Performance Capillary Electrophoresis Research Articles

Hemoglobin A2 and Heterogeneous Diagnostic Relevance Observed in Eight New Variants of the Delta Globin Gene.

Background: Hemoglobin A (Hb A) (α2β2) in the normal adult subject constitutes 96–98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the β-thalassemia carrier. Methods: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. Results: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1–2% during the screening tests in our laboratories. Conclusions: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

Online preconcentration of lysozyme in hen egg white using responsive polymer coating in CE

A mixed polymer brushes material based on poly (2-methyl-2-oxazoline)- and poly (acrylic acid)-coated capillary with switchable protein adsorption/desorption properties was applied for online preconcentration of lysozyme in hen egg white during capillary electrophoresis performance. First, lysozyme in simulated egg white was successfully online preconcentrated and the detection signal of lysozyme was amplified. Ovalbumin, ovomucoid, and conalbumin in egg white were verified show negligible interference on the online preconcentration of lysozyme according to the study on electroosmotic flow mobility. Second, a series validation procedure was carried out to evaluate the proposed method performance. There was a good linearity behavior range from 0.1 to 5.0ng/mL, limit of detection was 20 pg/mL, and limit of quantity was 50 pg/mL, the accuracy and robustness of this method were also excellent. Last, the proposed method has been successfully used to detect and analyze lysozyme in hen egg white, the determined amounts of lysozyme in hen egg white were consistent with reported normal levels and recoveries were in the range of 96.0-99.2%. After 75 consecutive runs, this prepared capillary was still stable for online preconcentration and determination of lysozyme in hen egg white without being affected by complex matrix.

Comparative study of atenolol in human plasma by high performance liquid chromatography and capillary zone electrophoresis

Atenolol is a beta-blocker that is cardioselective, meaning it only affects beta receptors. It is used to treat angina pectoris and high blood pressure. The HPLC and Capillary Zone Electrophoresis analytical technique was developed for the purpose of detecting and quantifying Atenolol in human plasma, according to the study paper you're reading right now. The internal standard and atenolol were recovered from the solution after being extracted from plasma using the Liquid-Liquid Extraction method. A mobile phase of 10mM sodium hydrogen phosphate, 7.3mM Sodium Lauryl sulphate (pH=3), methanol, and acetonitrile (40:57:3, v:v:v) is used, with a flow rate of 1.0ml/min. A fluorescence detector was used to detect the isolated materials, which had an excitation wavelength of 229 nm and an emission wavelength of 298 nm. With this in view, Atenolol's and the internal norm's survival times are observed to be 5.4 and 8.3 minutes, respectively. The linear correlation coefficient (R20.9992) was found in the Atenolol calibration curve. The recovery rate for atenolol and an internal norm was estimated to be between 76 and 87 percent. Solid-phase extraction was performed on an uncoated silica capillary with a diameter of 58.5 cm 75 m, and detection was performed at 194 nm in the Capillary Zone Electrophoresis procedure. For an electrolyte solution containing 50mM H3BO3 and 50mM Na2B4O7 (50:50 V/V), atenolol was determined to be present in the solution in less than 3 minutes. Energized with a voltage of 25kV and injected with a hydrodynamic configuration for 4S. Under various conditions, this method was used to assess the stability and capability of measuring Atenolol in human plasma.

An Overview on Analytical Methods for Quantitative Determination of Aristolochic Acids

Background: Aristolochic acids are chemically linked to nitrophenanthrene carboxylic acids which are found in aristolochia plants. These compounds are intrinsically carcinogenic, while they have been used in traditional medicine from a long time ago. Despite the beneficial effects of herbals for treating some diseases, they possess some side effects. Methods: Therefore, the development of a sensitive and selective procedure for the determination of these harmful components in various complicated samples is an important task for health systems and drug authorities. In the past years, ultra-pressure liquid chromatography, high performance liquid chromatography and capillary electrophoresis with different detection systems were used for determination of aristolochic acids in various samples. Results: In this review, different analytical methods have been discussed in brief and applications of them in diverse samples have been summarized. Conclusion: Different approaches are compared from point of sensitivity, selectivity, and extraction efficiency.

