A Simple, Accurate and Cost-Effective Capillary Electrophoresis Test with Computational Methods to Aid in Universal Microsatellite Instability Testing.

BACKGROUND: Microsatellite instability (MSI) testing is important for the classification of Lynch syndrome, as a prognostic marker and as a guide for adjuvant chemotherapy in colorectal cancer (CRC). The gold standard for determining MSI status has traditionally been fluorescent multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE). However, its use in the clinical setting has diminished and has been replaced by immunohistochemical (IHC) detection of loss of mismatch repair protein expression due to practicability and cost. The aim of this study was to develop a simple, cost-effective and accurate MSI assay based on CGE. METHOD: After amplification of microsatellites by polymerase chain reaction (PCR) using the National Cancer Institute (NCI) panel (BAT 25, BAT26, D5S346, D2S123, D17S250) of MSI markers, parallel CGE was utilized to classify colorectal cancers as MSI-H, MSI-L and MSS using the 5200 Fragment Analyzer System. Cell lines and patient cancer specimens were tested. DNA from 56 formalin-fixed paraffin-embedded cancer specimens and matched normal tissue were extracted and CGE was performed. An automated computational algorithm for MSI status determination was also developed. RESULTS: Using the fragment analyser, MSI status was found to be 100% concordant with the known MSI status of cell lines and was 86% and 87% concordant with immunohistochemistry (IHC) from patient cancer specimens using traditional assessment and our MSI scoring system, respectively, for MSI determination. The misclassification rate was mainly attributed to IHC, with only one (1.8%) sampling error attributed to CGE testing. CGE was also able to distinguish MSI-L from MSI-H and MSS, which is not possible with IHC. An MSI score based on total allelic variability that can accurately determine MSI status was also successfully developed. A significant reduction in cost compared with traditional fluorescent multiplex PCR and CGE was achieved with this technique. CONCLUSIONS: A simple, cost-effective and reliable method of determining MSI status and an MSI scoring system based on an automatic computational algorithm to determine MSI status, as well as degree of allelic instability in colorectal cancer, has been developed using the 5200 Fragment Analyzer System.

The profiling of microbiota in vaginal swab samples using 16S rRNA gene sequencing and IS-pro analysis.

BACKGROUND: 16S rRNA gene sequencing is currently the most common way of determining the composition of microbiota. This technique has enabled many new discoveries to be made regarding the relevance of microbiota to the health and disease of the host. However, compared to other diagnostic techniques, 16S rRNA gene sequencing is fairly costly and labor intensive, leaving room for other techniques to improve on these aspects. RESULTS: The current study aimed to compare the output of 16S rRNA gene sequencing to the output of the quick IS-pro analysis, using vaginal swab samples from 297 women of reproductive age. 16S rRNA gene sequencing and IS-pro analyses yielded very similar vaginal microbiome profiles, with a median Pearson's R2 of 0.97, indicating a high level of similarity between both techniques. CONCLUSIONS: We conclude that the results of 16S rRNA gene sequencing and IS-pro are highly comparable and that both can be used to accurately determine the vaginal microbiota composition, with the IS-pro analysis having the benefit of rapidity.

Low-cost and versatile analytical tool with purpose-made capillary electrophoresis coupled to contactless conductivity detection: Application to antibiotics quality control in Vietnam.

In this study, the development of our purpose-made capacitively coupled contactless conductivity detection (C4 D) for CE is reported. These systems have been employed as a simple, versatile, and cost-effective analytical tool. CE-C4 D devices, whose principle is based on the control of the ion movements under an electrical field, can be constructed even with a modest financial budget and limited infrastructure. A featured application was developed for quality control of antimicrobial drugs using CE-C4 D, with most recent work on determination of aminoglycoside and glycopeptide antibiotics being communicated. For aminoglycosides, the development of CE-C4 D methods was adapted to two categories. The first one includes drugs (liquid or powder form) for intravenous injection, containing either amikacin, streptomycin, kanamycin A, or kanamycin B. The second one covers drugs for eye drops (liquid or ointment form), containing either neomycin, tobramycin, or polymyxin. The CE-C4 D method development was also made for determination of some popular glycopeptide antibiotics in Vietnam, including vancomycin and teicoplanin. The best detection limit achieved using the developed CE-C4 D methods was 0.5 mg/L. Good agreement between results from CE-C4 D and the confirmation method (HPLC- Photometric Diode Array ) was achieved, with their result deviations less than 8% and 13% for aminoglycoside and glycopeptide antibiotics, respectively.

Fast and accurate quantification of insertion-site specific transgene levels from raw seed samples using solid-state nanopore technology.

