A simple voltammetric electronic tongue for the analysis of coffee adulterations.

This work presents a simple and low-cost analytical approach to detect adulterations in ground roasted coffee by using voltammetry and chemometrics. The voltammogram of a coffee extract (prepared as simulating a home-made coffee cup) obtained with a single working electrode is submitted to pattern recognition analysis preceded by variable selection to detect the addition of coffee husks and sticks (adulterated/unadulterated), or evaluate the shelf-life condition (expired/unexpired). Two pattern recognition methods were tested: linear discriminant analysis (LDA) with variable selection by successive projections algorithm (SPA), or genetic algorithm (GA); and partial least squares discriminant analysis (PLS-DA). Both LDA models presented satisfactory results. The voltammograms were also evaluated for the quantitative determination of the percentage of impurities in ground roasted coffees. PLS and multivariate linear regression (MLR) preceded by variable selection with SPA or GA were evaluated. An excellent predictive power (RMSEP = 0.05%) was obtained with MLR aided by GA.

Nitric oxide selective electrodes.

Since nitric oxide (NO) was identified as the endothelial-derived relaxing factor in the late 1980s, many approaches have attempted to provide an adequate means for measuring physiological levels of NO. Although several techniques have been successful in achieving this aim, the electrochemical method has proved the only technique that can reliably measure physiological levels of NO in vitro, in vivo, and in real time. We describe here the development of electrochemical sensors for NO, including the fabrication of sensors, the detection principle, calibration, detection limits, selectivity, and response time. Furthermore, we look at the many experimental applications where NO selective electrodes have been successfully used.

Molecular electronegativity distance vector model for the prediction of bioconcentration factors in fish.

Molecular electronegativity distance vector (MEDV) derived directly from the molecular topological structures was used to describe the structures of 122 nonionic organic compounds (NOCs) and a quantitative relationship between the MEDV descriptors and the bioconcentration factors (BCF) of NOCs in fish was developed using the variable selection and modeling based on prediction (VSMP). It was found that some main structural factors influencing the BCFs of NOCs are the substructures expressed by four atomic types of nos. 2, 3, 5, and 13, i.e., atom groups -CH(2)- or =CH-, -CH< or =C<, -NH(2), and -Cl or -Br where the former two groups exist in the molecular skeleton of NOC and the latter three groups are related closely to the substituting groups on a benzene ring. The best 5-variable model, with the correlation coefficient (r(2)) of 0.9500 and the leave-one-out cross-validation correlation coefficient (q(2)) of 0.9428, was built by multiple linear regressions, which shows a good estimation ability and stability. A predictive power for the external samples was tested by the model from the training set of 80 NOCs and the predictive correlation coefficient (u(2)) for the 42 external samples in the test set was 0.9028.

Detection of carbon monoxide production as a result of the interaction of five volatile anesthetics and desiccated sodalime with an electrochemical carbon monoxide sensor in an anesthetic circuit compared to gas chromatography.

OBJECTIVES: There is a continuing risk of production of toxic levels of carbon monoxide (CO) as a result of interaction of volatile anesthetics and desiccated strong base carbon dioxide absorbents like soda lime. The aim of this study is to establish the reliability of detection of CO levels by an electrochemical carbon monoxide sensor compared to gas chromatography. METHODS: Completely desiccated sodalime was conducted through a circle anesthesia system connected to an artificial lung. For different rates of CO production, a low flow anesthesia with a oxygen/nitrous oxide mixture was maintained using five volatile anesthetics. For quantification of CO production, a portable gas chromatograph (GC) was connected to this setup, as well as a Bedfont EC40 electrochemical carbon monoxide sensor (ES) with a claimed reliable sensitivity of 0-200 parts per million (ppm) and a maximum detection range of more than 5500 ppm. To assess the agreement between the GC and ES measurements the intra class correlation coefficient (ICC) and the 95% limits of agreement were calculated. Bland and Altman scatterplots were made to visualize the difference between measurements. RESULTS: For concentrations up to 200 ppm, no significant differences between the GC and ES mean CO measurements were found in the halothane experiments. However CO was not accurately measured at every moment during these experiments by the ES. For concentrations above 200 ppm the results of the two instruments differed significantly. The ES malfunctioned when exposed to sevoflurane and desiccated sodalime. CONCLUSIONS: From these data we conclude that the ES can only be used as an indicator of CO production. When this sensor is used with sevoflurane and desiccated sodalime it is not capable of normal operation. The use of a strong base free carbon dioxide absorbent is therefore recommended.

Dielectric measurement: error analysis and assessment of uncertainty.

