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PubMed2004

Monitoring multi-class pesticide residues in fresh fruits and vegetables by liquid chromatography with tandem mass spectrometry.

A new analytical method was developed using liquid chromatography with tandem mass spectrometry for the routine analysis of 31 multi-class pesticide residues and applied to approximately 50 fresh fruit and vegetable samples (green bean, cucumber, pepper, tomato, eggplant, watermelon, melon and zucchini). Extraction of the pesticides with ethyl acetate was carried out. The optimal ionisation conditions were selected for each pesticide in the same run. The procedure was validated and the values of some merit figures, such as recovery, precision, linear range, detection limit and quantification limit for each pesticide were calculated together with its calculated expanded uncertainty (U). The average recoveries in cucumber obtained for each pesticide ranged between 74 and 105% at two different fortification levels (n = 10 each) that ranged between 9 and 250 ng g(-1) (depending on the pesticide). The uncertainty associated to the analytical method was lower than 23% for all compounds tested. The calculated limits of detection and quantitation were typically <1 ng g(-1) that were much lower than the maximum residue levels established by European legislation.

Capsicum↗

PubMed2001

Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology.

BACKGROUND: Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. METHODS: Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. RESULTS: In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. CONCLUSIONS: By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.

Animals↗

PubMed2000

Analysis of catechins and caffeine in tea extracts by micellar electrokinetic chromatography.

Cancer chemotherapy is a new and important medical science and much interest has been focused on catechins, not only for their antioxidant activity, but also because of their known antimutagenic and antitumorigenic properties. Green tea and black tea, which are among the most popular beverages consumed worldwide, contain many different catechins. We developed an analytical method capable of separating six different catechins and caffeine in tea by micellar electrokinetic chromatography in only 20 min without extensive sample preparation. Furthermore, we compared the amount of catechins and caffeine in several teas and different preparation modes.

Caffeine↗

PubMed1988

Comparative analysis of the chemical composition of mixed and pure cultures of green algae and their decomposed residues by C nuclear magnetic resonance spectroscopy.

It is known that macromolecular organic matter in aquatic environments, i.e., humic substances, is highly aliphatic. These aliphatic macromolecules, predominantly paraffinic in structure, are prevalent in marine and lacustrine sediments and are believed to originate from algae or bacteria. A comparative study of mixed and pure cultures of green algae and their decomposed residues was performed by using solid-state C nuclear magnetic resonance spectroscopy as the primary analytical method. Results obtained in this study confirm the presence of components that are chemically refractory and that are defined as alghumin and hydrolyzed alghumin. These were detected in heterogeneous, homogeneous, and axenic biomasses composed of several genera of Chlorophyta. Although the chemical composition of algal biomass varied with culture conditions, the chemical structure of the alghumin and hydrolyzed alghumin, demonstrated by C nuclear magnetic resonance spectroscopy appeared to be constant for members of the Chlorophyta examined in this study. The alghumin was dominated by carbohydrate-carbon, with minor amounts of amide or carboxyl carbon and paraffinic carbon, the latter surviving strong hydrolysis by 6 N HCI (hydrolyzed alghumin). Bacterial decomposition of heterogeneous algal biomass labeled with C was conducted under both aerobic and anaerobic conditions to determine chemical structure and stability of the refractory material. The refractory fraction ranged from 33% in aerobic to 44% in anaerobic cultures. The refractory fraction recovered from either aerobic or anaerobic degradation comprised 40% alghumin, which represented an enrichment by 10% relative to the proportion of alghumin derived from whole cells of algae. The paraffinic component in the hydrolyzed alghumin of whole algal cells was found to be 1.8% and increased to 5.1 and 6.9% after aerobic and anaerobic bacterial degradation, respectively. It is concluded that members of the Chlorophyta contain a common insoluble structure composed of paraffinic carbon that is resistant to chemical and bacterial degradation under conditions used in this study. The paraffinic structure is identical to those constituting humin of aquatic origin. Thus, alga-derived macromolecular compounds deposited in aquatic environments (alghumin) probably contribute to sedimentary humic substances.

