV) and Se(VI) simultaneously at pH 6 and the retained inorganic selenium was eluted by concentrated ammonia. The eluent was used for FASS–CE–UV analysis after NH₃ evaporation. The factors affecting the preconcentration of both Se(IV) and Se(VI) by SPE and FASS were studied and the optimal CE separation conditions for Se(IV) and Se(VI) were obtained. Under the optimal conditions, the LODs of 57 ng L^?1 (Se(IV)) and 71 ng L^?1 (Se(VI)) were obtained, respectively. The developed method was validated by the analysis of a certified reference material of GBW(E)080395 environmental water and the determined value was in a good agreement with the certified value. It was also successfully applied to the speciation analysis of inorganic selenium in environmental water samples, including Yangtze River water, spring water, and tap water.     

On-line coupling of size exclusion and capillary zone electrophoresis via a reversed-phase C18 trapping column for the analysis of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebrospinal fluid (CSF) and tissues. The present study shows the feasibility of a recently developed system, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on an RP18 microcolumn with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE) for the quantification of structural-related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. After SEC separation of the enkephalins from large proteins present in CSF a heart-cut of 200 nuL, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution of the enkephalins with 80% acetonitrile, a fraction of the eluate is electrokinetically injected into the CZE system, where stacking and separation is achieved. The degradation of the peptides, caused by endogenous peptidases in the matrix, is sufficiently inhibited with imipramine HCl. The assay has a satisfactory linearity and intraday (9.70-16.3%) precision considering the complexity of this multidimensional separation system. The sensitivity of the method, with a concentration limit of quantification of 2.5 nug/mL, is comparable with other CZE assays for peptides and sufficient for the quantification of peptide drugs in biological matrices.     

Capillary electrophoresis with contactless conductivity detection for the quantification of fluoride in lithium ion battery electrolytes and in ionic liquids—A comparison to the results gained with a fluoride ion‐selective electrode

In this study, an optimized method using capillary electrophoresis (CE) with a direct contactless conductivity detector (C⁴D) for a new application field is presented for the quantification of fluoride in common used lithium ion battery (LIB) electrolyte using LiPF₆ in organic carbonate solvents and in ionic liquids (ILs) after contacted to Li metal. The method development for finding the right buffer and the suitable CE conditions for the quantification of fluoride was investigated. The results of the concentration of fluoride in different LIB electrolyte samples were compared to the results from the ion‐selective electrode (ISE). The relative standard deviations (RSDs) and recovery rates for fluoride were obtained with a very high accuracy in both methods. The results of the fluoride concentration in the LIB electrolytes were in very good agreement for both methods. In addition, the limit of detection (LOD) and limit of quantification (LOQ) values were determined for the CE method. The CE method has been applied also for the quantification of fluoride in ILs. In the fresh IL sample, the concentration of fluoride was under the LOD. Another sample of the IL mixed with Li metal has been investigated as well. It was possible to quantify the fluoride concentration in this sample.     

FET immunosensor for hemoglobin A1c using a gold nanofilm grown by a seed-mediated technique and covered with mixed self-assembled monolayers

A micro FET-based immunosensor was developed for the determination of hemoglobin-A1c (HbA1c). The HbA1c/hemoglobin ratio is an important index in diabetes control. The sensor was fabricated by Complementary Metal-Oxide-Semiconductor Transistor (CMOS) and Micro Electronic Mechanical System (MEMS) techniques. The antibodies were immobilized via mixed self-assembled monolayers (SAMs) on a gold nanofilm. The nanofilm was deposited on a gold electrode by seed-mediated growth and gave a uniform and well distributed coverage. Nonspecific sites and interferences by noise were eliminated by covering the AuNPs with mixed SAMs. Compared to the immunosensor fabricated via the mixed SAMs method without gold nanofilm, the immunosensor displays a more than 2-fold sensitivity. The immunosensor is capable of detecting HbA1c and hemoglobin in hemolyzed and diluted whole blood, and results showed good agreement with the established clinical method.

An automatic method for determination of glycosylated hemoglobins using low-pressure liquid chromatography

Several studies have revealed a correlation between blood levels of glucose and hemoglobin A1c (HbA1c), a minor form of hemoglobin (Hb) present at elevated concentrations in patients with diabetes mellitus. To facilitate a clinical study of the level of circulating HbA1c we have developed an automatic chromatographic system. An efficient separation of HbA1c from HbA0 and other rapid hemoglobins (HbA1a, HbA1b) was achieved on Bio-Rex-70 columns using three buffers. This system allows the daily analysis of 40 samples. The mean level of HbA1c in normal subjects was 5.4 +/- 0.4%. The method also detects the presence of elevated levels of HbF and the most frequent forms of abnormal hemoglobin (HbS, HbC).     

