Free Zone Capillary Electrophoresis Research Articles

Electrophoretic characterization of LNP/AAV-encapsulated nucleic acids: Strengths and weaknesses.

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs(>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.

An aptamer assay for aflatoxin B1 detection using Mg2+ mediated free zone capillary electrophoresis coupled with laser induced fluorescence

We described an aptamer based and Mg2+ mediated free zone capillary electrophoresis-laser induced fluorescence (CE-LIF) assay for aflatoxin B1 (AFB1) detection. This CE-LIF assay applied an anti-AFB1 aptamer with a single fluorescein (FAM) label at 5′ end and a short complementary DNA (cDNA). In the absence of AFB1, the cDNA hybridized with the aptamer probe and formed a duplex DNA. The use of running buffer containing MgCl2 allowed good isolation of the duplex DNA from the single stranded DNA in CE. We found introducing a biotin label on the cDNA further improved the isolation. When AFB1 existed in sample solution, the aptamer probe bound with AFB1, dissociating from the duplex DNA. Thus, the duplex DNA peak decreased, while the aptamer probe peak increased during CE-LIF analysis. We achieved detection of AFB1 by measuring the aptamer probe peak. The length of cDNA, the ratio of aptamer to cDNA, and the concentration of MgCl2 in sample buffer and separation buffer had great effect on the aptamer based CE-LIF assay. Under optimized conditions, the detection limit of AFB1 was 0.2 nM, and the dynamic range was from 0.2 nM to 500 nM. Limit of quantitation was 0.5 nM. This CE-LIF assay enabled detection of AFB1 spiked in diluted human serum, diluted human urine, and corn flour samples. This assay exhibits potential for wide application as it integrates the rapidity, high sensitivity, low sample consumption of CE-LIF analysis and the strengths of aptamer.

Capillary Electrophoresis of Free Amino Acids in Physiological Fluids Without Derivatization Employing Direct or Indirect Absorbance Detection.

Whole blood and/or plasma amino acids are useful for monitoring whole-body protein and amino acid metabolism in an organism under various physiological and pathophysiological conditions. Various methodological procedures are in use for their measurement in biological fluids. From the time when capillary electrophoresis was introduced as a technology offering rapid separation of various ionic and/or ionizable compounds with low sample and solvent consumption, there were many attempts to use it for the measurement of amino acids present in physiological fluids. As a rule, these methods require derivatization procedures for detection purposes.Here, we present two protocols for the analysis of free amino acids employing free zone capillary electrophoresis. Main advantage of both methods is an absence of any derivatization procedures that permits the analysis of free amino acid in physiological fluids. The method using direct detection and carrier electrolyte consisting of disodium monophosphate (10mM at pH2.90) permits determination of compounds that absorb in UV region (aromatic and sulfur containing amino acids, as well as some peptides such as carnosine, reduced, and oxidized glutathione). The other method use indirect absorbance detection, employing 8mM p-amino salicylic acid buffered with sodium carbonate at pH10.2 as running electrolyte. It permits quantification of 30 underivatized physiological amino acids and peptides. In our experience factorial design represents a useful tool for final optimization of the electrophoretic conditions if it is necessary.

Quantitative Assessment of Poorly Soluble Anticoagulant Rivaroxaban by Microemulsion Electrokinetic Chromatography.

