Perspective on element speciation.

During the second half of the 20th century it became evident that trace elements play a major role whenever biological activities, environmental chemistry, or material characteristics are discussed and the determination of trace elements has therefore gained outstanding importance in environmental and life sciences. Elements, even when present at minimal concentrations in biological and environmental matrices, can, in fact, exert fundamental influence on ecosystems and the vital functions of organisms. The study of, for example, pathophysiological processes in the human body requires the determination of elements down to the ng kg(-1) range. However, organic and inorganic matrix compounds at much higher concentration levels in a sample than trace elements make the determination of trace elements often rather difficult.

MicroNIR/Chemometrics: A new analytical platform for fast and accurate detection of Δ9-Tetrahydrocannabinol (THC) in oral fluids.

BACKGROUND: Δ9-Tetrahydrocannabinol (THC) is already considered one of the most addictive substances since an increasing number of consumers/abusers of THC and THC based products are observed worldwide. In this work, the capabilities of a novel miniaturized and portable MicroNIR spectrometer were investigated in order to propose a practical and intelligible test allowing the rapid and easy screening of Δ9-Tetrahydrocannabinol (THC) oral fluids without any pretreatment. METHODS: Specimens from volunteers were collected in order to consider any sources of variability in the spectral response and spiked with increasing amount of THC in order to realize predictive models to be used in real cases. Partial Least Square-Discriminant Analysis (PLS-DA) and Partial Least Square regression (PLSr) for the simultaneously detection and quantification of THC, were applied to baseline corrected spectra pre-treated by first derivative transform. RESULTS: Results demonstrated that MicroNIR/Chemometric platform is statistically able to identify THC abuse in simulated oral fluid samples containing THC from 10 to 100 ng/ml, with a precision and a sensitivity of about 1.51% and 0.1% respectively. CONCLUSIONS: The coupling MicroNIR/Chemometrics permits to simplify THC abuse monitoring for roadside drug testing or workplace surveillance and provides the rapid interpretation of results, as once the model is assessed, it can be used to process real samples in a "click-on" device.

Performance evaluation of thermal cyclers for PCR in a rapid cycling condition.

The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling. A temperature-sensitive multiplex PCR was run in parallel to assess undesirable PCR results caused by poor temperature control. Under the given conditions (20 s of annealing time and 20 microL reaction volume), a majority of the tested instruments showed prominent curving, undershooting, and/or overshooting in their temperature profiles, which substantially influenced the results of the temperature-sensitive multiplex PCR. Variations between wells were also observed in most instruments. It is strongly hoped that these problems will be addressed by manufacturers and that they will make substantial improvements in the precision and efficiency of thermal cyclers. In the meantime, users of thermal cyclers might be able to avoid unexpected poor outcomes of sensitive PCR-based assays by designing their PCR protocols with these findings in mind.

Verifying liquid-handler performance for complex or nonaqueous reagents: a new approach.

Multichannel volume dispensing devices, such as automated liquid handlers, are widely used in drug discovery assays and other high-throughput screening processes. The performance of these systems is heavily based on the ability to deliver proper volumes of specific reagents. Discussed herein is the recent research on broadening existing methods for accurately assessing liquid-handler performance when dispensing complex or nonaqueous reagents. Accurate and reliable adjustment of liquid-handler protocols for varied reagent types could have far-reaching adoption in all scientific communities.

Extending the scope of NMR spectroscopy with microcoil probes.

Capillary NMR (CapNMR) spectroscopy has emerged as a major breakthrough for increasing the mass-sensitivity of NMR spectroscopic analysis and enabling the combination of NMR spectroscopy with other analytical techniques. Not only is the acquisition of high-sensitivity spectra getting easier but the quality of CapNMR spectra obtained in many small-molecule applications exceeds what can be accomplished with conventional designs. This Minireview discusses current CapNMR technology and its applications for the characterization of mass-limited, small-molecule and protein samples, the rapid screening of small-molecule or protein libraries, as well as hyphenated techniques that combine CapNMR with other analytical methods.

