Area Relative Standard Deviation Research Articles

Liquid-phase microextraction of aromatic amines: hollow fiber–liquid-phase microextraction and parallel artificial liquid membrane extraction comparison

Aromatic amines (AA) are carcinogenic compounds that can enter the human body through many sources, one of the most important being tobacco smoke. They are excreted with the urine, from which they can be extracted and measured. To that end, hollow fiber-liquid-phase microextraction (HF-LPME) and parallel artificial liquid membrane extraction (PALME) were optimized for the analysis of representative aromatic amines, as alternatives to liquid–liquid extraction (LLE). Relevant extraction parameters, namely organic solvent, extraction time, agitation speed, and acceptor solution pH, were studied, and the two optimized techniques—HF-LPME: dihexyl ether, 45 min, 250 rpm, and pH 1; PALME: undecane, 20 min, 250 rpm and pH 1—were compared. Comparison of the optimized methods showed that significantly higher recoveries could be obtained with PALME than with HF-LPME. Therefore, PALME was further validated. The results were successful for nine different AA, with regression coefficients (R2) of at least 0.991, limits of detection (LOD) of 45–75 ng/L, and repeatability and peak area relative standard deviations (RSD) below 20%. Furthermore, two urine samples from smokers were measured as proof of concept, and 2-methylaniline was successfully quantified in one of them. These results show that PALME is a great green alternative to LLE. Not only does it use much smaller volumes of toxic organic solvents, and sample—enabling the study of samples with limited available volumes—but it is also less time consuming and labor intensive, and it can be automated.Graphical

Optimizing test approaches for the detection of exposed metal surfaces within a chromatographic flow path

It is critical to the success of any chromatography-based assay that the performance of the LC instrument be checked for its readiness and ability to perform the intended analysis. This includes gaging the suitability of a system to fulfill the purpose of different types of methods. One type of analysis that requires special consideration is the analysis of compounds which are prone to a particular form of non-specific binding, namely metal adsorption, where analytes interact and potentially adsorb to metal contained within the chromatographic flow path. For an analysis of compounds which are susceptible to metal adsorption, ideally a system suitability test would be performed to ensure there will not be any sample loss or detrimental peak shape effects resulting from potential analyte-to-metal interactions. To help chromatographers assess system inertness concerns like this, we have developed a method of testing LC systems for metal interactions using adenosine 5′-(α,β-methylene)diphosphate (AMPcP). This nucleotide analog has been confirmed to have a propensity to adsorb to titanium and stainless-steel frits and is resistant to hydrolysis and stable to long-term storage and repeat use (as is befitting of any reagent proposed for system suitability testing). AMPcP has been used in a flow injection test (no column in-line) to monitor for losses in recovery and peak shape perturbations that can potentially be present in any chromatography system manufactured with one or more metal based components. In this approach, sequential injections of AMPcP were made without a column and various peak attributes were monitored and ultimately correlated to the amount of metal surface area in the flow path. The ability of this method to discriminate between inert chromatographic surfaces versus exposed metal surfaces was verified by comparing peak areas, peak shapes, and injection repeatability for AMPcP using a UHPLC equipped with MP35N metal alloy components versus an equivalent UHPLC equipped with an ethylene bridged hybrid organic-inorganic surface (or so-called hybrid surface technology). Injections of caffeine were also explored to establish a negative control for this system suitability measurement. Caffeine does not interact with metal surfaces and can therefore give an instrument specific representation of peak shape and dispersion as well as an indication of overall mechanical system performance. Additionally, replicate injections of AMPcP and caffeine onto a UHPLC partially configured with hybrid surface technology (HST) readily identified exposed metal surfaces through an increased peak area relative standard deviation as well as a reduction in absolute recovery. Finally, a novel visualization tool was developed to provide an alternative method of determining system inertness without having to perform chromatographic calculations but instead a graphical peak shape comparison between a negative control, caffeine, and the metal sensitive AMPcP test probe.