Many modern crop varieties contain patented biotechnology traits, and an increasing number of these crops have multiple (stacked) traits. Fast and accurate determination of transgene levels is advantageous for a variety of use cases across the food, feed and fuel value chain. With the growing number of new transgenic crops, any technology used to quantify them should have robust assays that are simple to design and optimize, thereby facilitating the addition of new traits to an assay. Here we describe a PCR-based method that is simple to design, starts from whole seeds, and can be run to end-point in less than 5 minutes. Subsequent relative quantification (trait vs. non-trait) using capillary electrophoresis performed in 5% increments across the 0-100% range showed a mean absolute error of 1.9% (s.d. = 1.1%). We also show that the PCR assay can be coupled to non-optical solid-state nanopore sensors to give seed-to-trait quantification results with a mean absolute error of 2.3% (s.d. = 1.6%). In concert, the fast PCR and nanopore sensing stages demonstrated here can be fully integrated to produce seed-to-trait quantification results in less than 10 minutes, with high accuracy across the full dynamic range.

Cost-effective capillary electrophoresis with contactless conductivity detection for quality control of beta-lactam antibiotics.

A simple and inexpensive approach for determination of various antimicrobial drugs using a purpose-made compact capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C4D) is reported. The objective of the work is to propose an affordable and easily-implemented tool for quality control and detection of counterfeiting of antibiotic formulations in resource constrained developing countries. The design of the purpose-made CE-C4D system was improved according to the feedback from over 10 years of use of our previous instrument. CE-C4D methods were for the first time developed to analyze ß-lactam-based antibiotics commonly used in Vietnam, including single- ß-lactam antibiotics (i.e. Cephalexin, Cefotaxime Sodium, Cefixime and Sulbactam) as well as ß-lactams co-formulated with Sulbactam (i.e. Amoxicillin, Ampicillin, Cefoperazone and Sulbactam). Single ß-lactam antibiotics were analyzed using a background electrolyte (BGE) composed of Tris/Ace (10 mM, pH 7.8) whereas ß-lactam - Sulbactam combinations were simultaneously separated using a BGE containing Tris/Ace (10 mM, pH 7.5). The best achieved detection limits were 2.0 mg/L and 1.0 mg/L for these two groups, respectively. Good agreement between results obtained from CE-C4D and standard confirmation methods (LCMS) was achieved, with a coefficient of determination, r2, of 0.9991. The applicability of the developed CE-C4D method was demonstrated for quality control of 24 ß-lactam-based antimicrobial drugs available in Vietnam.

Screening gut microbial trimethylamine production by fast and cost-effective capillary electrophoresis.

The present work is aimed to develop a simple, rapid, and cost-effective CE method for the determination of trimethylamine (TMA) from bacterial origin. Optimum separation of TMA from the other components of the bacterial culture was achieved using a fused silica capillary (27 cm × 75 µm ID) and a background electrolyte solution that consisted of 0.75 M formic acid at pH 2.05. Analytical characteristics of the proposed method were evaluated through the study of its specificity, linearity, precision, accuracy, robustness, and detection/quantitation limit values. The method was linear over the range 25-2000 µM (R2 = 0.9998). The LOD and LOQ were 9 µM and 27 µM, respectively. Intra-day and inter-day RSD were ≤ 0.24% and ≤ 1.3% for migration time, respectively. Intra-day and inter-day RSD for peak area were ≤ 2.44% and ≤ 3.51%, respectively. The method showed a good accuracy with recovery percentages ranging from 95.45 to 102.21%. The method was successfully applied for the determination of microbial conversion of L-carnitine to TMA. The method shows great potential in high-throughput screening applications to assess the functionality of the gut microbiota to produce TMA. Graphical abstract.

Recent advances in the capillary electrophoresis analysis of antibiotics with capacitively coupled contactless conductivity detection.

This review describes briefly the high rate of counterfeiting of antimicrobial drugs with focus upon its immediate health consequences. The major part of this review encompasses accounts of the improvements achieved in the domain of miniaturization of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D). The application of this principle into the development of portable devices as well as its application to counter the health-system-crippling phenomenon of counterfeit antibiotic formulations, are discussed in the context of developing countries.

