Quantification of analytes in overlapping peaks from capillary electrophoresis using multivariate curve resolution-alternating least squares methods

Application of multivariate curve resolution with alternating least squares (ALS) methods to second-order data from capillary electrophoresis diode array detector (CE-DAD) is shown. Second-order data are easily obtained by setting individual data matrix of CE run one in top of the other. Initial qualitative solutions obtained by evolving factor analysis can be further optimized by simultaneous analysis of multiple electrophoresis run data with ALS regression. Quantification is achieved by the comparison of the analyte peak areas with that of pure standards. During the ALS regression procedure, the following constraints were applied: (i) both concentrations and unit pure spectra of the resolved components must be positive; (ii) elution profiles have an unimodal shape; (iii) correspondence exists between common species in the different data matrices; (iv) the pure spectrum of each species is the same in all runs where it is present. The above methods were applied for the determination of dinitrotoluene (DNT) isomeric compounds in overlapping peaks from CE.     

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.     

Resolution of Overlapped Capillary Electrophoresis Peaks by Using Heuristic Evolving Latent Projections to Quantify Chloroquine Phosphate and Promethazine Hydrochloride

Chloroquine phosphate and promethazine hydrochloride are two main components in compound reserpine tablets. It is difficult to separate the two compounds with capillary electrophoresis (CE). Heuristic evolving latent projections (HELP) is a chemometric algorithm which is suitable for resolving overlapped peaks from CE and HPLC. In this paper, HELP was applied to resolving the completely overlapped peaks of chloroquine phosphate and promethazine hydrochloride from CE. Mathematical separation and satisfactory quantification results can be achieved easily without too much time involved in the procedure.     

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.     

Determination of carbohydrates in juices by capillary electrophoresis, high-performance liquid chromatography, and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

The objective of this work was to develop a sample preparation procedure for determination of the carbohydrate profiles in commercial juice samples by three principally different analytical methods: capillary electrophoresis (CE) with indirect detection, high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The preparation and purification of juice samples prior to analysis is described. The method using Carrez reagents was found to be an efficient preparation tool for all three methods. The addition of Carrez reagents to the samples for mass analysis improved the quality of the mass spectra of oligosaccharides. The amounts of glucose, fructose, and sucrose as major carbohydrates in fruit juices measured by CE using a simple instrument are in good agreement with the HPLC values and the data declared by the producers of the juices. The results from both methods are critically evaluated and their impact for studies of authenticity is discussed. The decrease of sucrose amount during the storage of samples was explained by acid hydrolysis of this disaccharide.     

Comparative analysis of CE‐SSCP to standard RFLP‐CE‐FLA method in quantification of known viral variants within an RNA virus quasispecies

RNA viruses display the highest replication error rate in our biosphere, leading to highly diverse viral populations termed quasispecies. The gold standard method for detection and quantification of variants in a quasispecies is cloning and sequencing, but it is expensive, laborious and time consuming. Therefore, other mutation detection approaches, including SSCP, are often used. In this study, we demonstrate development and the usage of a CE‐SSCP method for quantification of two nearly identical viral variants in heterogenic population of a mumps virus strain and its comparison to RFLP‐CE‐fragment length analysis (RFLP‐CE‐FLA). Analyzed PCR fragments were of the same size (245 bp) with one difference in their nucleotide sequence. The limit of detection of both methods was at 5% of the minor variant. When PCR amplicons of the two variants were pooled, methods' results were very similar. On the contrary, the quantification results of samples in which variants were mixed prior to PCR showed substantial difference between the two methods. Our results indicate that although both methods can be used for detection and monitoring of a specific mutation within a viral population, caution should be taken when quantitative analysis of complex samples is based solely on results of one method.     