Assessing accuracy, precision and selectivity using quality controls for non-targeted analysis

The benchmarks to assess reproducibility are not well defined for non-target analysis. Parameters to evaluate analytical performance, such as accuracy, precision and selectivity, are well defined for target analysis, but remain elusive for non-target screening analysis. In this study, quality control (QC) guidelines are proposed to assure reliable data in non-target screening methodologies using a simple set of standards. Workflow reproducibility was assessed using an in-house QC mixture containing selected compounds with a wide range of polarity that can be detected either by electrospray ionization (ESI) in positive or negative mode. The analysis was performed by online solid phase extraction (SPE) liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Data processing was done by a commercially available software, Compound Discoverer v. 3.0 using an environmental working template, which searched a multitude of databases, including Chemspider, EPA Toxcast, MzCloud among others. We have specifically evaluated method specificity, precision, accuracy and reproducibility in terms of peak area and retention time variability, true positive identification rate, intraday (within days) and interday (consecutive days) variations and the use of QC samples to reduce false positives. The method showed a satisfactory accuracy with an identification rate of ≥70% for most of the QC compounds. Precision estimated based on peak area relative standard deviation (RSD) ranged between 30 and 50% for most of the compounds. Data normalization to a single internal standard did not improve peak area variability. Retention time precision showed great repeatability and reproducibility (RSD ≤ 5%). In addition, a simple model of RT vs log Kow was designed based on our QC mixtures to efficiently reduced false positives by an average of 49.1%.

Miniature and fully portable gradient capillary liquid chromatograph

A robust, portable and miniature battery powered gradient capillary liquid chromatograph (total weight ∼2.7 kg, without battery ∼2.0 kg), with integrated microfluidic injection, column heating and high sensitivity low-UV absorbance detection is presented. The portable capillary chromatograph, was applied with a packed reversed-phase capillary column (100 mm × 300 μm I.D., 5 μm ODS), housed within an integrated capillary column heater controlled by a proportional-integral-derivative (PID) chip module. The system delivered retention time and peak area relative standard deviation in isocratic mode of <0.7% (n = 10) and <3.3% (n = 10), respectively, and <0.1% (n = 10) and <2.3% (n = 10) respectively, for gradient elution mode. Detection was based upon a 255 nm light-emitting diode (LED) using one of two commercial capillary flow-cell options, namely a high sensitivity 12 nL Agilent capillary z-cell (HSDC) and a 45 nL Thermo Fisher Scientific UZ-View™ flow cell (UZFC). The HSDC, housed within a 3D printed detector arrangement, gave an effective pathlength of 1.01 mm (84% of nominal pathlength) and stray light of only 0.2%. Limits of detection for four test small molecule pharmaceuticals ranged from 65 to 101 μg L−1 based upon a 316 nL injection volume, with separation efficiencies of between 18,000 and 29,700 N m−1, with sub-4 min run times. The portable capillary LC system was successfully coupled to a small footprint portable mass spectrometer (Microsaic 4500 MiD) to demonstrate compatibility and ‘point-of-need’ miniaturised LC-MS capability.

Nuclear Magnetic Resonance Spectroscopy to Identify Metabolite Biomarkers of Nonresponsiveness to Targeted Therapy in Glioblastoma Tumor Stem Cells.

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15-20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.

Tantala-based sol-gel coating for capillary microextraction on-line coupled to high-performance liquid chromatography

A sol-gel organic-inorganic hybrid sorbent, consisting of chemically integrated tantalum (V) ethoxide (TaEO) and polypropylene glycol methacrylate (PPGM), was developed for capillary microextraction (CME). The sol-gel sorbent was synthesized within a fused silica capillary through hydrolytic polycondensation of TaEO and chemical incorporation of PPGM into the evolving sol-gel tantala network. A part of the organic-inorganic hybrid sol-gel network evolving in the vicinity of the capillary walls had favorable conditions to get chemically bonded to the silanol groups on the capillary surface forming a surface-bonded coating. The newly developed sol-gel sorbent was employed to isolate and enrich a variety of analytes from aqueous samples for on-line analysis by high-performance liquid chromatography (HPLC) equipped with a UV detector. CME was performed on aqueous samples containing trace concentrations of analytes representing polycyclic aromatic hydrocarbons, ketones, alcohols, amines, nucleosides, and nucleotides. This sol-gel hybrid coating provided efficient extraction with CME-HPLC detection limits ranging from 4.41pM to 28.19 pM. Due to direct chemical bonding between the sol-gel sorbent coating and the fused silica capillary inner surface, this sol-gel sorbent exhibited enhanced solvent stability. The sol-gel tantala-based sorbent also exhibited excellent pH stability over a wide pH range (pH 0–pH 14). Furthermore, it displayed great performance reproducibility in CME-HPLC providing run-to-run HPLC peak area relative standard deviation (RSD) values between 0.23% and 3.83%. The capillary-to-capillary RSD (n=3), characterizing capillary preparation method reproducibility, ranged from 0.24% to 4.11%. The results show great performance consistency and application potential for the sol-gel tantala-PPGM sorbent in various fields including biomedical, pharmaceutical, and environmental areas.