α-Thalassemia Intermedia Results from Interactions of Unstable Hb Prato [α31(B12)Arg→Ser (HBA1 or HBA2 c.96G>T or C)] with the α-Thalassemia-1 [– –SEA (Southeast Asian)] Deletion in Thailand

The clinical consequences of many abnormal hemoglobins (Hbs) interacting with α- or β-thalassemia (α- or β-thal) or other hemoglobinopathies have not been described. We evaluated a 75-year-old Thai woman and her 45-year-old daughter. Hematological data was obtained on an automated cell counter. Hemoglobin (Hb) analysis was carried out using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) assays. Mutations and globin haplotypes were identified by appropriated DNA techniques. The proband presented with moderate anemia and inclusion bodies in most of the red blood cells (RBCs), while altered RBC parameters were absent in her daughter. Hemoglobin analysis showed an abnormal Hb peak only in the proband. DNA analysis identified a G>T substitution at codon 31 of the α1-globin gene, corresponding to Hb Prato [α31(B12)Arg→Ser (HBA1 or HBA2 c.96G>T or C)] in both subjects. The α-thal-1 [– –SEA (Southeast Asian)] deletion was also identified in the proband, but not in her daughter. These mutations could be identified using newly developed allele-specific polymerase chain reaction (ASPCR) assays. The α haplotypic analysis demonstrated the Thai Hb Prato allele was associated with haplotype [+ – S + – + –] [the S represents the inter ζ hypervariable region (HVR)]. The combination of the unstable Hb Prato with α-thal-1 result in α-thal intermedia (α-TI) phenotypes. A simple DNA method is essential for detection, and a haplotypic α-globin gene cluster are presented.

Hb Guangxi [β65(E9)Lys→Glu (AAG>GAG); HBB: c.196A>G]: A Novel β-Globin Variant

We report a novel variant found in a female from Guilin municipality in the Guangxi Zhuang Autonomous Region of the People’s Republic of China. This variant can be readily detected by both cation exchange high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Sanger sequencing revealed a novel mutation on the β-globin gene [β65(E9)Lys→Glu (AAG>GAG); HBB: c.196A>G]. We named this novel hemoglobin (Hb) variant Hb Guangxi for the place of origin of the proband.

Preparation of diazoresin/graphene oxide covalent coated capillary for separation of proteins by capillary electrophoresis

Protein adsorption on the inner surface of capillary is the main reason for the poor separation performance of capillary electrophoresis. Currently, many ways have been reported to inhibit protein adsorption, while they have numerous disadvantages, such as complicated, toxic and unstable. Here, by using photosensitive diazoresin (DR) as coupling agents and graphene oxide (GO) as coating, we found a simple, economic and environmental friendly approach to inhibit protein adsorption. Through layer by layer (LBL) self-assembly technique and unique photochemistry reaction of DR, a stable and efficient capillary coating could be obtained. The bare capillary, non-covalently GO coated capillary and covalently GO coated capillary were all characterized. Results indicated that the DR/GO covalently linked capillary coatings showed great separation performance of proteins with excellent stability and repeatability. Compared with bare capillary and non-covalent, better separation efficiency was achieved by covalent GO capillary column.

The comparison of ion exchange-high performance liquid chromatography (IE-HPLC) and capillary electrophoresis (CE) for HbA1C measurement

The comparison of ion exchange-high performance liquid chromatography (IE-HPLC) and capillary electrophoresis (CE) for HbA1C measurement

Compound Heterozygosity for Hb D-Ibadan (HBB: c.263C>A) and Hb C (HBB: c.19G>A)

We report an individual with a compound heterozygosity for Hb D-Ibadan (HBB: c.263C>A) and Hb C (HBB: c.19G>A), a hemoglobin (Hb) combination not previously identified. The compound hemoglobinopathy was detected in a young woman during routine prenatal screening. Variant Hbs were identified and confirmed by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) followed by Sanger DNA sequencing. Hb D-Ibadan was present in significant excess over Hb C (70.3 to 24.4%). A complete blood count (CBC) revealed moderate microcytosis with slight anemia. The history suggests the Hb combination is clinically silent. The findings indicate the compound hemoglobinopathy demonstrates thalassemia minor-like red cell indices with an unequal distribution of the variant Hbs. Comparison with other Hb D-like heterozygous conditions is reviewed.

