Sample Injection Method Research Articles

SEC x IP two-dimensional LCMS for the analysis of non-denatured and denatured cyclic-peptide siRNAs in a single step

The increased effectiveness of small interfering RNAs (siRNAs) to induce gene silencing has brought a great therapeutic promise to many diseases. siRNAs are under highly active current research and development. Ligand conjugation and chemical modifications of the sense (SS) and antisense (AS) strands of the siRNA duplex improve stability and facilitate delivery, but significantly increase the complexity of the analytical requirements. Two chromatographic methods are needed to guide synthesis and formulation: (1) a non-denaturing method to analyze the duplex, residual sense and antisense strands, their impurities, and those of the duplex, and (2) a denaturing method for each strand and its impurities. In this work, ion-pair reversed phase (IP-RP) and strong anion exchange (SAX) methods were not successful in the analysis of a cyclic-peptide (CP) siRNA, in the non-denaturing mode. Selection of the most appropriate chromatographic method is greatly challenged by the chemical properties of the conjugated ligands. However, separation was possible by size exclusion chromatography (SEC). The non-denaturing SEC method was implemented, using a 2D-LC system, in the 1D dimension of the analysis, coupled with a denaturing IP-RP method in the 2D dimension. The 2D-LC system greatly simplified the siRNA analysis by combining, for the first time, the non-denaturing and denaturing methods into a single-instrument, one sample injection method. An additional benefit of the 2D-LC system is the interfacing of MS-incompatible methods (e.g., SAX, SEC) to a mass spectrometer, broadening thus the analytical options, by coupling with MS-compatible methods (IP-RP, HILIC) in the 2D dimension. Application of the approach was exemplified in a CP-siRNA duplex formulation study to determine the optimal mixing ratio of the individual strands. A duplex maximum was reached at a sample solution AS:SS ratio of 0.9. The method was found to be independent of the amount and concentration of sample injected. A duplex annealing study found no significant temperature or salt effects in the formulation of the CP-siRNA duplex.

Differential Colorimetric pH Measurement With a Microfluidic Chip Using a Novel Sample Injection Method

Differential Colorimetric pH Measurement With a Microfluidic Chip Using a Novel Sample Injection Method

Improvement of an Automated Sample Injection System for Pillar Array Columns to Increase Analytical Reproducibility.

In our previous study, we developed an automatic sample injection system for pillar array columns for quantitative analysis. An autosampler was used to maintain a constant sample injection volume. However, the sample was diluted during injection using the autosampler, thus deteriorating the analytical reproducibility. In this study, we have substituted the autosampler with a syringe pump to overcome the abovementioned problem and improve the system. Sample dilution was avoided by filling the entire capillary with the sample at a constant rate. This improved system also increased the analytical reproducibility. In the previous system, the relative standard deviation (RSD) exceeded 17% of the peak height for coumarin dyes. In contrast, the improved system decreased the RSD to the range 1.2–1.8%. The analytical reproducibility was evaluated by using five types of amino acids. The RSD of each peak height was within 3.0%, confirming good reproducibility. These results indicate that the sample injection method developed in this study can be applied to biological sample analyses as a simple quantitative analysis method for pillar array columns.

Sensitive Analysis of Metoprolol Tartrate and Diltiazem Hydrochloride in Human Serum by Capillary Zone Electrophoresis Combining on Column Field-Amplified Sample Injection.

A sensitive capillary zone electrophoresis (CZE) combined with field-amplified sample injection (FASI) method was investigated to simultaneously determine diltiazem (DI) hydrochloride and metoprolol (ME) tartrate in human serum. This method was validated by linearity, limits of detection (LOD), stability, precision and accuracy, and the related parameters are linearity (r2>0.9980), intra- and inter-day precisions (intra-day relative standard deviation (RSD) is 2.3-3.4%, and inter-day RSD is 3.8-7.5%) and the LOD (0.02ng/mL for ME tartrate and 0.01ng/mL for DI hydrochloride). So the proposed method is rapid, sensitive and accurate for determination of these two anti-anginal drugs in human serum. As for enrichment conditions, it was helpful to add phosphoric acid and isopropanol into samples for enrichment efficiency. A model was established to illustrate the possible enrichment mechanism of analytes, and a theory of half-width was also applied in CZE combined with FASI method to evaluate the peak performance.

