Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (alpha) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (beta) and delta (delta) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave's disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for alpha globin gene mutations showed a single -alpha(-3.7) deletion and Hb CS mutation. These findings were suggestive of compound heterozygosity of Hb CS and a single -alpha(-3.7) deletion with a concomitant heterozygous deltabeta thalassaemia. Co-inheritance of Hb CS and a single -alpha(-3.7) deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modification of alpha thalassemia presumptively as a result of co-inheritance with deltabeta chain abnormality as suggested by the high Hb F level.
A capillary electrophoretic method for the separation of the aminoglutethimide (AGT) enantiomers using methylated-beta-cyclodextrin (M-beta-CD) as chiral selector is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 9 min with resolution factor Rs=2.1, using a fused-silica capillary and a background electrolyte (BGE) of tris-phosphate buffer solution (50 mmol L(-1), pH 3.0) containing 30 mgm L(-1) of M-beta-CD. The separation was carried out in normal polarity mode at 25 degrees C, 16 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy/recovery were included. The proposed method was successfully applied to the assay of AGT enantiomers in pharmaceutical formulations. The computational calculations for the inclusion complexes of the R- and S-AGT-M-beta-CD rationalized the reasons for the different migration times between the AGT enantiomers.
Capillary electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE-C(4)D) has been employed for the determination of the β-blocker drugs (atenolol and amiloride) in pharmaceutical formulations. 150 mM acetic acid was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature, and injection time) was studied. Non-UV absorbing L-valine was used as an internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28 °C, 25 kV, and using hydrodynamic injection (25 s). The separation was effected in a bare fused-silica capillary 75 μm × 52 cm. The CE-C(4)D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision, and selectivity. Calibration curves were linear over the range 5-250 μg mL(-1) for the studied analytes. The relative standard deviations of intra- and inter-day precisions of migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of the β-blocker drugs in different pharmaceutical tablets.
A three-phase hollow fiber liquid-phase microextraction method coupled with CE was developed and used for the determination of partition coefficients and analysis of selected nitrophenols in water samples. The selected nitrophenols were extracted from 14 mL of aqueous solution (donor solution) with the pH adjusted to pH 3 into an organic phase (1-octanol) immobilized in the pores of the hollow fiber and finally backextracted into 40.0 microL of the acceptor phase (NaOH) at pH 12.0 located inside the lumen of the hollow fiber. The extractions were carried out under the following optimum conditions: donor solution, 0.05 M H(3)PO(4), pH 3.0; organic solvent, 1-octanol; acceptor solution, 40 microL of 0.1 M NaOH, pH 12.0; agitation rate, 1050 rpm; extraction time, 15 min. Under optimized conditions, the calibration curves for the analytes were linear in the range of 0.05-0.30 mg/L with r(2)>0.9900 and LODs were in the range of 0.01-0.04 mg/L with RSDs of 1.25-2.32%. Excellent enrichment factors of up to 398-folds were obtained. It was found that the partition coefficient (K(a/d)) values were high for 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol and 2,6-dinitrophenol and that the individual partition coefficients (K(org/d) and K(a/org)) promoted efficient simultaneous extraction from the donor through the organic phase and further into the acceptor phase. The developed method was successfully applied for the analysis of water samples.
This article reviews progress in the application of electrophoretic techniques for the separation of nanoparticles. Numerous types of nanoparticles have recently been synthesised and integrated into different products and procedures. Consequently, analytical methods for the efficient characterisation of nanoparticles are now required. Several studies have revealed that gel electrophoresis can readily be used for separating nanoparticles according to their size or shape. However, many other studies focused on separation of nanoparticles by CE. In some cases nanoparticles could be separated by CZE, simply using pure buffer as the BGE. In other studies, buffer additives (most often SDS) were used, enabling fast separations of metallic nanoparticles by size. Other CE methods also allowed for separation of nanoparticle conjugates with biomolecules. Dielectrophoresis is yet another electrophoretic technique useful in separation and characterisation of nanoparticles; particularly nanotubes. Detection methods often used after electrophoretic separation include UV/Vis absorption and fluorescence spectroscopy. Examples of recent and relevant older reports are presented here. The authors conclude that electrophoretic methods for nanoanalysis can provide inexpensive and efficient tools for quality assurance and safety control; and as a consequence, they can augment transfer of nanotechnologies from research to industry.