Chiral analysis of macromolecules

Around us, we have many chiral macromolecules such as protein, carbohydrate, nucleic acid, steroids, peptides and phospholipids. Hence, these compounds exist in enantiomeric forms and we know that only one of the enantiomeric forms is important to us and another may or may not be useful. Therefore, chiral analysis of macromolecules has taken a unique place in analytical chemistry. The chiral separation of such macromolecules by analytical techniques is very difficult so only few papers were found in the literature, which has been discussed in this chapter. The presented chapter describes chiral analysis of macromolecule only by two analytical techniques i.e. high performance liquid chromatography and capillary electrophoresis. Besides, the efforts were made to discuss the future perspective regarding enantio-separation of such chiral macromolecules.

Complex Interaction of Hb Q-Thailand (HBA1: c.223G>C) with β-Thalassemia/Hb E (HBB: c.79G>A) Disease

Hb Q-Thailand [α74(EF3)Asp→His (α1), GAC>CAC, HBA1: c.223G>C] is an abnormal hemoglobin (Hb) frequently found in Thailand and Southeast Asian countries. The association of the αQ-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report how to diagnose the coinheritance of Hb Q-Thailand with β-thalassemia (β-thal)/Hb E disease in four Thai samples from high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) testing results. Understanding of the HPLC chromatogram and CE electropherogram patterns of this complex mutation is important for interpretation of testing results and providing genetic counseling.

Method-dependent Discrepancies in Fetal Hemoglobin Quantification in Patients With Hemoglobin S.

Hemoglobin F (HbF) concentration is used in the diagnosis of certain hemoglobinopathies and accurate quantification is central to treatment of patients with sickle cell disease. The 2 most commonly used methods to quantify HbF are high performance liquid chromatography and capillary zone electrophoresis. This study reports discrepancies in HbF quantification between these methods when hemoglobin S is present in the sample. Clinicians and investigators should be mindful of the method used for HbF quantification when evaluating and treating patients who produce hemoglobin S.

Exploring the Versatility of an Atomic Absorption Spectrometer: Application to Direct Molecular Determination of Caffeine and Propranolol

A simple strategy using an atomic absorption spectrometer (AAS) for the direct determination of molecular compounds is proposed. The determination of caffeine and propranolol employing atomic absorption instrumentation was conducted, monitoring the emission lines of Fe (271.9 nm) and Mg (285.2 nm), respectively, using a quartz cuvette (1.0 cm) positioned in the burner head. Samples or standards were inserted into the cuvette in order to obtain the absorbance measurements. The parameters of merit were evaluated and the limits of detection were 0.46 and 0.56 mg L-1 for caffeine and propranolol, respectively, with a coefficient of determination R2 > 0.999. The methods developed were applied to the determination of caffeine and propranolol in real samples of beverages and pharmaceutical formulations, respectively. To evaluate the accuracy of the proposed methodologies the results obtained by atomic absorption spectrometer were compared with those obtained by molecular absorption spectrophotometry (UV-Vis) and high performance liquid chromatography (HPLC) or capillary electrophoresis (CE).

Comparison of Gas Chromatography and Liquid Chromatogram Detecting Pesticide Residue

The existing methods of detecting pesticide residue include gas chromatography, high performance liquid chromatography, gas chromatograph-mass, liquid chromatograph-mass, capillary electrophoresis, radioimmunoassay, biosensor and rapid detection on the spot. The paper analyzes the comparison of gas chromatography and liquid chromatogram detecting pesticide residue, for achieving the development tendency and the future goal of analyzing pesticide residue.