Microemulsion electrokinetic chromatography (MEEKC) is an electrophoretic methodology based on the separation of compounds by a microemulsionated electrolyte. There are few options for the evaluation of the stability and content of the oral anticoagulant rivaroxaban (RIV) in pharmaceutical formulations. RIV has low water solubility and undergoes ionization only under restricted pH conditions (pH < 1 or pH > 13), thus, hindering the application of free zone capillary electrophoresis as an analytical method. Therefore, the work aimed at developing and validating a stability-indicating MEEKC method for the analysis of RIV in pharmaceutical formulations. Separation was performed in a fused-silica capillary applying a voltage of 30 kV. The microemulsion system consisted of 13 mM tetraborate, pH 9.75 + 1.2% SDS + 1.0% ethyl acetate + 2.4% butanol. The linearity range was 25-150 μg mL-1, with r = 0.9982. Drug degradations were performed in acid and basic media (HCl 1 M and NaOH 0.1 M, respectively), oxidation with 3%H2O2, 60°C temperature and exposure to UV-C radiation. No interferences with RIV or internal standard peaks were detected. Method robustness was accessed through Plackett-Burman experimental design, after evaluation of model validity. Trueness values between 100.49 and 100.68% were obtained with repeatability. The method developed was found appropriate for quality control of RIV tablets, as a consistent analytical technique that is considered less damaging to the environment due to its low consumption of organic reagents.

Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009-2014).

This review of capillary electrophoresis methods for DNA analyses covers critical advances from 2009 to 2014, referencing 184 citations. Separation mechanisms based on free-zone capillary electrophoresis, Ogston sieving, and reptation are described. Two prevalent gel matrices for gel-facilitated sieving, which are linear polyacrylamide and polydimethylacrylamide, are compared in terms of performance, cost, viscosity, and passivation of electroosmotic flow. The role of capillary electrophoresis in the discovery, design, and characterization of DNA aptamers for molecular recognition is discussed. Expanding and emerging techniques in the field are also highlighted.

Effect of β-lactoglobulin A and B whey protein variants on cheese yield potential of a model milk system

Cheese yield mainly depends on the amount and proportion of milk constituents; however, genetic variants of the proteins present in milk may also have an important effect. The objective of this research was to study the effect of the variants A and B of β-lactoglobulin (LG) on cheese yield using a model system consisting of skim milk powder fortified with different levels of a mixture containing α-lactalbumin and β-LG genetic variants (A, B, or A-B) in a 1:2 ratio. Fortified milk samples were subjected to pasteurization at 65°C for 30min. Miniature cheeses were made by acidifying (pH=5.9) fortified milk and incubating with rennet for 1h at 32°C. The clot formed was cut, centrifuged at 2,600×g for 30min at 20°C and drained for determining cheese yield. Cheese-yielding capacity was expressed as actual yield (grams of cheese curd per 100g of milk) and dry weight yield (grams of dried cheese curd per 100g of milk). Free-zone capillary electrophoresis was used for determining β-LG A or B recovery in the curd during rennet-induced coagulation. The presence of β-LG variant B resulted in a significantly higher actual and dried weight cheese yield than when A or A-B were present at levels ≤0.675% of whey protein (WP) addition. Results of free-zone capillary electrophoresis allowed us to infer that β-LG B associates with the casein micelles during renneting, as shown by an increase in the recovery of this variant in the curd when β-LG B was added up to a maximum at 0.45% (equivalent to 0.675% WP). In general, actual or dried weight cheese yield increased as WP addition was increased from 0.225 to 0.675%. However, when WP addition ranged from 0.675 to 0.90%, a drastic drop in cheese yield was observed. This behavior may be because an increase in the aggregation of casein micelles with a concomitant inclusion of whey protein in the gel occurs at low levels of WP addition, whereas once the association of WP with the casein micelles reach a saturation point at addition levels higher than 0.675%, rearrangements of the gel network result in larger whey expulsion and syneresis. This knowledge is expected to be useful to maximize cheese yield and optimize processing conditions during cheese and cheese analogs manufacturing.