A sample pre-treatment method based on a dynamic mixed matrix membrane tip extraction followed by capillary electrophoresis with contactless conductivity detection (CE-C4D) was evaluated for the determination of tobramycin in human plasma. The extraction tip device consisted of a cellulose triacetate membrane tip wall immobilised with 15% (w/w) of hydrophilic lipophilic balance (HLB) nanoparticles as adsorbent. The extraction was performed dynamically by withdrawing/dispensing the plasma sample through the tip device followed by desorption into 20 μL of acidified aqueous solution at pH 3 prior to the CE-C4D analysis. Under the optimum conditions, the detection limit of the method for tobramycin was 10 ng/mL, with intraday and interday repeatability RSDs of 3.5% and 4.5%, respectively. Relative recoveries in spiked human plasma were 99.6%-99.9%. The developed approach was successfully demonstrated for the quantification of tobramycin in human plasma samples.
Peptides with biological properties, that is, bioactive peptides, are a class of biomolecules whose health-promoting properties are increasingly being exploited in food and health products. However, research on targeted techniques for the detection and quantification of these peptides is still in its infancy. Such information is needed in order to enhance the biological and chemometric characterization of peptides and their subsequent application in the functional food and pharmaceutical industries. In this review, the role of classic techniques such as electrophoretic, chromatographic, and peptide mass spectrometry in the structure-informed detection and quantitation of bioactive peptides are discussed. Prospects for the use of aptamers in the characterization of bioactive peptides are also discussed. PRACTICAL APPLICATIONS: Although bioactive peptides have huge potential applications in the functional foods and health area, there are limited techniques in enhancing throughput detection, quantification, and characterization of these peptides. This review discusses state-of-the-art techniques relevant in complementing bioactive detection and profiling irrespective of the small number of amino acid units. Insights into challenges, possible remedies and prevailing areas requiring thorough research in the extant literature for food chemists and biotechnologists are also presented.
The accumulation of paralytic shellfish toxins (PSTs) in contaminated shellfish is a serious health risk making early detection important to improve shellfish safety and biotoxin management. Capillary electrophoresis (CE) has been proven as a high resolution separation technique compatible with miniaturization, making it an attractive choice in the development of portable instrumentation for early, on-site detection of PSTs. In this work, capillary zone electrophoresis (CZE) with capacitively coupled contactless conductivity detector (C(4)D) and UV detection were examined with counter-flow transient isotachophoresis (tITP) to improve the sensitivity and deal with the high conductivity sample matrix. The high sodium concentration in the sample was used as the leading ion while l-alanine was used as the terminating electrolyte (TE) and background electrolyte (BGE) in which the toxins were separated. Careful optimization of the injected sample volume and duration of the counter-flow resulted in limit of detections (LODs) ranging from 74.2 to 1020 ng/mL for tITP-CZE-C(4)D and 141 to 461 ng/mL for tITP-CZE-UV, an 8-97 fold reduction compared to conventional CZE. The LODs were adequate for the analysis of PSTs in shellfish samples close to the regulatory limit. Intra-day and inter-day repeatability values (percentage relative standard deviation, n=3) of tITP-CZE-C(4)D and tITP-CZE-UV methods for both migration time and peak height were in the range of 0.82-11% and 0.76-10%, respectively. The developed method was applied to the analysis of a contaminated mussel sample and validated against an Association of Official Analytical Chemists (AOAC)-approved method for PSTs analysis by high performance liquid chromatography (HPLC) with fluorescence detection (FLD) after pre-column oxidation of the sample. The method presented has potential for incorporation in to field-deployable devices for the early detection of PSTs on-site.
A capillary electrophoresis (CE)-capacitively coupled contactless conductivity detection (C(4)D) method for the simultaneous separation of eleven underivatized fatty acids (FAs), namely, lauric, myristic, tridecanoic (internal standard), pentadecanoic, palmitic, stearic, oleic, elaidic, linoleic, linolenic and arachidic acids is described. The separation was carried out in normal polarity mode at 20 °C, 30 kV and using hydrodynamic injection (50 mbar for 1 s). The separation was achieved in a bare fused-silica capillary (70 cm × 75 μm i.d.) using a background electrolyte of methyl-β-cyclodextrin (~6 mM) and heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin (~8 mM) dissolved in a mixture of Na2HPO4/KH2PO4 (5 mM, pH 7.4):ACN:MeOH:n-octanol (3:4:2.5:0.5, v/v/v/v). C(4)D parameters were set at fixed amplitude of 100 V and frequency of 1000 kHz. The developed method was validated. Calibration curves of the ten FAs were well correlated (r(2)>0.99) within the range of 5-250 μg mL(-1) for lauric acid, and 3-250 μg mL(-1) for the other FAs. The method was simple and sensitive with detection limits (S/N=3) of 0.9-1.9 μg mL(-1) and good relative standard deviations of intra- and inter-day for migration times and peak areas (≤9.7%) were achieved. The method was applied to the determination of FAs in margarine samples. The proposed method offers distinct advantages over the GC and HPLC methods, especially in terms of simplicity (without derivatization) and sensitivity.