Simultaneous Separation and Determination of Aconitine Compounds in <i>Radix aconiti praeparata</i> by High Performance Capillary Zone Electrophoresis

A simple and inexpensive high performance capillary zone electrophoresis (CZE) was applied to separate and determine three alkaloids in Radix Aconiti Praeparata simultaneously. The optimized conditions for separation were obtained using 20 mmol/L borax buffer (pH 9.0) containing 2 mmol/L beta-cyclodextrin (β-CD) and 20% methanol. Online UV detection was performed at 235 nm. A voltage of 16 kV was carried at 25°C. Injection was performed at 0.5psi for 10 s. The separation and determination were satisfactory and quick.

Screening of Thalassaemia Carriers and Its Limitations

The α and β-thalassaemias (thal) are common genetic disorders of globin chain synthesis where the carriers have deficiency of α or β globin chain respectively. In resource limited settings, the naked eye single tube red cell osmotic fragility (NESTROFT) has been used for screening of thalassaemia. It has low specificity compared with its sensitivity. Screening of carriers encompasses automated/semi-automated techniques and manual methods for presumptive diagnosis of thalassaemia. The current strategy in screening involves a step by step process simplified into the following acronym, BHES. B, refers to blood counts and a film; H, haemoglobin (Hb) subtyping; E, Hb electrophoresis; and S, stability (H-inclusion and Sickle cell test). Blood counts and red blood cell indices are now available by automated instrumentation where a cut-off value of mean corpuscular Hb (MCH)<27 pg necessitates further investigation for thalassaemia or iron deficiency. The distribution of normal Hb subtypes (HbA, HbA2 and HbF) is done commonly by high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). The thalassaemia trait phenotype is indicated by a near normal Hb, microcytic and hypochromic red cell indices. In classical β-thalassaemia trait, the cut-off value of HbA2 value is 4% and above. However, milder mutations can result in HbA2 values between 3.2 to 3.9%. In alpha thalassaemia trait the HbA2 falls within the normal range. In a carrier of α0-thalassaemia, occasional or few H-inclusions suggests its presence. However, absence of H-inclusions does not rule out its presence. Recently, it has been shown that the current screening method of thalassaemia carriers has its limitations. Mild mutations may show normal or near normal red cell indices and be missed in the screening process. Carriers with these mutations can cause thalassaemia intermedia. In addition there are mutations that have more profound effects resulting in more severe outcome but have carriers with normal red cell indices. In α-thalassaemia intermedia, HbH disease, those with nondeletional mutations are clinically more severe. These non-deletion mutations include Hb Constant Spring, Hb Pakse and Hb Adana. Carriers with these non-deletional α-thal mutations have normal red cell indices. Cases of hydrops foetalis have been reported with a homozygous state of Hb Adana and also in α0 with Hb Adana. Coinheritance of α or β-thal also effects the red cell indices making them near normal. Thalassaemia intermedia and major have serious implications and prevention requires strategies to detect couples at risk. Mutational spectrum of the globin genes is updated continuously with improved diagnostic technology. Clinical programs preventing thalassaemia are presently based upon prospective screening and prenatal diagnosis. In order for prevention to be effective, there is a need to be aware that mutations can exist in thalassaemia carriers where the red cell indices are normal.

Evaluation of major methods of hemoglobin analysis

Hemoglobinopathy is a kind of hereditary hematonosis caused by the disproportion among normal hemoglobins or formation of abnormal hemoglobins.Hemoglobin analysis plays an important role in screening and diagnosing hemoglobinopathy.Emerging methods have been applied continuously to hemoglobinometry since the early 20th century.Nowadays,agarose gel electrophoresis,high performance liquid chromatography and capillary electrophoresis are widely used with mass spectrometry as the latest applied method.All these methods have their own features.Appropriate combination of these methods will lead to more scientific and reliable results.(Chin J Lab Med,2013,36:950-953) Key words: Hemoglobinometry; Electrophoresis; agar gel; Chromatography; high pressure liquid; Electrophoresis; capillary; Mass spectrometry