Sub part-per-million mass accuracy by using stepwise-external calibration in fourier transform ion cyclotron resonance mass spectrometry.

A new external calibration procedure for FT-ICR mass spectrometry is presented, stepwise-external calibration. This method is demonstrated for MALDI analysis of peptide mixtures, but is applicable to any ionization method. For this procedure, the masses of analyte peaks are first accurately measured at a low trapping potential (0.63 V) using external calibration. These accurately determined (< 1 ppm accuracy) analyte peaks are used as internal calibrant points for a second mass spectrum that is acquired for the same sample at a higher trapping potential (1.0 V). The second mass spectrum has a approximately 10-fold improvement in detection dynamic range compared with the first spectrum acquired at a low trapping potential. A calibration equation that accounts for local and global space charge is shown to provide mass accuracy with external calibration that is nearly identical to that of internal calibration, without the drawbacks of experimental complexity or reduction of abundance dynamic range. For the 609 mass peaks measured using stepwise-external calibration method, the root-mean-square error is 0.9 ppm. The errors appear to have a Gaussian distribution; 99.3% of the mass errors are shown to lie within three times the sample standard deviation (2.6 ppm) of their true value.

Development of a micro-fluidic manifold for copper monitoring utilising chemiluminescence detection.

The progressive development of a micro-fluidic manifold for the chemiluminescent detection of copper in water samples, based on the measurement of light emitted from the Cu(ii) catalysed oxidation of 1,10-phenanthroline by hydrogen peroxide, is reported. Micro-fluidic manifolds were designed and manufactured from polymethylmethacrylate (PMMA) using three micro-fabrication techniques, namely hot embossing, laser ablation and direct micro-milling. The final laser ablated design incorporated a reagent mixing channel of dimensions 7.3 cm in length and 250 x 250 microm in width and depth (triangular cross section), and a detection channel of 2.1 cm in length and 250 x 250 microm in width and depth (total approx. volume of between 16 to 22 microL). Optimised reagents conditions were found to be 0.07 mM 1,10-phenanthroline, containing 0.10 M cetyltrimethylammonium bromide and 0.075 M sodium hydroxide (reagent 1 delivered at 0.025 mL min(-1)) and 5% hydrogen peroxide (reagent 2 delivered at 0.025 mL min(-1)). The sample stream was mixed with reagent 1 in the mixing channel and subsequently mixed with reagent 2 at the start of the detection channel. The laser ablated manifold was found to give a linear response (R(2) = 0.998) over the concentration ranges 0-150 microg L(-1) and be reproducible (% RSD = 3.4 for five repeat injections of a 75 microg L(-1) std). Detection limits for Cu(ii) were found to be 20 microg L(-1). Selectivity was investigated using a copper selective mini-chelating column, which showed common cations found in drinking waters did not cause interference with the detection of Cu(ii). Finally the optimised system was successfully used for trace Cu(ii) determinations in a standard reference freshwater sample (SRM 1640).

Increased speed and sensitivity, and reduced sample size of a micro-radioallergosorbent test (MRAST).

Performance of the radioallergosorbent test (RAST) in small volumes (MRAST) increases the efficiency with which both IgE antibody and 125I-anti-IgE bind. The rate at which antibody binds is increased up to 290-fold so that the assay can be completed in 60 min with comparable sensitivity to RAST. The increased speed of binding is achieved by the use of small incubation volumes which does not affect the rate at which bound antibody is dissociated. The resulting change in the equilibrium in favour of antibody binding yields a 10-fold increase in sensitivity which makes it possible to use as little as 5 microliter of sample compared with 50 microliter which is normal for RAST. MRAST can be used to measure IgE antibody in heel prick blood samples taken from babies, in rat sera and in other situations where the sample is in short supply.

A standard operating procedure for assessing liquid handler performance in high-throughput screening.