The advantages and limitations of using partial differential analysis to assess the methodological uncertainty associated with the measurement of the dielectric properties of a material are discussed and an alternative pragmatic approach is proposed. It relies on repeat measurements of the dielectric properties of reference liquids and an analysis to estimate random and systematic uncertainties. Examples of measurement uncertainty are provided for well-defined monomolecular materials and for less homogeneous materials at microwave frequencies. All examples relate to measurement with an open-ended coaxial probe but the methodology is not specific to this technique. Examination of the components of uncertainty in the dielectric properties of biological tissue shows that, where the system is free of methodological bias, random fluctuations originating from sampling and natural inhomogeneity dominate the uncertainty budget. In such cases, the mean value of the measured parameter and the standard error of the mean can be taken as a good measure of the true value and its associated uncertainty.

Intra- and intermolecular interactions in small bioactive molecules: cooperative features from experimental and theoretical charge-density analysis.

The topological features of the charge densities, rho(r), of three bioactive molecules, 2-thiouracil [2,3-dihydro-2-thioxopyrimidin-4(1H)-one], cytosine [4-aminopyrimidin-2(1H)-one] monohydrate and salicylic acid (2-hydroxybenzoic acid), have been determined from high-resolution X-ray diffraction data at 90 K. The corresponding results are compared with the periodic theoretical calculations, based on theoretical structure factors, performed using DFT (density-functional theory) at the B3LYP/6-31G** level. The molecules pack in the crystal lattices via weak intermolecular interactions as well as strong hydrogen bonds. All the chemical bonds, including the intra- and intermolecular interactions in all three compounds, have been quantitatively described by topological analysis based on Bader's quantum theory of 'Atoms In Molecules'. The roles of interactions such as C-H...O, C-H...S, C-H...pi and pi...pi have been investigated quantitatively in the presence of strong hydrogen bonds such as O-H...O, N-H...O and N-H...S, based on the criteria proposed by Koch and Popelier to characterize hydrogen bonds and van der Waals interactions. The features of weak intermolecular interactions, such as S...S in 2-thiouracil, the hydrogen bonds generated from the water molecule in cytosine monohydrate and the formation of the dimer via strong hydrogen bonds in salicylic acid, are highlighted on a quantum basis. Three-dimensional electrostatic potentials over the molecular surfaces emphasize the preferable binding sites in the structure and the interaction features of the atoms in the molecules, which are crucial for drug-receptor recognition.

Picoliter-volume aqueous droplets in oil: electrochemical detection and yeast cell electroporation.

An electrochemical detection method was introduced for aqueous droplet analysis in oil phase of microfluidic devices. This method is based on the electrochemical signal difference between aqueous and oil. Applying a low alternating current (AC) voltage to a couple of Au microelectrodes, this method can offer size information and ion concentration range from 0.02 mmol/L to 1 mol/L of tens of picoliter to nanoliter aqueous droplets. Alternatively, applying a relative high AC voltage (18 Vpp) at a frequency of 1 kHz leads to electroporation of yeast cells encapsulated into picoliter droplets. We believe that this simple technique is useful for a number of aqueous droplet-based chemical and biological analyses as well as cell electroporation.

Biogenic amines in the human neocortex in patients with neocortical and mesial temporal lobe epilepsy: identification with in situ microvoltammetry.

Biogenic amines in well defined subtypes of human temporal lobe epilepsy (TLE) have not been well characterized. Specimens from five patients with neocortical TLE (NTLE) and nine with mesial TLE (MTLE) were immediately placed in Ringer's lactate; stearate indicator microelectrodes were placed in temporal gray matter, Ag/AgCl reference microelectrodes and auxiliary microelectrodes were placed 3-7 mm contralaterally to the indicator microelectrode. Dopamine (DA), ascorbic acid (AA), norepinephrine (NE) and serotonin (5-HT) were identified by their characteristic oxidative potentials in vitro. Four of five patients with NTLE had NE depletion in temporal neocortex while eight of nine patients with MTLE had high concentrations of NE (chi-square P<0.01). Significant concentrations of DA were present in the temporal lobes of three of five NTLE patients but in only one of the nine MTLE patients (chi-square P<0.05). 5-HT was present in the neocortex of both NTLE and MTLE patients in similar concentrations. AA was found in the neocortex of one NTLE patient. These data support an association between NE depletion and NTLE. The relative NE deficiency along with the consistent presence of DA in NTLE patients suggest an impairment in the catecholamine pathway. The presence of AA, a co-factor in NE synthesis, in the neocortex of one NTLE patient may also be related since AA is a cofactor in NE synthesis.

Soft- and hard-modeling approaches for the determination of stability constants of metal-peptide systems by voltammetry.