Simultaneous Determination of Trace Fluorine and Chlorine in Iron Ore by Combustion-Ion Chromatography (C-IC)

The determination of trace fluorine and chlorine in iron ore commonly requires complicated and time-consuming pretreatment involving alkali fusion, combustion in bombs, and pyrohydrolysis that prevent analysis of large numbers of samples. In this study, a rapid combustion-ion chromatography (C-IC) method for the simultaneous determination of fluorine and chlorine in iron ore is reported for the first time. The solid sample injection method, combustion time, sample mass, and absorption solution were optimized. The limits of detection for fluorine and chlorine were 0.029 mg g−1 and 0.031 mg g−1, respectively, which were similar to those for GB/T 6730.69-2010. Eight certified reference materials and three iron ore samples were used to evaluate the accuracy and precision of C-IC. The fluorine and chlorine content in the iron ore samples were from 0.086 to 1.773 mg g−1 and 0.050 to 3.10 mg g−1. The relative standard deviations for fluorine and chlorine were from 0.77% to 4.27% and 1.36% to 3.74%. The recoveries ranged from 95.3% to 104.6% and 91.9% to 104.3%. The results of iron ore samples determined by GB/T 6730.69-2010 and C-IC were in good agreement. C-IC is environmentally friendly and its suitability for automation may improve the analytical throughput.

MÉTODOS DE INJEÇÃO DA AMOSTRA EM MICROSSISTEMAS ELETROFORÉTICOS

SAMPLE INJECTION METHODS IN ELECTROPHORETIC MICROSYSTEMS. Electrophoresis is by far the most popular separation method implemented in microscale, most probably due to its instrumental simplicity, low cost and portability. Due to the increasing use of miniaturized electrophoretic systems, the study of fundamental aspects can improve the development of methodologies for several applications. One of the major challenges related to electrophoresis chips refers to the sample injection mode, in this way, this study presents a review on sample injection methods for microchip electrophoresis covering electrokinetic and hydrodynamic approaches, describing theoretic and instrumental aspects. Since the sample volume affects the analytical performance, the precise and reproducible control of the sample amount to be introduced into the separation channel is highly desirable to ensure reliable chemical analysis. Electrokinetic modes based on floating, pinched and gated protocols are presented and discussed providing an overview about the electrokinetic control of the sample through three methodologies. In the same way, hydrodynamic techniques including the use of microfabricated valves and pumps, syringe pumps, electronic micropipettes, rubber suction bulb and acupuncture needle are approached, thus expanding the view of hydrodynamic injectors based on classical and alternative methods.

Thermally Induced Actinidine Production in Biological Samples.

Actinidine, a methylcyclopentane monoterpenoid pyridine alkaloid, has been found in many iridoid-rich plants and insect species. In a recent research on a well-known actinidine- and iridoid-producing ant species, Tapinoma melanocephalum (Fabricius) (Hymenoptera: Formicidae), no actinidine was detected in its hexane extracts by gas chromatography-mass spectrometry analysis using a common sample injection method, but a significant amount of actinidine was detected when a solid injection technique with a thermal separation probe was used. This result led us to hypothesize that heat can induce the production of actinidine in iridoid-rich organisms. To test our hypothesis, the occurrence of actinidine was investigated in four iridoid-rich organisms under different sample preparation temperatures, including two ant species, T. melanocephalum and Iridomyrmex anceps Roger (Hymenoptera: Formicidae), and two plant species, Actinidia polygama Maxim (Ericales: Actinidiaceae) and Nepeta cataria L. (Lamiales: Lamiaceae). Within a temperature range of 50, 100, 150, 200, and 250 °C, no actinidine was detected at 50 °C, but it appeared at temperatures above 100 °C for all four species. A positive relationship was observed between the heating temperature and actinidine production. The results indicate that actinidine could be generated at high temperatures. We also found that the presence of methylcyclopentane monoterpenoid iridoids (iridodials and nepetalactone) was needed for thermally induced actinidine production in all tested samples. These results suggest that the presence of actinidine in iridoid-rich plants and ants might be a consequence of using high temperatures during sample preparation.