Western Blotting Using Microchip Electrophoresis Interfaced to a Protein Capture Membrane

Western blotting is a commonly used assay for proteins. Despite the utility of the method, it is also characterized by long analysis times, manual operation, and lack of established miniaturized counterpart. We report a new way to Western blot that addresses these limitations. In the method, sodium dodecyl sulfate (SDS)-protein complexes are separated by sieving electrophoresis in a microfluidic device or chip. The chip is interfaced to a moving membrane so that proteins are captured in discrete zones as they migrate from the chip. Separations of SDS-protein complexes in the molecular weight range of 11-155 kDa were completed in 2 min with 4 × 10(4) theoretical plates at 460 V/cm. Migration time and peak area relative standard deviations were 3-6% and 0.2%, respectively. Detection limit for actin was 0.7 nM. Assays for actin, AMP-kinase, carbonic anhydrase, and lysozyme are shown to demonstrate versatility of the method. Total analysis time including immunoassay was 22-32 min for a single sample. Because processing membrane for immunoassay is the slow step of the assay, sequential injections from different reservoirs on the chip and capture in different tracks on the same membrane allow increased throughput. As a demonstration, 9 injections were collected on one membrane and analyzed in 43 min (~5 min/sample). Further improvements in throughput are possible with more reservoirs or parallel channels.

Universal Anion Detection by Replacement-Ion Chromatography with an Atmospheric-Pressure Solution-Cathode Glow Discharge Photometric Detector

A new method of detection for ion chromatography (IC) is described that couples replacement-ion chromatography (RIC) with an atmospheric-pressure solution-cathode glow discharge (SCGD). In the new instrument, a conventional suppressed IC arrangement is followed by a "replacement column" that consists of a cation-exchange micromembrane suppressor continuously regenerated with Li(2)SO(4). In this arrangement analytes are stoichiometrically converted to Li salts when they pass through the replacement column and are introduced into the SCGD, where they are detected indirectly by atomic emission spectrometry. Though found to be unsuitable for cation determinations, the SCGD-RIC instrument shows good repeatability (<3.5% peak area relative standard deviation), approximately 4 orders of linear dynamic range, universal calibration (similar molar sensitivity for anions of the same charge), and detection limits between 0.08 and 0.64 μg·mL(-1) (25-μL injection) for several mono- and divalent anions. Deviations from the universal calibration were also observed and are critically evaluated. Optimal operating conditions are described, analytical figures of merit are presented, and background sources present in the system are characterized and explained.

Generation of Nonbiased Hydrodynamic Injections on Microfluidic Devices Using Integrated Dielectric Elastomer Actuators

Sample introduction is a crucial, yet often overlooked step in chemical analysis. Its importance is clearly portrayed in the case of electrokinetic injection for electrophoretic separations, where sampling bias favors the introduction of the fastest moving analytes in a mixture. To this end, a poly(dimethylsiloxane) (PDMS)-based microfluidic device that incorporates miniaturized and fully integrated dielectric elastomer actuators (IDEAs) in order to perform sample injection for electrophoresis is reported. These electromechanical actuators produce hydrodynamic fluid pulses within the channel network without the need for any modifications to the channel design and without the use of large, off-chip equipment. Separations of Fluorescein thiocarbamyl-labeled amino acids reveal that IDEA-derived injections have a more stable chemical composition than electrokinetic injections, with peak area relative standard deviations (RSDs) less than 1.1% over 30 injections at six different volumes. Moreover, the efficiency and resolution of separations with IDEA-derived injections are not significantly different from electrokinetic injections under similar separation conditions. The reproducibility of peak heights and peak areas over the course of 64 consecutive injections reveal that the actuation mechanism is very stable with peak area RSDs less than 1.8%. These results, coupled with facile fabrication and operation of IDEA devices, suggest that widespread adaptation of this technology could be very advantageous for many types of miniaturized analysis systems.