Electrophoretic Heterogeneity Limits the Utility of Streptavidin-β-Galactosidase as a Probe in Free Zone Capillary Electrophoresis Separations

Single molecule assays were performed on streptavidin-β-galactosidase using a capillary electrophoresis-based protocol in order to assess the suitability of single molecule β-galactosidase assays for adaptation to the detection of single copies of target DNA. The conjugate was found to have a heterogeneous catalytic rate, showing an average rate of 44,000 ± 24,000 min(-1), which is similar to that of the unmodified enzyme. Electrophoretic mobility was also measured on individual molecules and determined to be -1.32 × 10(-4) ± 0.19 × 10(-4) cm(2)V(-1)s(-1). The variance in mobility was several times that reported for the unmodified enzyme. The electrophoretic heterogeneity was found to result in the formation of a broad window of peaks in the resultant electropherograms of free zone separations of small plugs of streptavidin-β-galactosidase. This range of mobilities largely overlapped with that of the conjugate bound to primer and plasmid containing a target DNA sequence. This overlap suggests that the separation of free conjugate from that bound to target DNA, which is a requirement for application of the single enzyme molecule assay to the detection of target DNA sequences, is not plausible using free zone capillary electrophoresis.

Advances of Modern Electrophoresis Methods with Laser-Induced Fluorescence

Capillary electrophoresis (CE), especially free zone CE, offers a relatively simple separation with moderate selectivity based on the mobility of ions in solution. Laser-induced fluorescence (LIF) detection, an extremely sensitive technique, can be coupled with a variety of separation conditions to achieve sensitive and quantitative results. When these techniques are combined, CE/LIF provides the sensitivity and increased selectivity that makes trace level environmental analysis of fluorescent compounds possible at or below levels typical for GC/MS. We offer a panoramic review of the role of these tools in solving environmental and related analytical problems before providing a detailed experimental protocol.

Technological characterisation by free zone capillary electrophoresis (FCZE) of the vegetable rennet (Cynara cardunculus) used in “Torta del Casar” cheese-making

The purpose of this work was to develop a procedure based on protein analysis by free zone capillary electrophoresis (FZCE) that can be used as an alternative to other methods in the determination of the technological quality of vegetable rennet to use in “Torta del Casar” cheese-making. Samples of cardoon flowers (Cynara cardunculus) grouped according to location, harvest year, and ripening stages were used in the study. For the FZCE, a protocol for extracting the methanol-soluble proteins was tested. This method was found to give good repeatability of the corrected migration time (CMT), and showed higher effectiveness in discriminating the technological properties (milk-clotting and casein degradation activities) of vegetal rennets than the SDS–PAGE technique. In addition, three peaks found in the FZCE electropherograms were examined as a good tool to predict the impact of vegetable rennet on the creaminess and overall acceptability of the “Torta del Casar” cheese.

Kinetic studies of unmodified individual Escherichia coli β-galactosidase molecules in free solution

Assays were performed on individual Escherichia coli beta-galactosidase molecules at 2 different concentrations of the substrate DDAO-beta-D-galactoside using a free zone capillary electrophoresis-based protocol with an in-laboratory-constructed instrument utilizing laser-induced fluorescence detection. In a typical run, 2 enzyme molecules were injected into the capillary. They were separated from each other by a brief period of electrophoresis and incubated on the capillary in the presence of the substrate. They were then mobilized on the capillary into a zone of substrate at a different concentration, re-incubated, and the product peaks mobilized past the detector . The relative change in activity as the concentration was increased differed between molecules, suggesting differences in Km. In a different experiment, the capillary was filled with on average 13 enzyme molecules per run, incubated, and the activities of the individual molecules determined. The shapes of the distribution curves of single molecule activities obtained at different concentrations of the substrate resorufin-beta-D-galactoside were indistinguishable, suggesting a homogeneous Km. To explain why individual enzyme molecules behaved as if they were heterogeneous with respect to Km but the population behaved as if it were homogeneous, theoretical Michaelis-Menten curves were constructed. The curves for populations with heterogeneous Km values were found to be indistinguishable from that of a homogeneous population.