Solid phase extraction and capillary electrophoretic separation of racemic catecholamines by using magnetic particles coated with a copolymer prepared from poly(3,4-dihydroxyphenylalanine) and polyethyleneimine.

A method is presented for chiral separation of catecholamines present in bovine and mice blood. It combines magnetic solid phase extraction (MSPE) and chiral capillary electrophoresis (ch-CE). A copolymer consisting of poly(3,4-dihydroxyphenylalanine) and polyethyleneimine was coated onto magnetic particles (MPs) by co-deposition using the CuSO4/H2O2 system as a polymerization initiator. The coated MPs are spherical and the average diameter is about 168 ± 4nm. The thickness of the coating is approximately 19nm. The functional MPs are used as sorbents in MSPE to simultaneously extract the catecholamines epinephrine, norepinephrine and isoprenaline. Under the optimum conditions, the extraction efficiencies for those catecholamines are in the range from 92.3 to 98.3%, with relative standard deviations (RSDs) of <5.3%. The extraction can be performed within 4min. The extracts were then submitted to ch-CE. A method for field-enhanced sample injection (FESI) was used to enhance the detection sensitivities of the enantiomers. The limits of detection for catecholamine enantiomers range from 400 to 600pgmL-1. In comparison with the FESI-ch-CE method, the sensitivity enhancement factors of the MSPE/ch-CE method for catecholamines are about 10-fold. The method was applied to the determination of trace levels of catecholamine enantiomers in (spiked) bovine and mice blood. The recoveries ranged from 88.2 to 93.8%, with RSDs of <5.5%. The whole detection procedure takes less than 30min. Graphical abstract Schematic representation of the separation and detection of catecholamine enantiomers in blood by combination of polyDOPA/PEI-magnetic particles-based solid phase extraction and chiral-capillary electrophoresis.

Low-cost capillary electrophoresis instrumentation for assessment of rain water ionic composition

The objective of this Short Term Scientific Mission was to test the suitability of capillary electrophoresis for determination of rain water chemical composition. The fully functional, open-hardware, low-cost instrument was assembled and tested. An original protocol for determination of the chemical content of rain water was established and validated in the pilot test. Capillary electrophoresis proofed its highest suitability for the detection and quantification of ions in different samples, indicating substantial differences between clean and dirty snow waters. The hydrostatic sample injection method has been found very efficient and straightforward in manual and laboratory routines. An important deliverable of the Short Term Scientific Mission was a set of multimedia materials for the promotion of CE technique and especially low-cost CE instrumentation.

Coupling of capillary electrophoresis with electrospray ionization multiplexing ion mobility spectrometry.

In this work, ion mobility spectrometry (IMS) function as a detector and another dimension of separation was coupled with CE to achieve two-dimensional separation. To improve the performance of hyphenated CE-IMS instrument, electrospray ionization correlation ion mobility spectrometry is evaluated and compared with traditional signal averaging data acquisition method using tetraalkylammonium bromide compounds. The effect of various parameters on the separation including sample introduction, sheath fluid of CE and drift gas, data acquisition method of IMS were investigated. The experimental result shows that the optimal conditions are as follows: hydrodynamic sample injection method, the electrophoresis voltage is 10 kilo volts, 5 mmol/L ammonium acetate buffer solution containing 80% acetonitrile as both the background electrolyte and the electrospray ionization sheath fluid, the ESI liquid flow rate is 4.5 μL/min, the drift voltage is 10.5 kilo volts, the drift gas temperature is 383 K and the drift gas flow rate is 300 mL/min. Under the above conditions, the mixture standards of seven tetraalkylammoniums can be completely separated within 10 min both by CE and IMS. The linear range was 5-250 μg/mL, with LOD of 0.152, 0.204, 0.277, 0.382, 0.466, 0.623 and 0.892 μg/mL, respectively. Compared with traditional capillary electrophoresis detection methods, the developed CE-ESI-IMS method not only provide two sets of qualitative parameters including electrophoresis migration time and ion drift time, ion mobility spectrometer can also provide an additional dimension of separation and could apply to the detection ultra-violet transparent compounds or none fluorescent compounds.