The isolation of basic proteins by solid-phase extraction with multiwalled carbon nanotubes

Multiwalled carbon nanotubes (MWCNTs) have been employed for the first time as sorbents for the isolation of basic proteins from other protein species in biological sample matrices by solid-phase extraction (SPE). A microcolumn packed with MWCNTs was incorporated after appropriate pretreatment into a sequential injection system, which facilitates online selective sorption of basic protein species (hemoglobin and cytochrome c in this particular case). The retained protein species were afterwards separated from each other by sequential elution from the microcolumn through the employment of appropriate eluents. A 0.025 mol L(-1) phosphate buffer solution of pH 8.0 facilitated the efficient collection of hemoglobin, while a 0.5 mol L(-1) NaCl solution ensured the quantitative recovery of the retained cytochrome c. With a sample loading volume of 2.0 mL, enrichment factors of 11 and 15 were derived for hemoglobin and cytochrome c, along with retention efficiencies of 100% for both species and recovery rates of 98 and 90%, respectively. A sampling frequency of 8 h(-1) was achieved, and the precisions were 3.0% and 0.8% (RSD) for hemoglobin and cytochrome c at a concentration of 5.0 microg mL(-1). The practical applicability of this system was demonstrated by processing of human whole blood for isolation of hemoglobin, and satisfactory results were obtained by assay with SDS-PAGE.     

Determination of pentachlorophenol and tribromophenol in sawdust by ultrasound-assisted extraction and MEKC

An ultrasonic bar-assisted extraction and CE separation procedure for the determination of pentachlorophenol (PCP) and 2,4,6-tribromophenol (TBP) residues in sawdust was developed and applied. For this purpose, micellar electrokinetic capillary chromatography (MEKC) was used and compared with a GC/MS methodology. This methodology allowed the quantification of PCP and TBP in a concentration range of 2.5-12.0 mg/kg for TBP and 2.8-12.0 mg/kg for PCP. Different sample treatment processes were evaluated in order to extract these compounds from sawdust. Better results were obtained when the residues were extracted with ultrasound-assisted hexane, filtered, evaporated, dissolved in Na(2)CO(3), and injected into the CE equipment. The optimal option for GC/MS was extraction with Na(2)CO(3 )followed by a derivation using acetic anhydride and liquid-liquid extraction with hexane. This method allowed the quantification of TBP and PCP in sawdust in a concentration range of 0.19-12.00 mg/kg and 0.14-12.00 mg/kg, respectively. The CE method was compared with the GC/MS as reference method. The results were shown to be statistically similar by both methods for PCP as well as for TBP.     

Spectroelectrochemical study of hemoglobin A, alpha- and beta-fumarate crosslinked hemoglobins; implications to autoxidation reaction

The thermodynamics and kinetics of the reaction DeoxyHb-Fe(2+)<-->MetHb-Fe(3+) for human hemoglobin A (HbA), alpha- and beta-fumarate crosslinked hemoglobins were investigated by spectroelectrochemistry. Information from this study is used to determine what structural features and experimental conditions stabilize ferrous vs. ferric form of hemoglobin, and what implications this stabilization may have on the autoxidation reaction. Alpha- and beta-fumarate crosslinked hemoglobins, alphaXL-HbA and betaXL-HbA, were obtained by crosslinking deoxyhemoglobin and oxyhemoglobin, respectively, with bis(3,5-dibromosalicyl) fumarate (DBSF). Formal redox potentials, E(0), and reduction/oxidation rates were measured in the presence of mediator, hexammineruthenium(III) chloride. It was found that E(0) shifted positive for the alpha-, and negative for the beta-fumarate crosslinked hemoglobin compared to HbA for all experimental conditions investigated. This shift was consistent with stabilization of the tense (positive shift) or relaxed conformation (negative shift) conferred by crosslinking. Formal redox potentials shifted positive with addition of nitrate and chloride ions for alphaXL-HbA, indicating additional stabilization of the T quaternary. The slopes of the Nernst plots showed evidence of cooperativity as expressed by n(max). The data points (E(0), n(max)) were fitted by the MWC model which states that the electron transfer and the addition/removal of water are concerted. The set of K(R) and c values, where the parameter c is the ratio K(R)/K(T) and K(R) and K(T) are the ligand (water molecule and an electron-hole) dissociation constants for the R and T states, for the beta-crosslinked hemoglobin compared to that of HbA and alpha-crosslinked hemoglobin indicated that crosslinking of oxyhemoglobin affected differently the inner-coordination sphere at the heme site. By modulating the electrolyte concentration the reduction rates were measured as a function of DeltaE(0), the difference in E(0) between hemoglobin molecules and mediator. Linearization of the Marcus cross-relationship (based on the concerted water and electron transfer) was good for HbA, and poor for alphaXL-HbA and betaXL-HbA, consistent with results obtained by the MWC analysis. This may imply that the reduction of HbA is controlled by the driving force, DeltaE(0), whereas the reduction of alphaXL-HbA and betaXL-HbA occurs by a non-concerted mechanism controlled by structural features brought about by crosslinking. The autoxidation reaction, conversion of oxygen-bound ferrous hemoglobin to ferric hemoglobin, was found independent of E(0). Alpha-fumarate crosslinked hemoglobin showed the highest autoxidation rate despite its positive shift in formal redox potential as compared to HbA, followed by beta-fumarate crosslinked hemoglobin, and by native hemoglobin. These data suggest that the chemical mechanism of oxygen dissociation and accessibility of water and oxygen radicals to heme site control autoxidation.     