The Zn(2+)-glutathione system is studied as a model for metal-peptide systems where some critical factors must be considered when using voltammetric techniques for the determination of stability constants. These factors are the presence of side reactions (in this case, both the protonation of glutathione and the hydrolysis of Zn(2+)), the association-dissociation rates of the complexes compared with the time scales of the measurements (which makes the complexes electrochemically labile or inert), and the electron transfer kinetics on the electrode surface (which makes the metal ion reduction reversible or irreversible). For the study of these factors, three data treatment approaches have been applied: (i) the electrochemical hard-modeling approach (modelization of both chemical equilibrium and electrochemical processes), (ii) a chemical hard-modeling approach (modelization of chemical equilibria only, based on the least-squares curve-fitting program SQUAD), and (iii) a previously developed model-free soft-modeling approach based on multivariate curve resolution with a constrained alternating least-squares optimization. By analyzing differential pulse polarographic data obtained under different experimental conditions, the influence of the mentioned factors on every approach is discussed and, if possible, the corresponding stability constants are computed. The results of this study showed the potential usefulness of voltammetry in combination with hard- and soft-modeling data analysis for the study of peptide complexation equilibria of metal ions such as Zn which have neither relevant spectroscopic properties nor proper isotopes for NMR measurements.

Quantification of lipids, liposomes, amino acids, and proteins by thermal ultramicrodigestion and an ultramicrocoulometric assay, based on the reaction of hypobromite with ammonia.

The quantification of nitrogen in organic material is described. It is based on a novel thermal ultramicrodigestion in combination with an ultramicrocoulometric quantification. The lower detection limit of the coulometric measurement is 0.5 microg nitrogen, which corresponds to 20 microg lipid, 3 microg glycine, or 4 microg protein. Therefore it is as sensitive as the frequently used Lowry method. In contrast to the Lowry protein determination it is not disturbed by detergents and most other interfering substances.

Applications for the new electrochemiluminescent (ECL) and biosensor technologies.

Biosensor and electrochemiluminescent (ECL) assays are replacing enzyme-linked immunosorbent assays (ELISAs) at Schering-Plough as immunoassays of choice to monitor cytokine levels and detect anti-cytokine antibody responses during cytokine therapy. These new assays provide increased sensitivity and a better correlation with biological assays. Biosensor assays using the BIACORE 2000 (BIACORE, Uppsala, Sweden) are being adopted to support preclinical and clinical trials for the detection of antibodies capable of binding to IL-10 and IL-4. Significant advantages when using a biosensor assay are that real-time and label-free detection permit increased throughput and direct detection of binding interactions which enables detection of low affinity antibodies that are not detected by ELISA. The ECL assays using the ORIGEN Analyser (IGEN, Gaithersburg, MD) that we have implemented to replace existing ELISAs for quantification of serum IL-10 and serum interferon alfa levels are more sensitive and less subject to matrix effects. Data obtained during the validation of these assays are described.

Reversed-phase ion-pair HPLC method for the direct analysis of 1-OH midazolam glucuronide in human serum.

In recent years, it has become clear that the presence of high concentrations of 1-OH midazolam glucuronide is probably the cause of unexplained prolonged midazolam comas in patients with poor renal function. Until recently, only indirect methods for the analysis of this glucuronide were known, which had several disadvantages, such as a long analysis period (>6 hours). This article describes the validation of a method for the direct analysis of this compound in human serum, using reversed-phase ion-pair high-performance liquid chromatography (HPLC) in combination with solid phase extraction. The intraday and interday coefficients of variation have values below 6% for different possible serum concentrations. The limit of quantification (0.1 mg/L) is much lower than concentrations found in patients with a coma caused by the accumulation of 1-OH midazolam glucuronide. Recovery of 1-OH midazolam glucuronide is almost 100% at three different serum concentrations. Linearity is confirmed for normal serum levels (<1 mg/L) and for serum levels that might occur in patients with impaired renal function (<20 mg/L). Detection is performed at 254 nm with a diode array detector, which can also be used to check the peak purity in case of unexpected impurities.

Evaluation of Glucocard Memory 2 and Accutrend sensor blood glucose meters.