Affinity capillary electrophoresis for identification of active drug candidates in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominantly inherited degenerative disease with a slow progression. At the present, there is no commercially available treatment, but sustained effort is currently undertaken for the development of a promising lead compound. In the present paper we report the development of a fast, versatile, and cost-effective affinity capillary electrophoresis (ACE) method for the screening and identification of potential drug candidates targeting pathological ARN probes relevant for DM1. The affinity studies were conducted in physiologically relevant conditions using 50 mM HEPES buffer (pH 7.4) in a fused silica capillary dynamically coated with poly(ethylene oxide), by testing a library of potential ligands against (CUG)50 RNA as target probe with a total run time of 4-5 h/ligand. For the most promising ligands, their affinity parameters were assessed and some results formerly reported on the affinity of pentamidine (PTMD) and neomycin against CUG repeats were confirmed. To the best of the authors' knowledge, the estimated binding stoichiometry for some of the tested compounds (i.e., ~ 121:1 for PTMD against the tested RNA probe) is reported for the first time. Additionally, the potential of a novel pentamidine like compound, namely 1,2-ethane bis-1-amino-4-benzamidine (EBAB) with much lower in vivo toxicity than its parent compound has also been confirmed studying its effect on a live cell model by fluorescence microscopy. Further tests, such as the evaluation of the rescue in the mis-splicing of the involved genes, can be performed to corroborate the potential therapeutic value of EBAB in DM1 treatment. Graphical abstract ᅟ.

Online screening of α-amylase inhibitors by capillary electrophoresis.

Pancreatic α-amylase plays an important role in dietary starch hydrolysis in the small intestine and participates in enhanced glucose concentration after meals. It seems to be a problem for diabetic patients, who suffer from longer postprandial hyperglycemia after meal consumption than healthy people. There are commercially available drugs that inhibit α-amylase and thus reduce the postprandial hyperglycemia effect. However, these drugs may cause severe side effects. Conversely, some naturally occurring flavonoids were suggested to have an α-amylase-inhibiting effect without any side effects. There had been no rapid, undemanding method in terms of sample and reagent preparation that would enable screening of many potential inhibitors. Therefore, we developed an online capillary electrophoresis method to monitor α-amylase activity in the presence of an inhibitor. Each reaction constituent was introduced separately, directly into a capillary where the reagents were mixed by diffusion, which resulted in a 5-min analysis including conditioning of the capillary. We applied the method to test the inhibitory effect of flavonoid standards and their mixture and we investigated the inhibitory effect of ethanolic extract from Betula pendula bark. The developed method presents a faster and less expensive alternative to previously described offline methods. Graphical abstract Online CE screening of α-amylase inhibitors.

A simple, low-cost and robust capillary zone electrophoresis method with capacitively coupled contactless conductivity detection for the routine determination of four selected penicillins in money-constrained laboratories.

A simple and robust capillary zone electrophoresis method was developed and validated for the determination of amoxicillin and clavulanate, ampicillin, phenoxymethyl penicillin (Pen V) as well as flucloxacillin. Capacitively coupled contactless conductivity detection was employed as detection mode that makes CE a simple and economic tool for money-constrained laboratories. The developed method is straightforward and user-friendly. It offers good sensitivity and sufficient selectivity for the routine assay of the selected penicillins. The repeatabilities were <1.9% RSD for relative peak areas and <1% RSD for migration times for all the analytes. The method showed good linearity (R2  > 0.995) within the 80-120% range of the target concentration (0.5 mg/mL) for each antibiotic. The accuracy of the method, evaluated by standard fortification at three levels, was good for all the analytes. An extended robustness study was performed by varying ±10% of the optimum value of TRIS concentration, l-histidine concentration and temperature in a full factorial design at two levels. This was to evaluate larger than usual variability of factors on the assay value, in order to better cover potential global variance in lab conditions and equipment. Finally, the method was applied for the determination of percent (%) content of all antibiotics in available formulations.

Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection.

The branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) play a pivotal role in the human body. Herein, we developed capillary electrophoresis (CE) coupled with conventional UV detector to quantify underivatized BCAAs in two kinds of sport nutritional supplements. For direct UV detection at 195 nm, the BCAAs (Leu, two enantiomers of Ile and Val) were separated in a background electrolyte (BGE) consisting of 40.0 mmol/L sodium tetraborate, and 40.0 mmol/L ß-cyclodextrin (ß-CD) at pH 10.2. In addition, the indirect UV detection at 264 nm was achieved in a BGE of 2.0 mmol/L Na2HPO4, 10.0 mmol/L p-aminosalicylic acid (PAS) as UV absorbing probe, and 40.0 mmol/L ß-CD at pH 12.2. The ß-CD significantly benefited the isomeric separation of Leu, L- and D-Ile. The optimal conditions allowed the LODs (limit of detections) of direct and indirect UV absorption detection to be tens µmol/L level, which was comparable to the reported CE inline derivatization method. The RSDs (relative standard deviations) of migration time and peak area were less than 0.91% and 3.66% (n = 6). Finally, CE with indirect UV detection method was applied for the quantitation of BCAAs in two commercial sport nutritional supplements, and good recovery and precision were obtained. Such simple CE method without tedious derivatization process is feasible of quality control and efficacy evaluation of the supplemental proteins.