Journal Article↗

PubMed2002

Direct determination of free amino acids and sugars in green tea by anion-exchange chromatography with integrated pulsed amperometric detection.

Determination of amino acids and sugars in green tea by anion-exchange chromatography with integrated pulsed amperometric detection was developed. Amino acids and sugars were separated on an anion-exchange column at a flow-rate of 0.25 ml/min by using ternary gradient elution consisting of deionized water, 0.25 M sodium hydroxide, and 1.0 M sodium acetate. Under optimized conditions, theanine was separated from glutamine and three sugars (glucose, fructose, and sucrose) were eluted earlier than the neutral amino acids to avoid their interference with each other. RSDs of the peak area of analytes were lower than 4.6%. Detection limits for the analytes ranged from 0.12 to 4.9 pmol. The linearities for all analytes were two or three orders of magnitude with the correlation coefficients greater than 0.99. This method was applied to determination of amino acids and sugars in green tea with satisfactory results.

Amino Acids↗

PubMed2005

Ultrasound-assisted extraction of phenolic compounds from strawberries prior to liquid chromatographic separation and photodiode array ultraviolet detection.

Ultrasound-assisted extraction was used for the determination of phenolic compounds present in strawberries. The optimization study of the extraction was carried out using spiked samples (100 mg/kg). The sample immersed in an aqueous solution containing hydrochloric acid (0.4 M) was sonicated for 2 min (duty cycle 0.2 s, output amplitude 20% of the nominal amplitude of the converter, applied power 100 W with the probe placed 1cm from the bottom of the water bath and 5 cm from the walls of the precipitate glass). Subsequent separation was carried out by liquid chromatography (LC) with photodiode array UV detection. Calibration curves using the standard addition in green strawberries typically gave linear dynamic ranges of 2-300 mg/l for all analytes; R(2) values exceeded 0.996 in all cases. The method was applied to two types of strawberries to demonstrate the applicability of the proposed method, which is much faster and produces less analyte degradation than methods as solid-liquid, subcritical water and microwave-assisted extraction.

Chromatography, Liquid↗

PubMedDate not supplied

The non-photosynthetic plastid in malarial parasites and other apicomplexans is derived from outside the green plastid lineage.

The discovery of a non-photosynthetic plastid genome in Plasmodium falciparum and other apicomplexans has provided a new drug target, but the evolutionary origin of the plastid has been muddled by the lack of characters, that typically define major plastid lineages. To clarify the ancestry of the plastid, we undertook a comprehensive analysis of all genomic characters shared by completely sequenced plastid genomes. Cladistic analysis of the pattern of plastid gene loss and gene rearrangements suggests that the apicomplexan plastid is derived from an ancestor outside of the green plastid lineage. Phylogenetic analysis of primary sequence data (DNA and amino acid characters) produces results that are generally independent of the analytical method, but similar genes (i.e., rpoB and rpoC) give similar topologies. The conflicting phylogenies in primary sequence data sets make it difficult to determine the the exact origin of the apicomplexan plastid and the apparent artifactual association of apicomplexan and euglenoid sequences suggests that DNA sequence data may be an inappropriate set of characters to address this phylogenetic question. At present we cannot reject our null hypothesis that the apicomplexan plastid is derived from a shared common ancestor between apicomplexans and dinoflagellates. During the analysis, we noticed that the Plasmodium tRNA-Met is probably tRNA-fMet and the tRNA-fMet is probably tRNA-Ile. We suggest that P. falciparum has lost the elongator type tRNA-Met and that similar to metazoan mitochondria there is only one species of methionine tRNA. In P. falciparum, this has been accomplished by recruiting the fMet-type tRNA to dually function in initiation and elongation. The tRNA-Ile has an unusual stem-loop in the variable region. The insertion in this region appears to have occurred after the primary origin of the plastid and further supports the monophyletic ancestory of plastids.