The performance and practicability of 2 blood glucose meters (Glucocard Memory 2 and Accutrend sensor) were evaluated. Both glucose meters produced acceptably precise results in the hyper- and normoglycaemic concentration ranges. In the hypoglycaemic concentration range, the imprecision of Accutrend sensor was much higher than recommended by the American Diabetes Association. Within-run coefficients of variation for Glucocard Memory 2 were 6.3%, 3.9% and 2.4% at glucose concentrations of 1.7 mmol/l, 5.8 mmol/l and 11.7 mmol/l, respectively: for Accutrend sensor these were 15.2%, 5.0% and 1.2% at respective concentrations of 0.9 mmol/l, 4.2 mmol/l and 19.6 mmol/l. Between-day coefficients of variation for Glucocard Memory 2 were 4.8% and 3.5% at glucose concentrations of 3.9 mmol/l and 17.2 mmol/l, respectively and for Accutrend sensor they were 3.8% and 2.9% at glucose concentrations of 3.8 mmol/l and 18.7 mmol/l, respectively. Results were linear over a range of 1.6 mmol/l -29.7 mmol/l for Glucocard Memory 2 and 1.6 mmol/l -33.3 mmol/l for Accutrend sensor. Results of both blood glucose meters correlated closely with the hexokinase/glucose-6-phosphate dehydrogenase laboratory method. Ninety-eight percent of both Glucocard Memory 2 and Accutrend sensor results were within 20% of the comparison method values. Ninety-three percent of the Glucocard Memory 2 and 96% of the Accutrend sensor results were within 15% of the comparison method results. An inverse relation between the glucose readings and haematocrit values was observed for both blood glucose meters in the hyperglycaemic range and this effect was more pronounced for Accutrend sensor. In the normo- and hypoglycaemic ranges the effect was insignificant and absent, respectively. Minimum sample volume for Glucocard Memory 2 was 3 microliters and for Accutrend sensor it was 9 microliters. Lower sample volumes gave erroneous results. Presenting more than the required volume had no effect on results.

Electrochemical detection and quantification of the acetylated and deacetylated C8-deoxyguanosine DNA adducts induced by 2-acetylaminofluorene.

The genotoxic agent 2-acetylaminofluorene induces, upon metabolic activation, two main types of DNA adducts in animal tissue, i.e., (deoxyguanine-8-yl)-aminofluorene (dG-C8-AF) and N-(deoxyguanine-8-yl)-acetylaminofluorene (dG-C8-AAF). Quantification of the frequency of these adducts usually relies on the use of radioactively labeled 2-acetylaminofluorene. Here, we report the development of a sensitive, non-radioactive method for the quantification of dG-C8-AF and dG-C8-AAF. Essentially, the modified DNA bases are separated by high-performance liquid chromatography (HPLC) and quantified by electrochemical detection. We established that both modified bases guanine-C8-aminofluorene and guanine-C8-acetylaminofluorene are electrochemically active. Subsequently, a procedure was developed to quantify dG-C8-AF and dG-C8-AAF in genomic DNA. Following DNA hydrolysis the adducted bases were extracted by ethyl acetate, separated by HPLC, and detected electrochemically. This procedure has been applied in the analysis of dG-C8-AAF in N-acetoxy-2-acetylaminofluorene-modified calf thymus DNA and in the detection of dG-C8-AAF and dG-C8-AF in liver DNA of mice injected intraperitoneally with 150-450 mg N-hydroxy-2-acetylaminofluorene/kg. The quantification of relatively low dG-C8-AF and dG-C8-AAF adduct levels (i.e., 0.1-1 adduct/10(6) nucleotides) in mouse liver DNA demonstrates the sensitivity of this electrochemical detection procedure. The detection limit of the method is 1 adduct per 10(6) nucleotides for both adducts using 20 micrograms of DNA and 4 adducts per 10(8) nucleotides using 500 micrograms DNA.

Validated high-performance liquid chromatography-electrochemical method for determination of glutathione and glutathione disulfide in small tissue samples.

Glutathione (GSH) and glutathione disulfide (GSSG) are biologically important intracellular thiols; alterations in the GSH/GSSG ratio are often used to assess exposure of cells to oxidative stress. Although several methods are available for measuring GSH and GSSG, all have some disadvantages including the need to generate derivatives, the inability to conveniently measure both GSH and GSSG, and a lack of sufficient sensitivity to allow detection in very small samples/cells of extrahepatic tissue. These studies present a rapid, validated HPLC-electro-chemical method for determining GSH and GSSG in small samples such as those from microdissected airways of the mouse containing 50-200 micrograms protein which is suitable for routine use. GSH and GSSG can be measured at levels of 1 and 2 pmol on column, respectively, with acceptable accuracy and precision and without the need to generate derivatives. In microdissected airways from the mouse, the intraday assay coefficient of variation for GSH varied from 4.7 to 5.9% and for GSSG was 4.4 to 5.7%. The interday assay coefficient of variation ranged from 6.0 to 7.6% for GSH and 5.5 to 23% for GSSG. The effects of repeated freezing and thawing on the concentrations of GSH and GSSG indicate that multiple cycles do not significantly alter the GSH or GSSG concentration as the number of cycles increases. Addition of GSH or GSSG to samples increased the peak areas appropriately, without altering the peak shape, retention time, or peak area of the corresponding reduced (oxidized) thiol. The ratio of GSH/GSSG in freeze-clamped liver ranged from 46 to 248, while liver tissue which was homogenized fresh had GSH/GSSG ratios of 62-150. The technique appears to be capable of reproducibly measuring GSH and GSSG in small quantities of nonhepatic tissue.