A dynamic supported liquid membrane tip extraction (SLMTE) procedure for the effective extraction and preconcentration of glyphosate (GLYP) and its metabolite aminomethylphosphonic acid (AMPA) in water has been investigated. The SLMTE procedure was performed in a semi-automated dynamic mode and demonstrated a greater performance against a static extraction. Several important extraction parameters such as donor phase pH, cationic carrier concentration, type of membrane solvent, type of acceptor stripping phase, agitation and extraction time were comprehensively optimized. A solution of Aliquat-336, a cationic carrier, in dihexyl ether was selected as the supported liquid incorporated into the membrane phase. Quantification of GLYP and AMPA was carried out using capillary electrophoresis with contactless conductivity detection. An electrolyte solution consisting of 12 mM histidine (His), 8 mM 2-(N-morpholino)ethanesulfonic acid (MES), 75 microM cetyltrimethylammonium bromide (CTAB), 3% methanol, pH 6.3, was used as running buffer. Under the optimum extraction conditions, the method showed good linearity in the range of 0.01-200 microg/L (GLYP) and 0.1-400 microg/L (AMPA), acceptable reproducibility (RSD 5-7%, n=5), low limits of detection of 0.005 microg/L for GLYP and 0.06 microg/L for AMPA, and satisfactory relative recoveries (90-94%). Due to the low cost, the SLMTE device was disposed after each run which additionally eliminated the possibility of carry-over between runs. The validated method was tested for the analysis of both analytes in spiked tap water and river water with good success.
A three-phase hollow fiber liquid-phase microextraction (HF-LPME) coupled either with capillary electrophoresis (CE) or high performance liquid chromatography (HPLC) with UV detection methods was successfully developed for the determination of trace levels of the anti-diabetic drug, rosiglitazone (ROSI) in biological fluids. The analyte was extracted into dihexyl ether that was immobilized in the wall pores of a porous hollow fiber from 10 mL of aqueous sample, pH 9.5 (donor phase), and was back extracted into the acceptor phase that contained 0.1M HCl located in the lumen of the hollow fiber. Parameters affecting the extraction process such as type of extraction solvent, HCl concentration, donor phase pH, extraction time, stirring speed, and salt addition were studied and optimized. Under the optimized conditions (extraction solvent, dihexyl ether; donor phase pH, 9.5; acceptor phase, 0.1M HCl; stirring speed, 600 rpm; extraction time, 30 min; without addition of salt), enrichment factor of 280 was obtained. Good linearity and correlation coefficients of the analyte was obtained over the concentration ranges of 1.0-500 and 5.0-500 ng mL(-1) for the HPLC (r(2)=0.9988) and CE (r(2)=0.9967) methods, respectively. The limits of detection (LOD) and limits of quantitation (LOQ) for the HPLC and CE methods were (0.18, 2.83) and (0.56, 5.00) ng mL(-1), respectively. The percent relative standard deviation (n=6) for the extraction and determination of three concentration levels (10, 250, 500 ng mL(-1)) of ROSI using the HPLC and CE methods were less than 10.9% and 13.2%, respectively. The developed methods are simple, rapid, sensitive and are suitable for the determination of trace amounts of ROSI in biological fluids.
One of the major drawbacks of electrophoresis in both capillary and microchip is the unsatisfactory sensitivity. Online sample preconcentration techniques can be regarded as the most common and powerful approaches commonly applied to enhance overall detection sensitivity. While the advances of various online preconcentration strategies in capillary and microchip employing aqueous background electrolytes are well-reviewed, there has been limited discussion of the feasible preconcentration techniques specifically developed for capillary and microchip using nonaqueous background electrolytes. This review provides the first consolidated overview of various online preconcentration techniques in nonaqueous capillary and microchip electrophoresis, covering the period of the last two decades. It covers developments in the field of sample stacking, isotachophoresis, and micellar-based stacking. Attention is also given to multi-stacking strategies that have been used for nonaqueous electrophoresis.