Animals↗

PubMed2002

Unsupervised technique for robust target separation and analysis of DNA microarray spots through adaptive pixel clustering.

MOTIVATION: Microarray images challenge existing analytical methods in many ways given that gene spots are often comprised of characteristic imperfections. Irregular contours, donut shapes, artifacts, and low or heterogeneous expression impair corresponding values for red and green intensities as well as their ratio R/G. New approaches are needed to ensure accurate data extraction from these images. RESULTS: Herein we introduce a novel method for intensity assessment of gene spots. The technique is based on clustering pixels of a target area into foreground and background. For this purpose we implemented two clustering algorithms derived from k-means and Partitioning Around Medoids (PAM), respectively. Results from the analysis of real gene spots indicate that our approach performs superior to other existing analytical methods. This is particularly true for spots generally considered as problematic due to imperfections or almost absent expression. Both PX(PAM) and PX(KMEANS) prove to be highly robust against various types of artifacts through adaptive partitioning, which more correctly assesses expression intensity values. AVAILABILITY: The implementation of this method is a combination of two complementary tools Extractiff (Java) and Pixclust (free statistical language R), which are available upon request from the authors.

Algorithms↗

PubMed2001

Evaluation of a polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruits and vegetables.

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.

Colony Count, Microbial↗

PubMed2002

Analytical study on bioheat transfer problems with spatial or transient heating on skin surface or inside biological bodies.

Several closed form analytical solutions to the bioheat transfer problems with space or transient heating on skin surface or inside biological bodies were obtained using Green's function method. The solutions were applied to study several selected typical bioheat transfer processes, which are often encountered in cancer hyperthermia, laser surgery, thermal comfort analysis, and tissue thermal parameter estimation. Thus a straightforward way to quantitatively interpret the temperature behavior of living tissues subject to constant, sinusoidal, step, point or stochastic heatings etc. both in volume and on boundary were established. Further solution to the three-dimensional bioheat transfer problems was also given to illustrate the versatility of the present method. Implementations of this study to the practical problems were addressed.

Body Temperature↗

PubMed1984

Approximate-analytical solution of the diffusion, convection and reaction problem in homogeneous media.

In convex homogeneous domains, the diffusion, convection and reaction (DCR) problem may be solved by applying Green's function solution technique. When this technique is applied, the solution to the DCR problem consists of the sum of a set of integrals whose integrands involve the Green's function. The Green's function is singular at the upper limit of the time integral and is nonuniformly convergent at the boundaries of the domain. Due to this behaviour, numerical evaluation of the integrals is prohibitively expensive and in some cases, the integrals are incorrectly evaluated. The method presented in this work circumvents all the difficulties inherent with the numerical quadrature of the intergrals and in preliminary case studies (in rectangular coordinates) has reduced the required computation time by up to five orders of magnitude while increasing the accuracy of the results by as much as eight orders of magnitude. The method involves transforming the function in the integrand, which multiplies the Green's function, into a series of Legendre polynomials. The integral of the product of the Green's function and Legendre polynomials can be evaluated analytically. This produces both a rapid and accurate evaluation of the integral and subsequently the solution to the DCR problem.

Biological Transport, Active↗

PubMed1997

Double-stacked dielectric resonator for sensitive EPR measurements.

A new approximate method for predicting the resonant frequencies and for solving the field distribution problem of a cylindrical dielectric resonator (DR) is developed. The model proposed in this paper bridges the gap between rigorous and accurate finite-element or Green function-based numerical methods on the one hand and on the other hand, simple approximate solutions in which the field distribution can be described analytically, but the resulting frequency is accurate within a few percent only. In the method described here, the approximate solution for the microwave field distribution is modified by substituting different values of the radial separation constants inside and outside of the diskshaped DR. The model is generalized for the double-stacked DR structure and enables one to introduce corrections that take into account the presence of the shielding walls and of the cylindrical sample hole. Good agreement is found between experimental and calculated results for both the single and double-stacked structures that are designed around commercially available X-band DRs (9-10 GHz). For the resonant frequency of the lowest transverse-electric TEzero1 delta mode that is commonly used for EPR measurements, the accuracy of the method is better than 1%. Experimentally measured resonator filling factors are also in good agreement with those theoretically estimated. Both the theory and the experimental results suggest that the double-stacked DR structure with finite spacing between the ceramic cylinders is the most suitable for EPR measurements of long lossy samples.