A capillary electrophoresis assay for the fibrinolytic agent,eminase

Summary Capillary electrophoresis (CE) has been used to develop a method that allows analysis for all the main components of Eminase. This technique has also resolved the two glycosylated forms of lys-plasminogen. Quantitative CE analysis of concentration levels of this enzyme complex have been directly compared to those obtained from a bio-assay. A linear relation between the two methods has been demonstrated. Moreover, the CE method is highly reproducible, combines precision with speed, and allows quantitative analysis of the other protein components (streptokinase and human serum albumin) in Eminase.EMINASE is a trade name of SmithKline Beecham.     

色谱技术在药物-血浆蛋白相互作用研究中的应用进展

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α₁ -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。     

Separation and quantification of viral double-stranded RNA fragments by capillary electrophoresis in hydroxyethylcellulose polymer solutions

Capillary electrophoresis (CE) is an analytical technique widely utilized to resolve complex mixtures of nucleic acids. CE uses a variety of polymers in solution that act as a molecular sieve to separate nucleic acid fragments according to size. It has been shown previously that purified dsDNA can be resolved efficiently by solutions of hydroxyethylcellulose (HEC) polymer, providing a rapid and high resolution method of separation. We have applied this separation technique to viral double-stranded (ds) RNA segments derived from rotavirus process samples. HEC polymers of various molecular masses and concentrations were identified and compared for their ability to separate dsRNA based on the extent of expected polymer network formation. The HEC polymer exhibiting the most desirable separation characteristics was then used for subsequent optimization of various method parameters, such as, injection time, electric field strength, dye concentration and capillary equilibration. The optimized method was then applied to the quantification of genome concentration based on a representative segment of the rotavirus genome. This study demonstrated that purified viral dsRNA material of known concentration could be used to generate an external standard curve relating concentration to peak area. This standard curve was used to determine the concentration of unknown samples by interpolation. This novel RNA quantification assay is likely to be applicable to other types of virus, including those containing dsDNA.     

Investigation of a capillary electrophoretic approach for direct quantification of apolipoprotein A-I in serum

In the present study a rapid, reproducible and robust capillary electrophoresis (CE) procedure for the quantification of apolipoprotein A-I (Apo A-I) in serum without pretreatment has been developed (total run time, 11 min). The coefficients of variation (CV; n = 10) for the relative peak area are 1.8% at a concentration of 145 mg/dL and 1.6% at 196 mg/dL; and for the inter-assay 8.9% at 161 mg/dL (10 consecutive days), i.e., similar to the CVs of a high-throughput immunonephelometric routine assay. The CV for the migration time is 0.4% (n = 20). The robustness of the CE approach was tested in patient samples with hemolysis, hyperbilirubinemia and hyperlipidemia. A comparison of 99 Apo A-I serum values with results of a fixed-time immunonephelometric routine assay showed a positive constant bias of 60% (mean) for the immunonephelometric values, no deviation from linearity, but significant deviations in several samples. Investigations on interferences in the CE analyses gave no evidence that CE failed. Our study shows that CE is amenable to a fast analysis and a reproducible and reliable quantification of Apo A-I level in sera of various clinical samples.     

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.     

Determination of ephedrine alkaloid stereoisomers in dietary supplements by capillary electrophoresis

Three complementary capillary electrophoresis (CE) methods were developed for the separation and quantification of ephedrine and pseudoephedrine stereoisomers. Either single or dual cyclodextrin-based chiral selector systems provided enantioselective separation of the compounds of interest. The three methods were applied to the analysis of a suite of five standard reference materials (SRMs) containing ephedra. Use of a high-sensitivity UV detection cell enhanced quantification of the analytes of interest over the wide range of concentrations encountered in the SRMs. Results for (-)-ephedrine ranged from 0.31 to 76.43 mg/g, and for (+)-pseudoephedrine ranged from 0.049 to 9.23 mg/g in the materials studied. Results from the three methods agreed well with each other and with the results from other methods of analysis. The addition of known amounts of specific enantiomers was used to confirm the enantiomeric identity of the analytes. The results obtained by the three CE methods were utilized for value assignment of the ephedrine alkaloid content of these five SRMs.     