A new approach based on the integration of the free liquid membrane (FLM) into electrokinetic supercharging (EKS) was demonstrated to be a new powerful tool used in order to enhance online preconcentration efficiency in capillary electrophoresis (CE). A small plug of water immiscible organic solvent was used as a membrane interface during the electrokinetic sample injection step in EKS in order to significantly enhance the analyte stacking efficiency. The new online preconcentration strategy was evaluated for the determination of paraquat and diquat present in the environmental water samples. The optimised FLM-EKS conditions employed were as follows: hydrodynamic injection (HI) of 20mM potassium chloride as leading electrolyte at 50mbar for 75s (3% of the total capillary volume) followed by the HI of tris(2-ethylhexyl) phosphate (TEHP) as FLM at a 1mm length (0.1% of the capillary volume). The sample was injected at 10kV for 360s, followed by the HI of 20mM cetyl trimethylammonium bromide (CTAB) as terminating electrolyte at 50mbar for 50s (2% of the total capillary volume). The separation was performed in 12mM ammonium acetate and 30mM NaCl containing 20% MeOH at +25kV with UV detection at 205nm. Under optimised conditions, the sensitivity was enhanced between 1500- and 1866-fold when compared with the typical HI at 50mbar for 50s. The detection limit of the method for paraquat and diquat was 0.15-0.20ng/mL, with RSDs below 5.5%. Relative recoveries in spiked river water were in the range of 95.4-97.5%. A comparison was also made between the proposed approach with sole preconcentration of the field-enhanced sample injection (FASI) and EKS in the absence of the FLM.
A capillary electrophoresis (CE) method has been developed that allows the separation and estimation of primaquine enantiomers using hydroxypropyl-gamma-cyclodextrin (HP-gamma -CD) as a chiral selector. The influence of chemical and instrumental parameters on the separation, such as type and concentration of CD, buffer concentration, buffer pH, applied voltage, capillary temperature, and injection time, were investigated. Good separation of the racemic mixture of primaquine was achieved using a fused-silica capillary (52.5 cm effective length x 50 microm id) and a background electrolyte composed of tris-phosphate buffer solution (50 mM, pH 2.5) containing 15 mM HP-gamma-CD as a chiral selector. The recommended applied voltage, capillary temperature, and injection time were 15 kV, 25 degrees C, and 6 s, respectively. Within-day and interday reproducibility of peak area and migration time gave relative standard deviation values ranging from 1.05-3.30%. Good recoveries (range of 96.8-104.9%) were obtained from the determination of placebos that were spiked with 0.25-1.00 mg/L primaquine. The proposed CE method was successfully applied to the assay of primaquine diphosphate in pharmaceutical formulations (tablets).
Miniaturisation of microchip capillary electrophoresis (MCE) is becoming an increasingly important research topic, particularly in areas related to micro total analysis systems or lab on a chip. One of the important features associated with the miniaturised MCE system is the portable power supply unit. In this work, a very low electric field MCE utilising an amperometric detection scheme was designed for use in DNA separation. The device was fabricated from a glass/polydimethylsiloxane hybrid engraved microchannel with platinum electrodes sputtered onto a glass substrate. Measurement was based on a three-electrode arrangement, and separation was achieved using a very low electric field of 12 V/cm and sample volume of 1.5 µl. The device was tested using two commercial DNA markers of different base pair sizes. The results are in agreement with conventional electrophoresis, but with improved resolution. The sensitivity consistently higher than 100 nA, and the separation time approximately 45 min, making this microchip an ideal tool for DNA analysis.
Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre- or postcolumn oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on-site monitoring. In this study, CE methods were developed for C(4) D, FLD, UV absorption detection, and MS-making this first report of C(4) D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE-UV and CZE-C(4) D methods provide better resolution, selectivity, and separation efficiency compared to CZE-MS and MEKC-FLD. The sensitivity of the CZE-C(4) D and MEKC-FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE-C(4) D and 60.9 to 104 ng/mL for MEKC-FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C(4) D and MEKC-FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE-C(4) D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was successfully used for PST screening of a periodate-oxidized mussel sample, with results confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in shellfish samples.