Prolamin proteins alteration in durum wheat by species of the genus Eurygaster and Aelia (Insecta, Hemiptera)

Wheat bugs are widely distributed in various areas of Europe, Asia and North Africa. Species belonging to the genus Eurygaster and Aelia pierce wheat kernels affecting protein quality. The effects of these insects' feeding activity have been studied mainly in bread wheat (Triticum aestivum L.). This study provides information on the degradation of prolamin proteins (glutenins and gliadins) of bug-damaged durum wheat (Triticum turgidum L. var durum) in six cultivars grown in Sardinia (Italy). Samples of whole flour mixture of 70% sound wheat and 30% damaged wheat were hydrated and incubated at two temperatures (45 and 4 deg C), for different periods of time (0, 1 and 3 h). Glutenin and gliadin content was analysed using free zone capillary electrophoresis. The presence of bug-damaged kernels had influence on the quality of durum wheat proteins. Glutenins were rapidly degraded independently to incubation temperature. Gliadin degradation, however, took place with dependence on temperature and incubation time. Therefore glutenin degradation was possibly not due solely to the activity of proteolytic enzymes but also to some other as yet unknown factor linked to wheat bugs' feeding activity.

Authentication of “Cereza del Jerte” sweet cherry varieties by free zone capillary electrophoresis (FZCE)

The purpose of this work was to develop a procedure based on protein analysis by free zone capillary electrophoresis (FZCE) that can be used as an alternative to other methods in the determination of sweet cherry varieties for the authentication of “Cereza del Jerte”. Two autochthonous varieties of sweet cherry type “Picota”, ‘Ambrunés’ and ‘Pico Negro’, and the foreign variety ‘Sweetheart’ were used in the study. Two protocols for extracting the methanol-soluble proteins were tested. On the basis of the results, direct evaporation with nitrogen of a methanol extract was included in the extraction protocol for routine analysis. This method was found to give excellent repeatability of the corrected migration time (CMT), and showed greater effectiveness in discriminating sweet cherry varieties than the SDS–PAGE technique. Three peaks found in the FZCE electropherograms were investigated as a basis for discriminating between varieties. In addition, the FZCE analysis of methanol-soluble proteins provides information about the physico-chemical parameters relevant to the sensorial quality of the sweet cherries.

Hard Red Spring wheat/C-TRIM 20 bread: Formulation, processing and texture analysis

C-TRIM, a β-glucan-rich fraction, was added to Hard Red Spring wheat (HRSW) flour to increase soluble fiber content of bread, and to obtain a minimum of 0.75 g/bread serving (0.75 g/30 g or 2.5%) required by FDA for health claim. Three treatments or blends FGT0 (100% wheat flour – control), FGT1 (58% flour, 25% gluten and 17% C-TRIM) and FGT2 (60% flour, 22.5% gluten, and 17% C-TRIM) were used in the study. The total amount of soluble fiber from C-TRIM in FGT1 and FGT2 was 4.07–4.17% which was more than the amount required by FDA. The presence of C-TRIM increased both, the Farinograph water absorption and the arrival time. The dough mixing tolerance index (MTI) was also increased by C-TRIM. The FGT1 had higher stability than FGT2, whereas, the loaf volume of FGT1-B was also significantly higher than FGT0-B control and FGT2-B bread. The DSC results indicated that the amount of freezable-water in C-TRIM treated bread (FGT1-B and FGT2-B) was significantly higher than the control wheat flour bread (FGT0-B). This may be attributed to the higher amount of water absorbed by C-TRIM during bread dough (FGT1-D and FGT2-D) preparation and trapped or bound within the bread matrix after baking as compared to the control. After storage of FGT0-B, FGT1-B, and FGT2-B breads 2, 5, and 7 days storage at 25 °C, 4 °C, and −20 °C, the texture of bread were measured with a Texture Analyzer and the data analyzed statistically. The FTG0-B control bread firmness was significantly higher than FGT1-B and FGT2-B C-TRIM treated breads after 7 days storage at 25 °C. The amount of 0.1 M acetic acid-extractable protein was lower in FGT1-B than the control wheat flour (FGT0-B) sample. In addition, more protein was extracted at pH 7.0 than pH 4.5 because of less charges at neutral pH than pH 4.5. The free zone capillary electrophoresis analysis showed obvious differences in the protein charge and size between the dough and bread.