Ceramics↗

PubMed2004

Evaluation of rapid somatic cell counters under different analytical conditions in ovine milk.

A total of 31 individual ovine milk samples, ranging between 30 and 2600 x 10(3) cells/mL, were divided into 8 aliquots/milk with the objective of studying the overall accuracy of 2 rapid somatic cell count (SCC) counters, one based on cytometry on disk (Fossomatic 360) and the other on flow cytometry (Fossomatic 5000), under 4 types of preservation (without preservation, bronopol, sodium azide, and potassium dichromate) and 2 analytical temperatures (40 and 60 degrees C). All analyses were carried out in duplicate. In addition, each sample was analyzed in quadruplicate by reference microscopic method using Pyronin Y-methyl green as a stain. A second experiment using 13 samples divided into 20 aliquots/ sample, enabled repeatability to be studied depending on the values obtained in the SCC and on the SCC equipment used. Comparison of the methods was based on repeatability and accuracy studies (means comparison and regression studies vs. reference method). Both counters gave adequate repeatability and accuracy values in ovine milk, though the SCC obtained by Fossomatic 5000 was closer to the reference method and was somewhat more repeatable than Fossomatic 360. In the regression study, slope and intercept values were statistically different from their theoretical values (1.00 and 0.00, respectively) in the unpreserved samples but not in the preserved ones. In all cases, correlation coefficients very close to 1.00 were obtained. The preserved milk analyzed by flow cytometry gave optimal repeatability values (s(r) = 16.3 to 19.7 and s(r)% = 1.9 to 2.4), and their logSCC means (5.62 to 5.64) were not different from the reference value (5.63). Bronopol was the optimal preservative for the Fossomatic method. Analytical temperature did not contribute significantly to SCC variation, although disk cytometry gave slightly more repeatable SCC at 40 degrees than at 60 degrees C.

Animals↗

PubMed1986

Extraction and identification of different prostaglandins in Allium cepa.

Green onions (Allium cepa) were homogenized in a blender and extracted by normal extraction methods except that diethyl ether was used as the first extracting solvent. Different analytical procedures were used for the identification of the prostaglandins separated. TLC was applied using silica gel 60 F254 plates and a mixture of benzene, dioxane and acetic acid (20:10:1) as eluent, and the Rf values were compared with those of authentic samples. GC analysis on an SE 30 packed column and FID was applied; relative retention times of the onion extract components were measured and matched with authentic prostaglandin samples using cholesterol as an internal standard. GC-MS analyses using the same conditions adopted for GC analysis were conducted on a Finnigan MAT 112S instrument. Four peaks were identified. The prostaglandins identified were F1 alpha, E1, B1 and A2.

Allium↗

PubMed2005

Fluctuation spectrum of membranes with anchored linear and star polymers.

The effect of linear homopolymers, diblock copolymers, and star polymers anchored to a membrane on the membrane's fluctuation spectrum is investigated for low grafting densities. Due to the nonlocality of the polymer-membrane interaction, the effective bending rigidity kappa(eff)(q) of the composite membrane is found to depend strongly on the wave vector q of the membrane undulations. Analytical calculations for ideal linear chains and simulations for ideal and self-avoiding linear chains as well as for star polymers are presented. The analytical calculations are based on the Green's function approach of Bickel and Marques [Eur. Phys. J. E 9, 349 (2002)]; for the simulations the Monte Carlo method is used. The functional form of kappa(eff)(q) differs for end-grafted chains and diblock copolymers. In general, the polymer effect is most pronounced for undulations on length scales larger than or comparable to the polymer size, and decreases rapidly for smaller undulation wavelengths. Anchored linear chains always increase kappa; anchored star polymers may increase as well as decrease kappa, depending on whether they are anchored symmetrically or asymmetrically to the membrane.