Simultaneous electrochemiluminescence determination of galanthamine, homolycorine, lycorenine, and tazettine in Lycoris radiata by capillary electrophoresis with ultrasonic-assisted extraction.

After ultrasonic-assisted extraction, four lycoris radiata alkaloids: galanthamine, homolycorine, lycorenine, and tazettine were determined by capillary electrophoresis electrochemiluminescence. Polyvinylpyrrolidone was added to the running buffer (RB) to obtain better resolution. Experimental conditions influencing the determination were examined, including the additives, detection potential, separation voltage, injection voltage and time, and RB pH and concentration. Under optimal experimental conditions, the baseline separation of the four alkaloids occurred within 16min. The proposed method displayed the following linear ranges (in ng/mL): galanthamine [60-5000], homolycorine [40-5000], lycorenine [5.0-1500], and tazettine [8.0-2500]. The detection limits in ng/mL, (S/N=3), were galanthamine [14], homolycorine [11], lycorenine [1.8], and tazettine [3.1]. Intra-day and inter-day RSDs for the four alkaloids of the six replicates were less than 2.7% and 3.1%, respectively. The recoveries in% were: tazettine [102.5], lycorenine [98.20], galanthamine [97.30], and homolycorine [98.33].

A Rapid Procedure for the Purification of 8-Aminopyrene Trisulfonate (APTS)-Labeled Glycans for Capillary Electrophoresis (CE)-Based Enzyme Assays.

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene 1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, which utilizes equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel and subsequent solid-phase extraction (SPE), yields single glycan species free of excess labeling reagents and buffer components. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.

A rapid and simple method for the determination of psychoactive alkaloids by CE-UV: application to Peganum Harmala seed infusions.

The ß-carboline alkaloids of the harmala (HAlks) group are compounds widely spread in many natural sources, but found at relatively high levels in some specific plants like Peganum harmala (Syrian rue) or Banisteriopsis caapi. HAlks are a reversible Mono Amino Oxidase type A Inhibitor (MAOI) and, as a consequence, these plants or their extracts can be used to produce psychotropic effects when are combined with psychotropic drugs based on amino groups. Since the occurrence and the levels of the HAlks in natural sources are subject to significant variability, more widespread use is not clinical but recreational or ritual, for example B. caapi is a known part of the Ayahuasca ritual mixture. The lack of simple methods to control the variable levels of these compounds in natural sources restricts the possibilities to dose in strict quantities and, as a consequence, limits its use with pharmacological or clinical purposes. In this work, we present a fast, simple, and robust method of quantifying simultaneously the six HAlks more frequently found in plants, i.e., harmine, harmaline, harmol, harmalol, harmane, and norharmane, by capillary electrophoresis instruments equipped with the more common detector UV. The method is applied to analyze these HAlks in P. Harmala seeds infusion which is a frequent intake form for these HAlks. The method is validated in three different instruments in order to evaluate the transferability and to compare the performances between them. In this case, harmaline, harmine, and harmol were found in the infusion samples. Copyright © 2016 John Wiley & Sons, Ltd.

Cycling temperature capillary electrophoresis: A quantitative, fast and inexpensive method to detect mutations in mixed populations of human mitochondrial DNA.

Cycling temperature capillary electrophoresis has been optimised for mutation detection in 76% of the mitochondrial genome. The method was tested on a mixed sample and compared to mutation detection by next generation sequencing. Out of 152 fragments 90 were concordant, 51 discordant and in 11 were semi-concordant. Dilution experiments show that cycling capillary electrophoresis has a detection limit of 1-3%. The detection limit of routine next generation sequencing was in the ranges of 15 to 30%. Cycling temperature capillary electrophoresis detect and accurate quantify mutations at a fraction of the cost and time required to perform a next generation sequencing analysis.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis.

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

Study on the potential application of salivary inorganic anions in clinical diagnosis by capillary electrophoresis coupled with contactless conductivity detection.

A capillary electrophoresis approach with capacitively coupled contactless conductivity detection method has been developed for the determination of inorganic metabolites (thiocyanate, nitrite and nitrate) in human saliva. Field amplified sample injection, as a simple sample stacking technique, was used in conjunction for online preconcentration of above inorganic anions. A selective separation for the target anions from other coexisting constituents present in saliva could be obtained within 14min in a 10mmol/L His-90mmol/L HAc buffer (pH 3.70) at the separation voltage of -18kV. The limits of detection and limits of quantification of the three analytes were within the range of 3.1-4.9ng/mL (S/N=3) and 10-16ng/mL (S/N=10), respectively. The average recovery data were in the range of 81-108% at three different concentrations. This method provides a simple, rapid and direct approach for metabolite analyses of nitric oxide and cyanide based on noninvasive saliva sample, which presents a potential fast screening tool for clinical test.