The thrust of early drug discovery in recent years has been toward the configuration of homogeneous miniaturized assays. This has allowed organizations to contain costs in the face of exponential increases in the number of screening assays that need to be run to remain competitive. Miniaturization brings with it an increasing dependence on instrumentation, which over the past several years has seen the development of nanodispensing capability and sophisticated detection strategies. To maintain confidence in the data generated from miniaturized assays, it is critical to ensure that both compounds and reagents have been delivered as expected to the target wells. The authors have developed a standard operating procedure for liquid-handling quality control that has enabled them to evaluate performance on 2 levels. The first level provides for routine daily testing on existing instrumentation, and the second allows for more rigorous testing of new dispensing technologies. The procedure has shown itself to be useful in identifying both method programming and instrumentation performance shortcomings and has provided a means to harmonizing instrumentation usage by assay development and screening groups. The goal is that this type of procedure be used for facilitating the exchange of liquid handler performance data across the industry.

Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialysed protein solutions.

The quantization of small amounts of chemical denaturants as urea or guanidine hydrochloride in protein solutions after dialysis is a difficult task in the molecular biology laboratory practice. Refractometric methods are useful to quantify a denaturant in the molar range but this methodology is not helpful when the denaturant is present in small amounts. The method herein described is a new comparative method that requires, a priori, the quantification of the stock solutions of urea (8 M) and guanidine hydrochloride (6 M) by refractometry to prepare by sequential dilution the standards used for comparison in the spectropolarimeter. The method is based on the observation that the wavelengths, at which the absorbance of polarized light increases in the far-UV region, as observed by spectropolarimetry, is related to the concentration of the chemical denaturant present in the protein solution. In the quantitation method herein reported, the urea and guanidine hydrochloride detection limits range from 1.2 x 10(-4) to 6 x 10(-6) M depending on the protein dialysis buffer used for a standard cell path length of 1 cm. The sensibility of this method results to be comprised in a range 4-5 orders of magnitude higher than that measured by refractometry. The determinations in both the sample and the control preparations are virtually completed within approximately 10 min.

[Evaluation of pipetting systems. III. Micropipette precision in a routine task]. / Evaluación de sistemas de pipeteo. III. La precisión de una micropipeta en el trabajo rutinario.

OBJECTIVE: To establish a norm of the precision achievable with a micropipette in an IRMA assay under routine conditions. MATERIAL AND METHODS: A micropipette (Gilson) adjusted to dispense 100 microL was used by a single analyst with experience in its use. In each assay, ten aliquots of radioactive antiprolactin were pipetted in clean tubes (PRE-batch tubes), followed by pipetting of the tubes being processed in the assay, and at the end, a second pipetting of 10 aliquots in clean tubes (POST-batch tubes). The study includes the data of 15 consecutive batches during a seven month period with an overall mean of 283 tubes per batch. The PRE- and POST-tubes were read in a gamma counter (Crystal plus). The mean, SD and CV for PRE, POST and global (PRE+POST) tubes were calculated for each batch. RESULTS: The global CV of the 15 batches ranged from 1.6 to 6.9%, mean of 3.1%. We found no evidence of increased imprecision due to fatigue of the analyst, but surprisingly, we observed that in nine of the 15 batches there was a significant difference in the means of the PRE-tubes vs the POST-tubes (t test) without differences in precision. Thus, part of the global variability is due to what we have called pseudoimprecision (i.e. an increase in CV due to differences in means). In addition, the POST-tubes had higher values in the first 7 batches but the opposite occurred in the last 8 batches (table 2). This shift in the sign of the PRE-POST differences suggests the presence of opposite factors operating in time, i.e. one or more factors increased the volume of pipetting after using the pipette more than 150 times (batches 1-7) whereas other/others decreased it (batches 8-15). CONCLUSIONS: 1. Our first approximation to a norm of micropipetting precision in batches of 200-300 tubes was a CV of 3.1%. 2. This norm was influenced by a problem of pseudoimprecision detected ex-post-facto. 3. Our findings justify continuation studies to detect the pseudoimprecision and evaluate its causes prospectively.

Development and evaluation of a fluorescence microplate assay for quantification of heparins and other sulfated carbohydrates.