A highly resolved anion-exchange chromatographic method for determination of saccharidic tracers for biomass combustion and primary bio-particles in atmospheric aerosol

An improved high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) method is developed and validated for simultaneous determination of atmospherically relevant sugar alcohols, monosaccharides, and monosaccharide anhydrides. The improved method enables the separation of levoglucosan and arabitol which were not or insufficiently separated by the previous HPAEC–PAD methods. Reproducibility of the method was tested for both standard solutions and atmospheric aerosol samples. The peak area relative standard deviation (RSD%) of standard solutions were found to be lower than 1.5% for consecutive analyses ( n = 3) and lower than 4% for day to day variation ( n = 9). The peak area RSD% of atmospheric samples with typical European wintertime monosaccharide concentrations ( n = 9) was found to be similar to that of standard solutions. Limits of detection ranged from 0.002 mg L −1 for inositol to 0.08 mg L −1 for fructose. The developed method offers a simple, reliable and cost effective determination of atmospheric tracers for biomass combustion and for selected bio-aerosol components at sub-nanogram per cubic-meter-air concentration levels for routine analysis.

Microfluidic chip made of COP (cyclo-olefin polymer) and comparion to PMMA (polymethylmethacrylate) microfluidic chip

Hot embossing method was studied for the fabrication of microfluidic microchannels on COP (cyclo-olefin polymer) chip. The ending temperature of heating stage, i.e. embossing reference temperature ( T r), was determined by the viscoelastic property of the COP, according to variable temperature quasi-creep experiments. Taguchi method was used to decide the processing parameters in cooling and demolding stage. The optimized parameters of hot embossing are: embossing reference temperature 143 °C, temperature and pressure holding time 2 min, pressure at cooling and demolding stage 1.6 MPa, the demolding temperature in the lower heating plate 80 °C and temperature difference between the upper and lower heating plate 10 °C. Experiment shows that microchannel fabricated with the above parameters has high repeatability and low substrate deformation, average repeatability is 97.6% in width and 94.3% in depth. Experiments on background fluorescence, electrophoresis, and DNA separation and detection were carried out on COP and PMMA (polymethylmethacrylate) chips, respectively. Compared to the PMMA chip, the COP chip has higher signal to noise ratio, higher electrophoresis efficiency, and lower peak area relative standard deviation (R.S.D.). In separation 6 μg/ml ϕ X174 RF DNA Hinc II, 11 DNA fragments have been identified in less than 2 min on the COP chip, thus it has large application potential in biochemical analysis.

The potential of inductively coupled plasma‐mass spectrometric detection for capillary electrophoretic analysis of pesticides

In this work, the potential of inductively coupled plasma-mass spectrometry (ICP-MS) coupled to capillary electrophoresis (CE) to determine organophosphorus pesticides (OPPs) is demonstrated. Element specific detection of (31)P with ICP-MS is performed for the detection of OPPs. Three common OPPs, including glyphosate, glufosinate, and aminomethylphosphonic acid (AMPA), were analyzed by CE-ICP-MS to demonstrate its applicability for the analysis of OPPs. The advantages of using ICP-MS with respect to other common detectors, such as flame photometric detection (FPD), for CE analysis of OPPs are shown. Additionally, different CE separation conditions were studied to achieve complete baseline separation of the pesticide compounds in short migration times. Two CE buffer systems were evaluated for the separation of OPPs using ICP-MS detection. A buffer solution containing 40 mmol.L(-1) ammonium acetate at pH 9.0 and an applied voltage of +20 kV were finally selected leading to a separation time of 10.0 min. Both migration time and area relative standard deviations (%RSD) were evaluated and their respective values were in the intervals of 1.1-3.3% and 2.7-5.3%. Detection limits obtained with the CE-ICP-MS system were in the range of 0.11-0.19 mg.L(-1) (as compound) yielding an enhancement of 130- to 230-fold with respect to FPD. The proposed methodology was finally applied for the determination of the OPPs mentioned above in natural river water samples.

Microchip-based capillary electrochromatography using packed beds.

Integration of a packed column onto a microchip for performance of capillary electrochromatography (CEC) is described. The quartz device incorporated a cross-injector, and a double weir trapping design for formation of 1, 2 and 5 mm long CEC columns. Three fluorescent dyes were baseline-resolved with plate numbers of 330,(330,000 plates/m; height equivalent to a theoretical plate, H = 3.0 microm) for BODIPY 493/503, 360 (360,000 plates/m; H = 2.8 microm) for rhodamine 123 and 244 (244,000 plates/m; H = 4.1 microm) for acridine orange (AO) with 500 V applied on a 1 mm long column. The 2 mm column yielded approximately 1.8 times more theoretical plates than did the 1 mm column, when operated at the same flow rate. Van Deemter plots were obtained for the three column lengths, showing increased plate height for the 5 mm length. A 2 mm column gave peak height and area relative standard deviation (RSD) values of 2.5 and 3.3%, respectively, as averages for the three dyes (n = 15). The RSD for the dye retention times was 1% (n = 6) over one day, and 3% (n = 30) over five days. Indirect fluorescence detection of thiourea and of amino acids was possible using a neutral indicator dye (BODIPY 493/503), with a detection limit of 10 microM for amino acids.