Analysis of γ-hydroxy butyrate by combining capillary electrophoresis-indirect detection and wall dynamic coating: application to dried matrices.

γ-Hydroxybutyric acid (GHB) is a powerful central nervous system depressant, currently used in medicine for the treatment of narcolepsy and alcohol dependence. In recent years, it has gained popularity among illegal club drugs, mainly because of its euphoric effects as well as doping agent and date rape drug. The purpose of the present work was the development of a rapid analytical method for the analysis of GHB in innovative biological matrices, namely dried blood spots (DBSs) and dried urine spots (DUSs). The analytical method is based on capillary zone electrophoresis with indirect UV absorption detection at 210 nm and capillary wall dynamic coating. The background electrolyte is composed of a phosphate buffer containing nicotinic acid (probe for detection) and cetyltrimethylammonium bromide (CTAB, reversal of electroosmosis in wall dynamic coating). The influence of probe and CTAB concentration, together with buffer pH, on migration time and signal response was investigated. Under the optimized conditions, analytical linearity and precision were satisfactory; absolute recovery values were also high (>90 %); the use of dried matrices (DBSs and DUSs) was advantageous as an alternative matrix to classical ones. No interferences were found either from the most common exogenous or from endogenous compounds. This analytical approach can offer a rapid, precise and accurate method for GHB determination in innovative biological samples, which could be important for screening purposes in clinical and forensic toxicology. Graphical Abstract CE method, by combined indirect UV detection and dynamic coating, for GHB determination in DBSs and DUSs.

In-line coupling of microextractions across polymer inclusion membranes to capillary zone electrophoresis for rapid determination of formate in blood samples.

Polymer inclusion membranes (PIMs) have several important features, i.e., PIMs are dry and non-porous membranes, which can be prepared ahead of use and stored without noticeable deterioration in extraction performance. In this contribution, in-line coupling of microextractions across PIMs to a separation method for clinical purposes was demonstrated for the first time. Formate (the major metabolite in methanol poisoning) was determined in undiluted human serum and whole blood by capillary zone electrophoresis (CZE) with simultaneous capacitively coupled contactless conductivity detection (C(4)D) and UV-Vis detection. A purpose-made microextraction device with PIM was coupled to a commercial CZE instrument in order to ensure complete automation of the entire analytical procedure, i.e., of formate extraction, injection, CZE separation and quantification. PIMs for formate extractions consisted of 60% (w/w) cellulose triacetate as base polymer and 40% (w/w) Aliquat™ 336 as anion carrier. The method was characterized by good repeatability of peak areas (≤7.0%) and migration times (≤0.8%) and by good linearity of calibration curves (r(2) = 0.993-0.999). Limits of detection in various matrices ranged from 15 to 54 µM for C(4)D and from 200 to 635 µM for UV-Vis detection and were sufficiently low to clearly distinguish between endogenous and toxic levels of formate in healthy and methanol intoxicated individuals. In addition, PIMs proved that they may act as phase interfaces with excellent long-term stability since once prepared, they retained their extractions properties for, at least, two months of storage.

Fast analysis of glibenclamide and its impurities: quality by design framework in capillary electrophoresis method development.

A fast capillary zone electrophoresis method for the simultaneous analysis of glibenclamide and its impurities (I(A) and I(B)) in pharmaceutical dosage forms was fully developed within a quality by design framework. Critical quality attributes were represented by I(A) peak efficiency, critical resolution between glibenclamide and I(B), and analysis time. Experimental design was efficiently used for rapid and systematic method optimization. A 3(5)//16 symmetric screening matrix was chosen for investigation of the five selected critical process parameters throughout the knowledge space, and the results obtained were the basis for the planning of the subsequent response surface study. A Box-Behnken design for three factors allowed the contour plots to be drawn and the design space to be identified by introduction of the concept of probability. The design space corresponded to the multidimensional region where all the critical quality attributes reached the desired values with a degree of probability π ≥ 90%. Under the selected working conditions, the full separation of the analytes was obtained in less than 2 min. A full factorial design simultaneously allowed the design space to be validated and method robustness to be tested. A control strategy was finally implemented by means of a system suitability test. The method was fully validated and was applied to real samples of glibenclamide tablets.