Combination of field amplified sample injection and hydrophobic interaction electrokinetic chromatography (FASI-HIEKC) as a signal amplification method for the determination of selected macrocyclic antibiotics

In this study, a field amplified sample injection (FASI) and hydrophobic interaction electrokinetic chromatography (HIEKC) method has been developed for the separation of five macrolide antibiotics: spiramycin, ivermectin, tylosin, josamycin, rapamycin, and one ansamycin drug – rifamycin. By the manipulation of both the sample and separation buffer compositions, their pH values and molarity, a systematic approach has been achieved to maximize analyte differential electrophoretic mobility and signal amplification. The impact of the sample solution composition and the injection mode on the signal amplification effect of the six tested antibiotics was also investigated. Moreover, the influence of the injection of the sample and the water plug on the quantity, symmetry and height of the analyte signal was demonstrated. All the analytes were completely resolved in less than 8 min in an uncoated fused-silica capillary of 75 μm internal diameter (I.D.) x 50 cm length. The electrophoretic separations were performed in a 60% (v/v) acetonitrile and 20 mM phosphate electrolyte system (pH 7.1) with an applied voltage of 25 kV. The established method was validated and confirmed to be applicable for the determination of the active ingredients in a quality control analysis.

Evaluation of sample injection precision in respect to sensitivity in capillary electrophoresis using various injection modes.

A comparative study was conducted to assess the injection precision in capillary electrophoresis for cationic analytes (arecoline, codeine, papaverine). The precision was measured in respect to methods sensitivity in various injection modes in capillary electrophoresis: standard hydrodynamic injection (3.45kPa for 6 s), large volume sample stacking (3.45kPa for 40 s), and field-amplified sample injection (10 kV for 65 s). All measurements were conducted for aqueous solutions of standards to minimize the errors linked to the sample preparation step. The methods were submitted to precision assessment at three concentration levels: at the limit of quantification, three-fold and ten-fold of limit of quantification. The results were compared to those from high-performance liquid chromatography as a reference technique. The field-amplified sample injection method was shown to provide greatest sensitivity (quantification limits down to 4 ng/mL for all three tested compounds) but the lowest precision. High-performance liquid chromatography was established as the most reliable technique (coefficient of variation in all intraday experiments was below 1%). It was also shown that with a use of large volume sample injection technique, similar sensitivity as in high-performance liquid chromatography can be easily reached.

Fabrication of a 3D Teflon microdevice for energy free homogeneous liquid flow inside a long microchannel and its application to continuous-flow PCR

The 3D spiral PTFE microdevice was fabricated for performing continuous-flow PCR using a single heater and via semi-automated sample injection method.

Acupuncture injection for field amplified sample stacking and glass microchip-based capillary gel electrophoresis.

Acupuncture sample injection is a simple method to deliver well-defined nanoliter-scale sample plugs in PDMS microfluidic channels. This acupuncture injection method in microchip CE has several advantages, including minimization of sample consumption, the capability of serial injections of different sample solutions into the same microchannel, and the capability of injecting sample plugs into any desired position of a microchannel. Herein, we demonstrate that the simple and cost-effective acupuncture sample injection method can be used for PDMS microchip-based field amplified sample stacking in the most simplified straight channel by applying a single potential. We achieved the increase in electropherogram signals for the case of sample stacking. Furthermore, we present that microchip CGE of ΦX174 DNA-HaeⅢ digest can be performed with the acupuncture injection method on a glass microchip while minimizing sample loss and voltage control hardware.

Determination of DNA Methylation and Hydroxymethylation Levels in Biological Samples by Field-Amplified Sample Injection-Capillary Zone Electrophoresis with UV Detection

In this study, a method of field-amplified sample injection coupled with capillary zone electrophoresis with ultraviolet detection was established for evaluation of DNA methylation and hydroxymethylation levels in biological materials. By modifying an existing method, the separation of cytosine (C), 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) was performed on an uncoated capillary column (40 cm × 75 μm I.D.) using 300 mmol L−1 tris solution (pH 2.90) as running buffer and detected at 280 nm. The detection limits (S/N = 3) of the method were 0.004 ng mL−1 for cytosine (C), 0.01 ng mL−1 for 5-methylcytosine (5-mC), and 0.02 ng mL−1 for 5-hydroxymethylcytosine (5-hmC). The proposed method has been successfully applied to the evaluation of DNA methylation and hydroxymethylation levels of blood samples from 15 hepatocellular carcinoma patients and 5 liver cirrhosis patients and liver tissues from 50 pairs of tumor and matched tumor-adjacent samples.