Application of 2,3-naphthalenediamine in labeling natural carbohydrates for capillary electrophoresis

Neutral and acidic monosaccharide components in Ganoderma lucidum polysaccharide are readily labeled with 2,3-naphthalenediamine, and the resulting saccharide-naphthimidazole (NAIM) derivatives are quantified by capillary electrophoresis (CE) in borate buffer. Using sulfated-α-cyclodextrin as the chiral selector, enantiomers of monosaccharide-NAIMs are resolved on CE in phosphate buffer, allowing a simultaneous determination of the absolute configuration and sugar composition in the mucilage polysaccharide of a medicinal herb Dendrobium huoshanense. Together with the specific enzymatic reactions of various glycoside hydrolases on the NAIM derivatives of glycans, the structures of natural glycans can be deduced from the digestion products identified by CE analysis. Though heparin dissachrides could be successfully derived with the NAIM-labeling method, the heparin derivatives with the same degree of sulfation could not be separated by CE.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

Determination of ciprofloxacin and its impurities by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been elaborated for separation, identification and determination of ciprofloxacin and its impurities. The separation, phosphate buffer pH 6.0 was supplemented with 0.075 M pentane-1-sulfonic acid sodium salt. The elaborated method was validated. The selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision, and accuracy of capillary zone electrophoresis were evaluated. The results obtained by CZE were also compared with those obtained by liquid chromatography. Regarding the validation results the CE method fulfils the current European Pharmacopoeia (Eur. Ph.) requirements. The evaluated CE method could be applicable to the analysis of different medicinal products containing ciprofloxacin.     

Application of Multivariate curve resolution-alternating least square methods on the resolution of overlapping CE peaks from different separation conditions

Discussed in this paper is the development of a new strategy to improve resolution of overlapping CE peaks by using second-order multivariate curve resolution with alternating least square (second-order MCR-ALS) methods. Several kinds of organic reagents are added, respectively, in buffers and sets of overlapping peaks with different separations are obtained. Augmented matrix is formed by the corresponding matrices of the overlapping peaks and is then analyzed by the second-order MCR-ALS method in order to use all data information to improve the precision of the resolution. Similarity between the resolved unit spectrum and the true one is used to assess the quality of the solutions provided by the above method. 3,4-Dihydropyrimidin-2-one derivatives (DHPOs) are used as model components and mixed artificially in order to obtain overlapping peaks. Three different impurity levels, 100, 20, and 10% relative to the main component, are used. With this strategy, the concentration profiles and spectra of impurities, which are no more than 10% of the main component, can be resolved from the overlapping peaks without pure standards participant in the analysis. The effects of the changes in the components spectra in the buffer with different organic reagents on the resolution are also evaluated, which are slight and can thus be ignored in the analysis. Individual data matrices (two-way data) are also analyzed by using MCR-ALS and heuristic evolving latent projections (HELP) methods and their results are compared with those when MCR-ALS is applied to augmented data matrix (three-way data) analysis.     

多元曲线分辨-交替最小二乘方法在联用数据分辨中的应用

通过对模拟数据和高效毛细管电泳实验数据的分析,讨论了多元曲线分辨-交替最小二乘方法(MCR-ALS)在毛细管电泳-二极管阵列检测(CE-DAD)联用数据分辨中的应用.讨论了几种因素对MCR-ALS单个数据矩阵分辨结果的影响,包括待分析物光谱间的相似程度、浓度曲线的重叠程度以及由渐进因子分析(EFA)所得到的浓度初始值等.MCR-ALS还可用于多个数据矩阵的同时分析,即二阶MCR-ALS.结果表明,与一阶MCR-ALS相比,二阶MCR-ALS方法能够更好地解决各种分辨问题,得到合理和满意的分辨结果.     

Simplified hemoglobin chain detection by capillary electrophoresis

Hemoglobin (Hb) chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb chains also after sample extraction. We describe a CE method for analysis of Hb chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the hemoglobinopathies.