Preferential binding of sorghum tannins with γ-kafirin and the influence of tannin binding on kafirin digestibility and biodegradation

Kafirins, sorghum prolamins bind with sorghum condensed tannins (CTs). The binding of different kafirin species with sorghum CTs was investigated. Analysis by chemical assay and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), reversed-phase high-performance liquid chromatography (RP-HPLC), and free zone capillary electrophoresis (FZCE), showed that γ-kafirin bound more CTs than the other kafirin species. SDS–PAGE suggested that the γ-kafirin-bound tannins were in the form of aggregates of molecular size >200k. RP-HPLC and FZCE revealed that sample preparation and drying the kafirins prior to the binding assays had a significant impact on γ-kafirin solubility. The effect of tannin binding on kafirin and kafirin film digestibility and film biodegradation was determined. Kafirins bound to tannins had lower digestibilities than unbound kafirins. Films made from tannin-bound kafirin had much lower digestibility and were less biodegradable than films made from unbound kafirin. The increase in kafirin film life by tannin modification appears to be due to a decrease in protein digestibility caused by kafirin–tannin binding. These findings suggest that γ-kafirin content in sorghum may be manipulated to either reduce or increase tannin binding in order to change the functionality of the kafirin in food, feed or film applications.

Effect of β-Lactoglobulin A and B Whey Protein Variants on the Rennet-Induced Gelation of Skim Milk Gels in a Model Reconstituted Skim Milk System

The effect of fortifying reconstituted skim milk with increasing levels of the β-lactoglobulin (β-LG) genetic variants A, B, and an A-B mixture on rennet-induced gelation was studied by small deformation-sensitive rheology. Free-zone capillary electrophoresis and high-sensitivity oscillatory rheology were used to elucidate the role of potential heterotypic associative interactions between whey proteins and casein in a mixed colloidal system, subjected to moderate heating (65°C for 30min) prior to renneting, on the gelling properties of the system. Increasing levels of added whey protein, in the concentration range of 0.225 to 1.35% of added protein, led to a concomitant progressive increase in the equilibrium shear storage modulus, G′ (recorded after ∼10,800s), in the order β-LG B>β-LG A and β-LG A-B, as the general expected consequence of the setup of denser casein gel networks. The preferential effect of β-LG B over β-LG A on the mechanical strength of the gels may be due to the formation of cross-links and aggregates involving whey proteins and rennet hydrolysis products or an increase in the size of the casein micelle caused by the grafting of β-LG B to its surface, or both. The results of free-zone capillary electrophoresis were consistent with the notion that β-LG B (and not β-LG A) binds to the casein micelle under an optimal stoichiometry of 1:0.045 (mg/mg), even in the absence of heat treatment. The liquid-like character of the gel networks formed, tan δ, was a parameter sensitive to the level of addition of β-LG A in particular. At low concentrations (up to 0.45%) of β-LG A, tan δ increased by almost twice as much, which was interpreted as a result of the increase in the loss modulus, G″, of the sol fraction because of the presence of unbound β-LG A. At greater incremental concentrations of β-LG (>0.45%), the formation of smaller whey protein aggregates confined to the sol fraction may have led to a progressive decrease in tan δ. The critical gel time, tgel, was also affected by the concentration of added whey protein and described 3 zones of behavior, irrespective of the type of whey protein variant. The critical gel time was slightly shorter for β-LG B than for β-LG A at 0.45% of added whey protein, but this difference became larger at 0.67%. Even when only β-LG B was found to associate with casein prior to renneting, both β-LG A and β-LG B, either alone or mixed, had a profound influence on the mechanical strength and coagulation kinetics of the rennet-induced casein gels. This knowledge is expected to be useful to exert better control and optimize processing conditions during the manufacturing of cheese and cheese analogs.