Biopolymers↗

PubMedDate not supplied

Gas chromatographic/mass spectrometric confirmation of leucomalachite green in catfish (Ictalurus punctatus) tissue.

A gas chromatographic/mass spectrometric (GC/MS) method was developed to confirm the presence of leucomalachite green (LMG), a metabolite of the triphenylmethane dye malachite green (MG), in catfish tissue. Residues were isolated according to a previously described liquid chromatographic (LC)/VIS spectrometric analysis of MG and LMG in fish. In our isolation procedure, analytes are extracted from tissue with acetonitrile-buffer, partitioned into CH2Cl2, and applied to neutral alumina and propylsulfonic acid solid-phase extraction cartridges. Before GC/MS analysis, extracts prepared for the LC determinative method are eluted from a cyano solid-phase extraction cartridge, extracted into organic solvent, and concentrated for GC/MS analysis. Selected ion monitoring was performed by using 5 diagnostic ions (m/z 330, 329, 253, 210, and 165) of LMG. The method was validated by confirming LMG in tissue fortified with mixtures of MG and LMG (5 and 10 ng/g each) and in tissue from fish that had been exposed to low levels of MG.

Aniline Compounds↗

PubMed2005

Digital image processing-an alternate tool for monitoring of pigment levels in cultured cells with special reference to green alga Haematococcus pluvialis.

A method for analyzing carotenoid content in Haematococcus pluvialis, a green alga was developed using digital image processing (DIP) and an artificial neural network (ANN) model. About 90 images of algal cells in various phases of growth were processed with the tools of DIP. A good correlation of R(2)=0.967 was observed between carotenoid content as estimated by analytical method and DIP. Similar correlation was also observed in case of chlorophyll. Since the conventional methods of carotenoid estimation are time consuming and result in loss of pigments during analysis, DIP method was found to be an effective online monitoring method. This method will be useful in measurement of pigments in cultured cells.

Algorithms↗

PubMed2003

Fossomatic cell-counting on ewe milk: comparison with direct microscopy and study of variation factors.

Using the Fossomatic method (FSCC) a total of 23,003 analytical SCC observations were carried out on 6400 aliquots taken from 80 individual ewe milk samples with the objective of studying the influence of 4 preservation procedures (without preservation, potassium dichromate, azidiol, and bronopol), 2 storage temperatures (ambient and refrigeration), 10 milk ages (3,6,12, and 24h, and 2,3,4,5,7, and 9d postcollection), and two analytical temperatures (40 and 60 degrees C). In addition, each sample was analyzed with direct microscopic method (DMSCC), using 3 different stainings for each sample: methylene blue (MB), May-Grünwald-Giemsa (MGG) and Pyronin Y-methyl green (PGM). This allowed DMSCC and FSCC (at 24 h of age) to be compared. The reference DMSCC from MB staining was a reliable method in ewe milk, though more specific stainings such as MGG and PMG slightly improve the residual standard deviation for repeated SCC. Between DMSCC and FSCC, the highest coefficients of correlation (0.972 to 0.996) corresponded to preserved and refrigerated milk, and the lowest (0.708 to 0.919) to unpreserved and ambient stored aliquots. Except for the unpreserved and ambient stored aliquots, SCC values were similar in all aliquots. Under FSCC, preservation, storage and analytical temperature, milk age, and most of the interactions showed a significant effect on SCC variation. In preserved samples, logSCC values ranged between 5.67 (bronopol) and 5.62 (azidiol). The higest values (5.72) were for unpreserved milk, which showed false overestimation of SCC due to bacterial proliferation. LogSCC was higher at 60 degrees C (5.68) than at 40 degrees C (5.65). The interaction between age, preservation and storage temperature showed no cell degeneration in properly handled samples over the 9 d of study.

Animals↗