Evaluation of electrochemical nitric oxide and nitrogen dioxide analyzers suitable for use during mechanical ventilation.

OBJECTIVE: Inhaled nitric oxide (NO) is increasingly being used in the treatment of diseases characterized by hypoxemia and pulmonary hypertension. To avoid complications, accurate quantitative analysis of NO and NO2 is necessary during this therapy. We evaluated the accuracy of electrochemical NO and nitrogen dioxide (NO2) analyzers suitable for use during mechanical ventilation. METHODS: We evaluated six electrochemical NO analyzer brands (Bedfont, B & W, Dräger, EIT, Pulmonox, Saan). All were calibrated and used per manufacturer's specifications. An adult mechanical ventilator was used to produce serial dilutions of NO with O2 for [NO] of 0-80 ppm. F1O2 settings of 0.90, 0.70, 0.50, 0.30, and 0.21 were used. Settings of low, moderate, and high ventilation pressures were evaluated. Gas was sampled from the inspiratory limb of the ventilator circuit using either a sidestream or mainstream technique. [NO] was also measured using a calibrated chemiluminescence analyzer. For the analyzers that measured NO2, serial dilutions of 8.5 ppm NO2 with O2 were analyzed using chemiluminescence and the electrochemical analyzers. RESULTS: Bias +/- precision for [NO] by individual devices ranged from 1.8 +/- 1.9 ppm to -1.0 +/- 0.7 ppm. There were significant differences in the bias between analyzers (P < 0.001), pressure settings (P < 0.001), and NO level (P < 0.017). The difference in bias between levels of F1O2 was not significant (P = 0.062). Bias +/- precision for NO2 ranged from 0.18 +/- 0.12 ppm to -0.14 +/- 0.13 ppm, with a significant difference between analyzers (P < 0.001). CONCLUSIONS: The bias and precision of these analyzers was acceptable for clinical use. The devices tended to be most accurate at [NO] < or = 20 ppm-the clinical conditions at which NO is most commonly used.

MEPSIM: a computational package for analysis and comparison of molecular electrostatic potentials.

MEPSIM is a computational system which allows an integrated computation, analysis, and comparison of molecular electrostatic potential (MEP) distributions. It includes several modules. Module MEPPLA supplies MEP values for the points of a grid defined on a plane which is specified by a set of three points. The results of this program can easily be converted into MEP maps using third-parties graphical software. Module MEPMIN allows to find automatically the MEP minima of a molecular system. It supplies the cartesian coordinates of these minima, their values, and all the geometrical relationships between them (distances, angles, and dihedral angles). Module MEPCOMP computes a similarity coefficient between the MEP distributions of two molecules and finds their relative position that maximizes the similarity. Module MEPCONF performs the same process as MEPCOMP, considering not only the relative position of both molecules but also a conformational degree of freedom of one of them. The most recently developed module, MEPPAR, is another modification of MEPCOMP in order to compute the MEP similarity between two molecules, but only taking into account a particular plane. The latter module is particularly useful to compare MEP distributions generated by pi systems of aromatic rings. MEPSIM can use several wavefunction computation approaches to obtain MEP distributions. MEPSIM has a menu type interface to simplify the following tasks: creation of input files from output files of external programs (GAUSSIAN and AMPAC/MOPAC), setting the parameters for the current computation, and submitting jobs to the batch queues of the computer. MEPSIM has been coded in FORTRAN and its current version runs on VMS/VAX computers.

Coulometric electrochemical detection of hydroxyproline using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

A new method for detection of hydroxyproline has been developed. Hydroxyproline was derivatized using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; separation and detection were accomplished using HPLC with coulometric electrochemical detection. Derivatized hydroxyproline was initially detected using a 16 channel coulometric electrochemical array system. The assay was slightly modified for use with a simpler 2-channel coulometric electrochemical detector. Both detectors are sensitive into the upper fmol range. Derivatization of hydroxyproline occurs in 5 min and the derivative is stable for several hours at 0 degrees C. The techniques were used to quantitate hydroxyproline in purified type I collagen and other biological samples.