Enhancing resolution of free-flow zone electrophoresis via a simple sheath-flow sample injection.

In this work, a simple and novel sheath-flow sample injection method (SFSIM) is introduced to reduce the band broadening of free-flow zone electrophoresis separation in newly developed self-balance free-flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C-phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free-flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath-flow sample injection method.

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis (2013-2015).

This review updates and follows-up a previous review by highlighting recent advancements regarding capillary electromigration methodologies and applications in pharmaceutical analysis. General approaches such as quality by design as well as sample injection methods and detection sensitivity are discussed. The separation and analysis of drug-related substances, chiral CE, and chiral CE-MS in addition to the determination of physicochemical constants are addressed. The advantages of applying affinity capillary electrophoresis in studying receptor-ligand interactions are highlighted. Finally, current aspects related to the analysis of biopharmaceuticals are reviewed. The present review covers the literature between January 2013 and December 2015.

Determination of chloroacetic acids in water by capillary zone electrophoresis with field-amplified sample injection

ABSTRACTA simple and sensitive method has been developed for the determination of chloroacetic acids and acetic acid in water using capillary zone electrophoresis under modified electroosmotic flow with indirect UV detection. Potassium hydrogen phthalate at pH 5.40 was used as background electrolyte (BGE), and hexadecyltrimethylammonium bromide was used as electroosmotic flow modifier. Field-amplified sample injection (FASI) method was used to enhance the sensitivity. Results showed that the limit of detection for these analytes was enhanced more than 15-fold and the repeatabilities were good with relative standard deviations (RSDs %) of migration time and corrected peak areas being below 0.33%, 4.45% (intra-day) and 0.87%, 9.67% (inter-day), respectively. An off-line liquid–liquid extraction (LLE) process with methyl tert-butyl ether was carried out to detect these compounds in water samples. The dissociation constants of acetic acid and monochloroacetic acid (MCA) were determined with two methods and the results obtained were consistent with the reference values.

Optimization of extraction parameters for quantification of fermentation volatile by-products in industrial ethanol with solid-phase microextraction and gas chromatography

The rapid growth of the biofuels industry resulted in new research needs in chemical analysis. Methods for screening and quantification of impurities resulting from changes in feedstock, process and purification are needed. Direct sample injection methods are often not sensitive for lower concentrations. This research, developed an analytical method to simultaneously quantify fermentation volatile by-products in industrial corn-based ethanol. These include acetaldehyde, ethyl vinyl ether, 1,1-diethoxyethane, isoamyl alcohol, isoamyl acetate, styrene, 2-pentylfuran, ethyl hexanoate, ethyl octanoate, and ethyl decanoate. Headspace solid-phase microextraction (SPME) coupled with gas chromatography–mass spectrometry (GC–MS) and GC-FID were used. The effects of SPME coating, concentration, time, and salting out were tested. The optimized method used Carboxen/PDMS 85 µm coating with 10% (v/v) ethanol, 20 s headspace extraction, and no salt addition. The method had values of R2 between 0.93 and >0.99 and relative standard deviations between 0.10 and 11.96%. The method detection limits were between 9.5 × 10−4 to 9.7 × 10−8 mol/L.This is one of the most comprehensive quantification methods for volatile impurities in raw ethanol to date. This new method was used to quantify 10 prevalent impurities in corn-based industrial ethanol. Copyright © 2015 The Institute of Brewing & Distilling

Capillary zone electrophoresis for bottom-up analysis of complex proteomes.

Capillary zone electrophoresis (CZE) is emerging as a useful tool in proteomic analysis. Interest arises from dramatic improvements in performance that result from improvements in the background electrolyte used for the separation, the incorporation of advanced sample injection methods, the development of robust and sensitive electrospray interfaces, and the coupling with Orbitrap mass spectrometers with high resolution and sensitivity. The combination of these technologies produces performance that is rapidly approaching the performance of UPLC-based methods for microgram samples and exceeds the performance of UPLC-based methods for mid- to low nanogram samples. These systems now produce over 10000 peptide IDs in a single 100-min analysis of the HeLa proteome.