Due to their complex composition, quantification of heparins is difficult. On the one hand there are many biological tests, which only indirectly detect effects of the antithrombin-binding material. On the other hand direct quantitative methods are available but they are often insensitive, challenging, time-consuming or expensive. The aim of this study was to develop a sensitive, rapid, simple as well as inexpensive direct quantification assay suitable for routine analysis. Based on Polymer-H, a novel heparin complexing, fluorescent labeled synthetic polymer (lambda((ex)) 320nm, lambda((em)) 510nm), a microplate assay was developed and optimized. The specificity of the assay was evaluated by structure-assay response relationships studies using structurally defined glucan sulfates, heparins, and other natural and synthetic sulfated carbohydrates. The fluorescence intensity of Polymer-H (7.5microg/ml) showed to be concentration-dependently amplified by heparins as well as by other sulfated carbohydrates. The best sensitivity, accuracy and linearity were observed in a range from 0.63 to 5.0microg/ml heparins. No differences in the fluorescence between various heparins were observed, so that only one calibration curve is needed. In addition, all types of carbohydrates with a degree of sulfation (DS)> approximately 1.2 and a M(r)>3000 can be quantified as well. By own calibration curves also other sulfated carbohydrates like fondaparinux or other glycosaminoglycans (DS>0.4) can be determined.

Principle, calibration, and application of the in situ alkali chloride monitor.

The extended use of biomass for heat and power production has caused increased operational problems with fouling and high-temperature corrosion in boilers. These problems are mainly related to the presence of alkali chlorides (KCl and NaCl) at high concentrations in the flue gas. The in situ alkali chloride monitor (IACM) was developed by Vattenfall Research and Development AB for measuring the alkali chloride concentration in hot flue gases (less than or approximately 650 degrees C). The measurement technique is based on molecular differential absorption spectroscopy in the UV range. Simultaneous measurement of SO(2) concentration is also possible. The measuring range is 1-50 ppm for the sum of KCl and NaCl concentrations and 4-750 ppm for SO(2). This paper describes the principle of the IACM as well as its calibration. Furthermore, an example of its application in an industrial boiler is given.

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing.

Mass spectrometry has become an invaluable tool for the identification of prohibited peptide hormones and proteins in doping control analysis. Regulatory authorities have established criteria for identifying banned drugs in doping control specimens, but these criteria do not address the specific issues for high molecular weight protein drugs such as molecular weight determination of multiply charged molecules, analysis of chemically or enzymatically derived degradation products, identification of amino acid sequence tags, etc. Technical considerations such as sample preparation methods (e.g. immunoaffinity purification), resulting analytes (e.g. intact compounds vs. chemically or enzymatically derived peptides), ionization modes, analyzer resolution, and the information provided by respective techniques are discussed in light of sports drug testing requirements using typical application examples.

Regular arrays of microdisc electrodes: simulation quantifies the fraction of 'dead' electrodes.

Arrays of microdisc electrodes have found widespread use in electroanalysis. These are commonly produced lithographically and practical arrays may contain up to hundreds of individual disc electrodes (e.g. of gold, platinum, indium,...) to maximise sensitivity and minimise limits of detection. Typically, however, the lithographic fabrication process is imperfect resulting in a significant fraction (often tens of percent) of electrochemically inactive electrodes. We demonstrate that a 2-dimensional simulation based on the diffusion domain approximation in conjugation with simple experiments on the ferrocyanide redox couple in aqueous solutions can be used to rigorously 'count' the number of active electrodes in a non-destructive fashion. The agreement with an independent count in which active electrodes are identified via electro-plating with copper followed by ex situ microscopic examination is quantitatively excellent.

Quantification of gliadin levels to the picogram level by flow cytometry.

