Cost-effective Capillary Electrophoresis Research Articles

Whole exome sequencing analysis of canine urothelial carcinomas without BRAF V595E mutation: Short in-frame deletions in BRAF and MAP2K1 suggest alternative mechanisms for MAPK pathway disruption.

Molecular profiling studies have shown that 85% of canine urothelial carcinomas (UC) harbor an activating BRAF V595E mutation, which is orthologous to the V600E variant found in several human cancer subtypes. In dogs, this mutation provides both a powerful diagnostic marker and a potential therapeutic target; however, due to their relative infrequency, the remaining 15% of cases remain understudied at the molecular level. We performed whole exome sequencing analysis of 28 canine urine sediments exhibiting the characteristic DNA copy number signatures of canine UC, in which the BRAF V595E mutation was undetected (UDV595E specimens). Among these we identified 13 specimens (46%) harboring short in-frame deletions within either BRAF exon 12 (7/28 cases) or MAP2K1 exons 2 or 3 (6/28 cases). Orthologous variants occur in several human cancer subtypes and confer structural changes to the protein product that are predictive of response to different classes of small molecule MAPK pathway inhibitors. DNA damage response and repair genes, and chromatin modifiers were also recurrently mutated in UDV595E specimens, as were genes that are positive predictors of immunotherapy response in human cancers. Our findings suggest that short in-frame deletions within BRAF exon 12 and MAP2K1 exons 2 and 3 in UDV595E cases are alternative MAPK-pathway activating events that may have significant therapeutic implications for selecting first-line treatment for canine UC. We developed a simple, cost-effective capillary electrophoresis genotyping assay for detection of these deletions in parallel with the BRAF V595E mutation. The identification of these deletion events in dogs offers a compelling cross-species platform in which to study the relationship between somatic alteration, protein conformation, and therapeutic sensitivity.

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.

Determination of enantiomeric purity of esomeprazole by capillary electrophoresis

Abstract Proton pump inhibitors are the most effective agents used in gastric hyper-acidity-related disorders. Omeprazole is a benzimidazole-derivative compound with an asymmetric sulphur in its structure, which generates its chiral character. Esomeprazole (S-omeprazole) was the first proton pump inhibitor introduced as an enantiomerically pure compound in therapy, after the successful chiral switch of the racemic omeprazole. This work is aimed at performing a complementary study to an already published chiral separation method of omeprazole. As preliminary analysis, the electrophoretic behavior of omeprazole enantiomers and the possible mechanism of the chiral resolution was studied using different background electrolytes containing different β-cyclodextrin derivatives, as chiral selectors. The robustness of the chiral separation method was tested by applying a Plackett-Burman design. The method was validated according to current ICH guidelines and proved to be reliable, linear, precise and accurate for the determination of 0.2% R-omeprazole as chiral impurity in esomeprazole samples. The validated method was successfully used for the analysis of esomeprazole-containing gastro-resistant tablets. According to our results, valuable information on the mechanism of chiral separation of omeprazole was gained and the application area of the previously developed method was successfully enlarged. The presented rapid and cost-effective capillary electrophoresis method proved to be suitable for the determination of enantiomeric purity of esomeprazole from pharmaceutical preparations and could represent an alternative for the available compendial methods.

Comprehensive and quantitative stability study of ascorbic acid using capillary zone electrophoresis with ultraviolet detection and high-resolution tandem mass spectrometry.

Ascorbic acid is a powerful antioxidant compound involved in many biological functions, and a chronic deficiency is at the origin of scurvy disease. A simple, rapid, and cost-effective capillary electrophoresis method was developed for the separation and simultaneous quantification of ascorbic acid and the major degradation products: dehydroascorbic acid, furfural, and furoic acid. Systematic optimization of the conditions was performed that enabled baseline separation of the compounds in less than 10min. In addition to simultaneous quantification of ascorbic acid alongside to the degradation products, stability studies demonstrated the possibility using capillary electrophoresis to separate and identify the major degradation products. Thus, high-resolution tandem mass spectrometry experiments were conducted in order to identify an unknown degradation product separated by capillary electrophoresis and significantly present in degraded samples. Comparison of mass spectrometry data and capillary electrophoresis electropherograms allowed to identify unambiguously trihydroxy-keto-valeraldehyde. Finally, capillary electrophoresis was successfully applied to evaluate the composition of different pharmaceutical preparation of ascorbic acid. Results showed the excellent performance of the capillary electrophoresis method due to the separation of excipients from the compounds of interest, which demonstrated the relevance of using an electrophoretic separation in order to perform comprehensive stability studies of ascorbic acid.