Binding constants for the enantiomers of modafinil with the negatively charged chiral selector sulfated-β-CD (S-β-CD) using CE technique is presented. The calculations of the binding constants employing three different linearization plots (double reciprocal, X-reciprocal and Y-reciprocal) were performed from the electrophoretic mobility values of modafinil enantiomers at different concentrations of S-β-CD in the BGE. The highest inclusion affinity of the modafinil enantiomers were observed for the S-enantiomer-S-β-CD complex, in agreement with the computational calculations performed previously. Binding constants for each enantiomer-S-β-CD complex at different temperatures, as well as thermodynamic parameters for binding, were calculated. Host-guest binding constants using the double reciprocal fit showed better linearity (r(2)>0.99) at all temperatures studied (15-30°C) and compared with the other two fit methods. The linear van't Hoff (15-30°C) plot obtained indicated that the thermodynamic parameters of complexation were temperature dependent for the enantiomers.
Rapid and direct online preconcentration followed by CE with capacitively coupled contactless conductivity detection (CE-C(4)D) is evaluated as a new approach for the determination of glyphosate, glufosinate (GLUF), and aminophosphonic acid (AMPA) in drinking water. Two online preconcentration techniques, namely large volume sample stacking without polarity switching and field-enhanced sample injection, coupled with CE-C(4)D were successfully developed and optimized. Under optimized conditions, LODs in the range of 0.01-0.1 microM (1.7-11.1 microg/L) and sensitivity enhancements of 48- to 53-fold were achieved with the large volume sample stacking-CE-C(4)D method. By performing the field-enhanced sample injection-CE-C(4)D procedure, excellent LODs down to 0.0005-0.02 microM (0.1-2.2 microg/L) as well as sensitivity enhancements of up to 245- to 1002-fold were obtained. Both techniques showed satisfactory reproducibility with RSDs of peak height of better than 10%. The newly established approaches were successfully applied to the analysis of glyphosate, glufosinate, and aminophosphonic acid in spiked tap drinking water.
Micelle to solvent stacking was implemented for the recently established NACE-C(4) D method to determine tamoxifen and its metabolites in standard samples and human plasma of breast cancer patients. For stacking, the standard samples and extract after liquid-liquid extraction (LLE) were prepared in methanol and the resulting sample solution was pressure injected after a micellar plug of SDS. Factors that affected the stacking such as SDS concentration, micelle, and sample plug length were examined. The sensitivity enhancement factor (peak height from stacking/peak height from typical injection of sample in BGE) was 15-22. The method detection limits with LLE were in the range of 5-10 ng/mL, which was lower than the established method (where the LLE extract was also prepared in methanol) with reported method detection limits of 25-40 ng/mL. The intraday and interday repeatability were in the range of 1.0-3.4% and 3.8-6.5%, respectively.
Online preconcentration using electrokinetic supercharging (EKS) was proposed to enhance the sensitivity of separation for endocrine disrupting chemical (methylparaben (MP)) and phenolic pollutants (2-nitrophenol (NP) and 4-chlorophenol (CP)) in water sample. Important EKS and separation conditions such as the concentration of BGE; the choice of terminating electrolyte (TE); and the injection time of leading electrolyte (LE), sample, and TE were optimized. The optimum EKS-CE conditions were as follows: BGE comprising of 12 mM sodium tetraborate pH 10.1, 100 mM sodium chloride as LE hydrodynamically injected at 50 mbar for 30 s, electrokinetic injection (EKI) of sample at -3 kV for 200 s, and 100 mM CHES as TE hydrodynamically injected at 50 mbar for 40 s. The separation was conducted at negative polarity mode and UV detection at 214 nm. Under these conditions, the sensitivity of analytes was enhanced from 100- to 737-fold as compared to normal CZE with hydrodynamic injection, giving LOD of 4.89, 5.29, and 53 μg/L for MP, NP and CP, respectively. The LODs were adequate for the analysis of NP and CP in environmental water sample having concentration at or lower than their maximum admissible concentration limit (240 and 2000 μg/L for NP and CP). The LOD of MP can be suitable for the analysis of MP exists at mid-microgram per liter level, even though the LOD was slightly higher than the concentration usually found in water samples (from ng/L to 1 μg/L). The method repeatabilities (%RSD) were in the range of 1.07-2.39% (migration time) and 8.28-14.0% (peak area).