Dynamic capillary coatings with zwitterionic surfactants for capillary isoelectric focusing.

The use of surfactants as additives was demonstrated for the first time in capillary isoelectric focusing (CIEF) to dynamically modify the surfaces of bare fused silica capillaries. These surfactants were zwitterionic sulfobetaines: dodecyldimethyl (3-sulfopropyl) ammonium hydroxide (C12N3SO3), hexadecyldimethyl (3-sulfopropyl) ammonium hydroxide (C16N3SO3) and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U). They were added directly to the protein-ampholyte mixture, and remained in the capillary during isoelectric focusing and mobilization. The C16N3SO3 and CAS U coatings were shown effective in CEF. Separation of seven IEF protein standards was obtained, with significantly improved resolution compared to that from an uncoated silica capillary. The effect of these surfactants on the electroosmotic flow (EOF) in CIEF was determined. CAS U was effective in suppressing the EOF at neutral and alkaline pH conditions, C16N3SO3 was effective in suppressing EOF at acidic and neutral pH conditions. C12N3SO3 however had little effect on the EOF. The pH gradients formed inside these surfactant coated capillaries were recta-linear at pH 6 to 9 (R2 approximately equal to 0.99). Reproducibility of migration time and peak area was determined. For all three coatings, the migration time standard deviations were less than 1.6 min, and the relative standard deviations of area were below 10%. The protein recovery in the CAS U-modified capillary was quantitative or near-quantitative for five of the seven proteins studied.

Optimization of a capillary electrophoresis–electrospray mass spectrometry method for the quantitation of the 20 natural amino acids in childrens blood

This paper describes useful information on the capillary electrophoresis–electrospray ionization mass spectroscopy (CE–ESIMS) interfacing for the analysis of amino acids (AAs) in standard mixtures and in child plasma blood serum. The developed procedure allows quantitation of the 20 natural AAs, in a single run, without any derivatization. Limits of detection as low as 3–20 μmol/l (5–30 pg injected) per analyte were obtained with an efficiency of about 100 000 plates and a peak area relative standard deviation below 4%.

Separation of porphyrins by capillary electrophoresis in fused-silica and ethylene vinyl acetate copolymer capillaries with visible absorbance detection.

A mixture of 5 porphyrins were separated by capillary electrophoresis. A UV-VIS detector was used to detect the separated components. Separation was performed in 2 types of capillaries, i.e. fused-silica and ethylene vinyl acetate copolymer (EVA) plastic capillaries. The concentration limit of detection (CLOD), mass limit of detection (MLOD), and relative standard deviations (RSD) of migration time and area were investigated. LOD was comparable to that of epi-fluorescence detection. LOD was lower when separation was performed in EVA capillary compared to fused-silica capillary. RSD of migration times of the porphyrins when separated in fused-silica capillary ranges between 0.5 to 1.6% and in EVA capillary ranges between 0.3 to 1.2%. Area RSD in fused-silica capillary ranges between 7 to 20% and in EVA capillary ranges between 4.7 to 12.3%. Urine spiked with porphyrins was also analysed by CE using fused-silica and EVA capillaries. Analysis of urine sample spiked with porphyrins showed that stacking effect of porphyrins was observable only in fused-silica and not in EVA capillary. This led to similar LOD in fused-silica capillary to those for EVA capillary.

Comparison of ninhydrin and o-phthalaldehyde post-column detection techniques for high-performance liquid chromatography of free amino acids

A comparative study of two post-column derivatization techniques for amino acid determination has been completed. Using conventional high-performance liquid chromatography, amino acids were separated on either sodium- or lithium-form, polystyrene-based, strong cation exchangers, then derivatized with either o-phthalaldehyde or ninhydrin. Amino acid detection limits with o-phthalaldehyde are near 5 pmoles for primary amines and 100 pmoles for secondary amines such as proline. Detection limits with ninhydrin are near 100 pmoles. The o-phthalaldehyde system should be the system chosen whenever detection of secondary amino acids is unimportant. Retention time reproducibility averaged 1–4% relative standard deviation for both systems with a median peak area relative standard deviation of 3%. A substantial benefit of amino acid determination by high-performance liquid chromatography is the ease with which one can apply this type of chromatography to solve other separation problems.