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 β-lactam-based antibiotics commonly used in Vietnam, including single- β-lactam antibiotics (i.e. Cephalexin, Cefotaxime Sodium, Cefixime and Sulbactam) as well as β-lactams co-formulated with Sulbactam (i.e. Amoxicillin, Ampicillin, Cefoperazone and Sulbactam). Single β-lactam antibiotics were analyzed using a background electrolyte (BGE) composed of Tris/Ace (10 mM, pH 7.8) whereas β-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 β-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 (27cm × 75μm ID) and a background electrolyte solution that consisted of 0.75 Mformic acid at pH2.05. 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 25-2000μM (R2 = 0.9998). The LOD and LOQ were 9μM and 27μM, 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.

Letrozole Determination by Capillary Zone Electrophoresis and UV Spectrophotometry Methods

Abstract Objective: Letrozole is a highly potent oral nonsteroidal aromatase inhibitor triazole derivative. The aim of this study was to quantify letrozole from bulk, pharmaceutical formulation, and spiked urine samples by developing a simple, rapid and cost effective capillary electrophoresis method. Methods: A capillary zone electrophoresis method was optimized and validated. Additionally, an UV spectrophotometry method was used for comparing results. Results:The capillary zone electrophoresis method using a 90 mM sodium tetraborate background electrolyte proved to be an efficient method for determination of letrozole in a very short time, less than 2 minutes, using 20 kV voltage, 50 mbar/2 seconds pressure and 50°C temperature as optimum parameters. Additionally, the UV spectrophotometry method proved to be simple and efficient to quantify letrozole from bulk material and pharmaceutical formulation with linearity of response between 5 to 20 μg·mL-1 concentrations. For both methods, validation parameters, including linearity, detection and quantification limits were determined. Also we proved that our electrophoretic method has potential in analyzing letrozole from biological samples, obtaining encouraging results on estimation of letrozole from spiked urine samples without any special treatment. Conclusions: To quantify letrozole from bulk material, pharmaceutical preparations, and spiked urine samples the capillary zone electrophoresis method using a tetraborate sodium background electrolyte has proven to be simple and appropriate. Also a simple UV spectrophotometric method has been developed and validated for the same purposes.

Determination of NTBC in serum samples from patients with hereditary tyrosinemia type I by capillary electrophoresis☆

Hereditary tyrosinemia type I is a serious metabolic disorder leading to liver failure. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is a relatively new drug which is used to prevent the accumulation of toxic metabolites in patients with hereditary tyrosinemia type I. In the present study, we have developed a new, simple, fast, and cost-effective capillary electrophoresis method for the quantitative monitoring of this drug in serum samples. Micellar electrochromatographic separation of NTBC was performed using 20 mmol/L phosphate and 40 mmol/L sodium dodecylsulfate (SDS) at pH 12 as running electrolyte. Separation of NTBC was achieved in around 4 min. Reproducibilities of migration times and corrected peak areas of NTBC (as R.S.D.%) were found as 0.73 and 1.99, respectively. The detection limit was 3.17 and the quantification limit was 10.6 micromol/L for NTBC using UV detection at 278 nm. The utility of the method was demonstrated by the detection of NTBC in serum samples from patients with hereditary tyrosinemia type I using this drug.

PET-laminated thin-film capillary electrophoresis chips by sandwiching method

In this article, the sandwiching method, a novel, simple, and cost-effective capillary electrophoresis (CE) chip fabrication approach, is proposed. In this new approach, polymer PET-laminated film rather than the commonly used silicon wafer is chosen to be the material of the chip; a micro CNC machining center is adopted to cut the microchannels; and a temperature-controlled roll laminator is used to bond the microchannels in between two sealing PET films. Separation experiments using red Rhodamine B and blue Xylene Cyanole illustrate that the separation efficiency of the sandwiching CE chip is comparable to the semiconductor-based chip and the conventional capillary. However, the sandwiching method requires much less complicated fabrication processes and is more cost-effective.

92 The BPSU study of biliary atresia — outcome after 8 years

Hereditary tyrosinemia type I is a serious metabolic disorder leading to liver failure. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is a relatively new drug which is used to prevent the accumulation of toxic metabolites in patients with hereditary tyrosinemia type I. In the present study, we have developed a new, simple, fast, and cost-effective capillary electrophoresis method for the quantitative monitoring of this drug in serum samples. Micellar electrochromatographic separation of NTBC was performed using 20 mmol/L phosphate and 40 mmol/L sodium dodecylsulfate (SDS) at pH 12 as running electrolyte. Separation of NTBC was achieved in around 4 min. Reproducibilities of migration times and corrected peak areas of NTBC (as R.S.D.%) were found as 0.73 and 1.99, respectively. The detection limit was 3.17 and the quantification limit was 10.6 μmol/L for NTBC using UV detection at 278 nm. The utility of the method was demonstrated by the detection of NTBC in serum samples from patients with hereditary tyrosinemia type I using this drug.