A Solid-Phase Microcolumn Extraction Method for the Analysis of Acetic Acid and Furfural Emitted from Paper

A rapid non-destructive sampling technique for the analysis of volatile organic compounds emitted from paper sheets is presented. A capillary, which is connected to an adsorbent-packed microcolumn, is inserted between two sheets at the centre of a paper stack placed in a bag made of polyethylene terephthalate/aluminium/polyethylene. The other end of the microcolumn is connected to a gas-tight syringe and an appropriate volume (5 mL) of the gaseous phase is aspirated. The loaded microcolumn is transferred into a modified split/splitless inlet of the GC-FID for desorption and analysis. The method has been successfully applied to the analysis of acetic acid and furfural in artificially aged paper sheets and naturally aged old books. The method shows good repeatability with limits of quantification for acetic acid and furfural of 0.1 and 0.04 ng mL^?1 respectively in the gas phase.     

Development and validation of a capillary electrophoresis-indirect photometric detection method for the determination of the non-UV-absorbing 1,4-dideoxy-1,4-imino-D-arabinitol in active pharmaceutical ingredients,solutions and tablets using an internal standard

A high speed, selective, and robust capillary electrophoresis (CE) method with high capacity was developed and validated for determination of assay of 1,4-dideoxy-1,4-imino-D-arabinitol in active pharmaceutical ingredients, solutions, and tablets during the development work at preclinical and Phase I and II clinical studies. 1,4-Dideoxy-1,4-imino-D-arabinitol, tartrate has (almost) no UV absorption. Therefore, the developed CE method for quantification was based on indirect UV detection. A cation CE principle was chosen using an electrolyte at pH 4.0 containing dimethyldiphenylphosphonium hydroxide, which has a strong UV absorbance. The quantification was performed using internal standard technique, by which piperidine was used as internal standard. The method was validated. The validation results showed that the CE method was suitable for the assay (and dissolution) analysis.     

Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry

A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.     

Quantification of components in overlapping peaks from capillary electrophoresis by using continues wavelet transform method

The application of continues wavelet transform (CWT) for resolving overlapping peaks from capillary electrophoresis (CE) is described. Overlapping peaks can be resolved easily by transforming experimental signals into their wavelet coefficients. The proposed method was applied for the determination of benzoic acid and salicylic acid in overlapping peaks from CE. The composition of the two acids in Zuguangsan, a traditional Chinese patent medicine, was determined. The quantification results are satisfactory. CWT has been shown to be a practicable approach for resolving overlapping peaks and for quantitative determining coeluted compounds in overlapping peaks from CE. The quantification results obtained from CWT were compared with those obtained from numerical differentiation method. CWT has been shown prior to numerical differentiation method for processing experimental signals which contain noise.     

Comparison of HPLC and CE methods for the determination of cetirizine dihydrochloride in human plasma samples

Two methods, capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC), for analysis of cetirizine dihydrochloride in small sample volumes of human plasma were compared. The CE and HPLC assays were developed and validated by analyzing a series of plasma samples containing cetirizine dihydrochloride in different concentrations using these two methods. The extraction procedure is simple and no complicated purification steps or derivatization are required. The analysis time in the HPLC method was shorter than that in the CE method, but solvent consumption was considerably lower in the CE method. The calibration curve was linear to at least 10-1000 ng/mL both for CE and HPLC with r(2) = 0.9993 and r(2) = 0.9994, respectively. The detection limits for cetirizine dihydrochloride were 3 and 5 ng/mL with CE and HPLC (a UV detector was applied in the both cases), respectively. Both methods were selective, robust and specific, allowing reliable quantification of cetirizine dihydrochloride, and could be useful for clinical and biomedical investigations.     