BACKGROUND: Celiac disease is a widely prevalent enteropathy caused by intolerance to gliadin, one of the gluten proteins. We developed two methods for the analysis of gliadin levels. Both methods use flow cytometry and rat antibodies against a 16-residue peptide of gliadin. The peptide is common to the alpha-, beta-, gamma-, and omega-gliadins. METHODS: In the one-site assay, the antigen (gliadin standard or food extract) was adsorbed on 3-mum latex particles. Sensitized particles were then incubated, in this order, with rat anti-gliadin peptide antibodies and anti-rat immunoglobulin G antibodies labeled with fluorescein isothiocyanate. In the two-site assay, the antigen was trapped on the latex particles by rat anti-gliadin antibodies and then measured by the same antibodies labeled with fluorescein. RESULTS: Detection limits were 1 ng/ml for the one-site assay and 10 pg/ml for the two-site assay. The two-site assay displayed gliadin at concentrations above the limit proposed by the Codex Alimentarius in 2 of 40 gluten-free products. CONCLUSION: There is a growing concern that gliadin, even when present in gluten-free foods within the limit fixed by the Codex Alimentarius, over the long term may become toxic to patients with celiac disease. The techniques described in this study provide an opportunity to further decrease the acceptable limit of gliadin in gluten-free foods.

Improved alanine aminotransferase assay using the microtiter plate.

New procedures and new devices have been developed to improve the alanine aminotransferase (ALT) assay by using microtiter plates with 96 wells. Sera for ALT determination and reagents are distributed with a computer-controlled multiple syringe for simultaneous filling of the 96 wells. The volumes simultaneously distributed by this new multiple syringe show very little variation. The coefficient of variation (CV) is 0.6 percent (n = 96). Repeated filling of the wells with this syringe results in good reproducibility of distributed volumes (CV = 0.5%, n = 10). The temperature of sera, reagents, and mixtures, even during photometry, is actively regulated (down or up) to 25 degrees C in special chambers. Thus, the temperature varies minimally +/- 0.05 degrees C. ALT values in the low normal range as well as the high pathologic range show little variation (CV less than 2.3%) in aliquots of the same sample simultaneously or subsequently examined by the new procedure and with new devices. There is good correlation between the improved method using microtiter plates and the conventional methods (r greater than 0.9900, ALT values from 1-154 U/L). False-low and false-high values are excluded by computerized evaluation of many single determinations during the kinetic reactions in each sample. The new method is precise as well as simple, saves time, and may become important for the determination of other serum constituents on the basis of kinetics.

Bedside diagnostic blood testing. Its accuracy, rapidity, and utility in blood conservation.

OBJECTIVE: --Bedside diagnostic testing utilizing microchemistry instruments potentially offers physicians the opportunity to evaluate urgent blood test results rapidly and reliably using only drops (250 microL) of whole blood. The use of microchemistry technology may also represent an essential component of a blood conservation program in acutely ill patients. We tested the hypothesis that a microchemistry instrument would have important advantages in the intensive care unit, including rapid turnaround time, decreased iatrogenic blood loss, and the provision of accurate analyte results. DESIGN: --One-year prospective, random-sample comparative study. SETTING: --A tertiary, acute care, 1000-bed university teaching hospital and a 450-bed university-affiliated community hospital. PATIENTS: --Blood samples from 850 patients were analyzed. RESULTS: --There were strong correlations (for pH, R2 = .97; PCO2, R2 = .97; PO2, R2 = .99; sodium, R2 = .83; potassium, R2 = .94; chloride, R2 = .90; glucose, R2 = .98; and hematocrit, R2 = .92) when paired samples were analyzed simultaneously in the laboratory comparing the microchemistry instrument and currently accepted clinical laboratory instruments. There were significant correlations (for pH, R2 = .91; PCO2, R2 = .94; PO2, R2 = .97; sodium, R2 = .91; potassium, R2 = .86; chloride, R2 = .91; ionized calcium, R2 = .82; glucose, R2 = .96; and hematocrit, R2 = .75) but increased variability occurred in some analyte results when samples were first analyzed in the intensive care unit (using the microchemistry instrument) and then transported to the laboratory in the routine fashion for "stat" determinations. CONCLUSIONS: --The multichannel microchemistry instrument provided accurate analyte determinations when compared with accepted clinical laboratory instruments. Significant savings of time and blood were also realized by the use of a microchemistry instrument in the intensive care unit. Bedside microchemistry may provide clinically important advantages in emergency departments, operating rooms, and neonatal, pediatric, coronary, adult medical and surgical, trauma, and burn intensive care units.