Application of temperature-induced phase partition of proteins for the detection of smoked paprika adulteration by free zone capillary electrophoresis (FZCE)

A procedure for protein extraction was developed for use in the determination, by free zone capillary electrophoresis (FZCE), of smoked paprika “Pimentón de La Vera” adulteration with paprika elaborated from varieties of pepper foreign to the “La Vera” region, in the centre-west of Spain. Two autochthonous varieties of pepper, Jaranda and Bola, and the varieties Papri Queen, Papri King and Sonora, foreign to the “La Vera” region, were used in the study. Several Tris–HCl buffer concentrations and pH values were tested for the extraction of the hydrophilic and hydrophobic protein fractions obtained by temperature-induced phase partition with Triton X-114. On the basis of the results, 0.5 mM Tris–HCl buffer, pH 7.4, with 150 mM sodium chloride, was adopted as the optimal extraction buffer. Five peaks found in the FZCE electropherograms of the hydrophilic protein fraction were investigated as a basis for detecting and estimating the adulteration of smoked paprika. The adulteration detection limits varied from 10% to 40% of paprika elaborated from foreign varieties within a satisfactory working range of admixture (5–80%) sufficiently large to cover the adulteration levels of interest. In addition, a peak of this fraction was identified as a marker for the smoke-drying process. With respect to the hydrophobic proteins, the use of the peak denominated M and the ratio of peaks M and K as markers for determining adulteration gave the best results, with an adulteration detection limit of 5% (w/w), and correlation coefficients greater than 0.965.

Free-Zone Capillary Electrophoresis Analysis of Hordein Patterns at Different Stages of Barley Malting

We have carried out a comparison of hordein patterns at different stages of the malting process using free-zone capillary electrophoresis (FZCE). FZCE has proved to be a suitable technique for the separation and characterization of hordeins in barley seeds. Assays of protein extraction and electrophoretic procedures led us to conclude that hordeins were best extracted with 40% ethanol and analyzed using 50 mM phosphate-glycine, pH 2.5, containing 20% ACN and 0.05% HPMC, at 12.5 kV and 45 degrees C, with 10 s hydrodynamic injection at 0.5 psi and 50 microm i.d. x 31 cm uncoated fused-silica capillary. Our results afford useful information about changes in the composition of these proteins in barley during malting.

Detection of Smoked Paprika “Pimentón de La Vera” Adulteration by Free Zone Capillary Electrophoresis (FZCE)

The purpose of this work was to develop a procedure based on protein analysis by free zone capillary electrophoresis (FZCE) that can be used in the determination of smoked paprika "Pimentón de La Vera" adulteration with paprika elaborated from varieties of pepper foreign to the "La Vera" region, in central western Spain. Two autochthonous varieties of pepper, Jaranda and Bola, and the variety Papri Queen, foreign to the "La Vera" region, were used in the study. Several aqueous solutions for solubilization of the methanol-soluble proteins were tested, and the FZCE conditions of capillary dimensions, FZCE buffer concentrations, and detection wavelengths were optimized. On the basis of the results, 30% (v/v) acetonitrile was adopted as the suspending solution for routine analysis, and the optimal FZCE parameters were 75 microm inner diameter and 57 cm total length capillaries, 8.75 mM phosphate/20.6 mM tetraborate as run buffer, and 256 nm as detection wavelength. This method was found to give excellent repeatability of the corrected migration time (CMT) with coefficients of variation (RSD %; n = 5) of <1% for most of the proteinaceous compounds analyzed and showed greater effectiveness in discriminating paprika varieties than the SDS-PAGE technique. Four peaks found in the FZCE electropherograms were investigated as a basis for detecting and estimating the adulteration of smoked paprika with paprika elaborated from the Papri Queen variety. The adulteration detection limits varied from 5 to 40% of the Papri Queen variety within a satisfactory working range of mixture (5-80%) sufficiently large to cover the adulteration levels of interest. The use of peak 6 as a marker for determining adulteration gave the best results, with an adulteration detection limit of 5-10% (w/w).

Evaluation of a Preclinical Blood Test for Scrapie in Sheep Using Immunocapillary Electrophoresis

An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.

Plasma creatinine and creatine quantification by capillary electrophoresis diode array detector

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