Association of Hb Thailand [α56(E5)Lys→Thr] and Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] with α0-Thalassemia: Molecular and Hematological Features and Differential Diagnosis

We report the molecular and hematological characteristics of two rare hemoglobin (Hb) variants found in associations with a common α0-thalassemia (α0-thal) in Thai patients. The first case (P1) was a generally healthy 27-year-old man discovered during our ongoing thalassemia screening program. Hemoglobin and DNA analyses identified a previously undescribed condition of compound heterozygosity for Hb Thailand [α56(E5)Lys→Thr] and α0-thal (SEA deletion). The second case (P2) was a 4-year-old boy with hypochromic microcytic anemia. Hemoglobin and DNA analyses also identified a compound heterozygosity for Hb Phnom Penh [α117(GH5)-Ile-α118(H1)] in association with α0-thal (SEA deletion). Although Hb H (β4) inclusion bodies were observed in both cases, Hb analysis using both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) did not show Hb H. While the two Hb variants could be recognized on Hb HPLC analysis, their corresponding Hb A2 derivatives: the Hb A2-Thailand and Hb A2-Phnom Penh, were clearly observed on CE. Apparently, combination of these two Hb variants with α0-thal are not expressed as Hb H disease. The two Hb variants could be confirmed by polymerase chain restriction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR assays.

Stability-indicating methods for the enantioselective determination of dihydropyridines by high performance liquid chromatography and capillary electrophoresis

This paper presents simple, rapid, precise and accurate stability-indicating HPLC and CE methods, which were developed and validated for the determination of nitrendipine, nimodipine and nisoldipine. These drugs are calcium channel antagonists of the 1,4-dihydropyridine type which are used in the treatment of cardiovascular diseases. Experimental results showed a good linear correlation between the area and the concentration of drugs covering a relatively large domain of concentration in all cases. The linearity of the analytical procedures was in the range of 2.0–120.0 μg mL−1 for nitrendipine, 1.0–100.0 μg mL−1 for nimodipine and 100.0–600.0 μg mL−1 for nisoldipine, the regression determination coefficient being higher than 0.99 in all cases. The proposed methods were found to have good precision and accuracy. The chemical stability of these drugs was determined under various conditions and the methods have shown adequate separation for their enantiomers and degradation products. In addition, degradation products produced as a result of stress studies did not interfere with the detection of the drugs' enantiomers and the assays can thus be considered stability-indicating.

Direct detection of diverse metabolic changes in virally transformed and Tax-expressing cells by mass spectrometry

HTLV-1- induced transformation causes extensive changes at the gene, protein and metabolite levels. These changes are usually followed by gene-expression profiling and proteomic analysis. Exploring the metabolic consequences of viral transformation adds to the picture because the viruses rely on the metabolic network of their cellular hosts for survival and replication. Metabolites are small molecules of diverse physico-chemical properties with greatly different abundance levels that make their analysis challenging. Typically optical (e.g., Fourier transform infrared spectrometry), nuclear magnetic resonance (NMR) and mass spectrometric techniques in combination with separation techniques, such as gas chromatography, high performance liquid chromatography (HPLC) and capillary electrophoresis, have been used for metabolomic studies. Here we utilized a new and novel method called laser ablation electrospray ionization (LAESI) to detect metabolites without any processing of samples. When using the LAESI technique to identify metabolic changes in HTLV1 and Tax1 transformed T lymphocytes and in HTLV3 and Tax3 cells, we found virus type specific (HTLV1 vs. HTLV3), expression specific (Tax1 vs. Tax3) and cell type specific (T lymphocytes vs. kidney epithelial cells) changes in the metabolite profiles. We have identified a number of metabolites that are known in the literature to be deregulated in the viral transformation process (e. g., arginine, cAMP, glutathione) as well as multiple novel metabolites that may have implications in HTLV1-induced transformation (e. g., putrescine, N-acetyl aspartic acid, methoxytyramine). These new findings point to metabolic pathways that have a heretofore unexplored role in the viral transformation of host cells.