Use of large-volume sample stacking in on-line solid-phase extraction-capillary electrophoresis for improved sensitivity

We present a new system for the sensitive analysis of cephalosporins by CE using both on-line SPE and large-volume sample stacking (LVSS). Sample volumes of 250 muL were loaded onto the SPE microcolumn which was then desorbed with 426 nL of ACN. The SPE elution plug was injected into the CE system via an in-line valve interface filling approximately 60% of the volume of the separation capillary. Subsequently, LVSS was performed by applying a voltage of -5 kV, which resulted in the simultaneous removal of the elution solvent and the preconcentration of the analytes in a narrow zone. This way the amount of analyte loaded into the capillary could be considerably increased without serious loss of CE separation efficiency. LODs for cefoperazone and ceftiofur were in the ng/L range which represents an improvement of a factor of 8450 and 11 450 when compared with direct CE injection. The cephalosporin test compounds presented a good linear response (corrected peak area) between 0.5 and 10 mug/L with correlation coefficients higher than 0.995. The final method is compared with previously reported LVSS-CE and SPE-CE systems for the analysis of cephalosporins.     

Comparison between two different hemichromes of hemoglobins (HbA and HbS) induced by n-dodecyl trimethylammonium bromide: chemometric study

The interaction of n-dodecyl trimethylammonium bromide (DTAB) with oxyhemoglobin A and oxyhemoglobin S is investigated using UV–visible absorption spectra and chemometric resolution techniques. Oxyhemoglobins (A and S) induced to partial oxidized form (ferrihemoglobin) by DTAB and finally transform to fully oxidized hemichrome. Hemichrome mole fractions of HbS are more than HbA because of more hydrophobic interaction of DTAB–HbS in second set of binding site relative to DTAB–HbA. The visible spectra between 500 and 650 nm are used for identifying the present components in solution because each species of hemoglobin has a specific spectrum in this region. The number of components and mole fraction of mentioned species were determined by employing chemometric resolution techniques. Subspace comparison was used for determination of the number of components in each concentration of hemoglobin and DTAB. After the determination of components, multivariate curve resolution-alternating least square (MCR-ALS) by initial estimates of spectral profiles and proper constraints, was used to resolve the data matrix into pure concentration and spectral profiles. The results show that both number and mole fraction of components which were formed during hemoglobin (HbA and HbS) oxidation by DTAB were initial hemoglobin concentrations independent. Furthermore, in average the mole fraction of hemichrome of HbS is 14.4% more than HbA. On the other hand, the mole fraction of HbA ferrihemoglobin is 15.6% higher than HbS averagely.     

New analytical tools and epidemiological data for the identification of HbA2 borderline subjects in the screening for beta-thalassemia

The increase of HbA(2) is the most important feature in the identification of beta-thalassemia carriers. However, some carriers are difficult to identify, because the level of HbA(2) is not in the typical range. Few data are available concerning the prevalence of such unusual phenotypes, and knowing their expected prevalence could be helpful in detecting systematic drifts in the analytical systems for HbA(2) quantification. In this study we report a retrospective investigation in two centres with high prevalence of beta-thalassemia. The prevalence of borderline subjects was found to be 2.2 and 3.0%, respectively. The genotypes of a subgroup of these subjects were then analyzed and in about 25% of cases a mutation in the globin genes was identified. We conclude that the occurrence of HbA(2) borderline phenotypes is not a rare event. In order to obtain more accurate HbA(2) measurements the development of an international reference measurement system for HbA(2), based on quantitative peptide mapping, has been recently started. We believe that the innovative approach of our method could also be used as a model to develop accurate quantitative methods for other red cell proteins relevant to the biodynamic properties and the surface electrochemistry of erythrocytes.     

Membrane-immobilized haptoglobin as affinity matrix for a hemoglobin-A1c immunosensor

An amperometric immunosensor for hemoglobin-A1c (HbA1c) determination has been developed utilizing membrane-immobilized haptoglobin as affinity matrix fixed in front of a Pt-working electrode. The HbA1c assay was carried out in a two-step procedure including the selective hemoglobin enrichment on the sensor surface and the specific HbA1c detection by a glucose oxidase (GOx) labeled anti-HbA1c antibody. Hydrogen peroxide generated by the enzyme label was oxidized at +600 mV versus Ag/AgCl. A standard curve for HbA1c was obtained with a linear range between 0 and 25% HbA1c of total hemoglobin which correspond to 7.8–39 nM. ELISA studies confirmed the advantage of a sandwich-type format with haptoglobin as capture molecule for selective hemoglobin binding over the direct adsorption method. Results by the sandwich immunoassay showed a linear correlation within the clinically relevant range 5–20% (CV < 3). For sensor application the immobilization procedure of haptoglobin onto CDI-activated cellulose membranes was optimized.