Atomistic insight into chondroitin-6-sulfate glycosaminoglycan chain through quantum mechanics calculations and molecular dynamics simulation.

Chondroitin-6-sulfate (C6S) is a glycosaminoglycan (GAG) constituent in the extracellular matrix, which participates actively in crucial biological processes, as well as in various pathological conditions, such as atherosclerosis and cancer. Molecular interactions involving the C6S chain are therefore of considerable interest. A computational model for atomistic simulation was built. This work describes the design and validation of a force field for a C6S dodecasaccharide chain. The results of an extensive molecular dynamics simulation performed with the new force field provide a novel insight into the structure and dynamics of the C6S chain. The intramolecular H-bonds in the disaccharide linkage region are suggested to play a major role in determining the chain structural dynamics. Moreover, the unravelling of an additional H-bond involving the sulfate groups in C6S is interesting as changes in sulfation have been claimed to be an important factor in several diseases. The force field will prove useful for future studies of crucial interactions between C6S and various nanoassemblies. It can also be used as a basis for modeling of other GAGs.

Pressurised hot water extraction-microporous membrane liquid-liquid extraction coupled on-line with gas chromatography-mass spectrometry in the analysis of pesticides in grapes.

Pressurised hot water extraction-microporous membrane liquid-liquid extraction was coupled on-line with gas chromatography-mass spectrometry (PHWE-MMLLE-GC-MS) for the analysis of pesticides in grapes. MMLLE serves as a trapping step after PHWE. Water from PHWE is directed to the donor side of the membrane unit and the analytes are extracted to the acceptor solution on the other side. The role of MMLLE is to clean and concentrate the extract before on-line transfer to the GC via a sample loop and an on-column interface using partially concurrent solvent evaporation. The extraction conditions were investigated, and then the quantitative features such as linearity, limit of quantification (LOQ), extraction yield and enrichment factors. LOQs in the range 0.3-1.8 microg kg(-1) were achieved. Procymidone and tetradifon were found in the skins of the grapes. The results were in good agreement with those obtained by liquid-solid and ultrasonic extractions.

Determination of pesticides in red wines with on-line coupled microporous membrane liquid-liquid extraction-gas chromatography.

Microporous membrane liquid-liquid extraction (MMLLE) was coupled on-line with gas chromatography for the determination of pesticides in wine. The MMLLE-GC provided to be efficient and selective and the method was linear, repeatable and sensitive. The limits of detection ranged from 0.05 to 2.3 microg/l and the limits of quantification were 0.2-7.5 microg/l for all the analytes using FID as detector. With MS detection LODs in the range 0.03-0.4 and LOQs of 0.3-3.5 microg/l were achieved. The method was applied to the determination of pesticides in several red wines of different origin.

Analysis of PAH compounds in soil with on-line coupled pressurised hot water extraction-microporous membrane liquid-liquid extraction-gas chromatography.

Pressurised hot water extraction (PHWE) was coupled on-line with microporous membrane liquid-liquid extraction (MMLLE) and gas chromatography (GC) in the analysis of polycyclic aromatic hydrocarbon (PAH) compounds in soil. The MMLLE serves as a trapping device after the PHWE. Water from PHWE is directed to the donor side of the membrane unit and the analytes are extracted to the acceptor solution on the other side of the membrane. The role of MMLLE is to clean and concentrate the extract, which is then transferred on-line to the GC via a sample loop and an on-column interface using partially concurrent solvent evaporation. Separate optimisation of MMLLE and simulations of the PHWE-MMLLE connection were carried out before the actual on-line coupling. After optimisation of the whole on-line system, the efficiencies of the PHWE-MMLLE-GC and PHWE-solid-phase trap extractions were compared. The PHWE-MMLLE-GC method allowed on-line analysis of soil samples. The method was linear, with limits of detection in the range 0.05-0.13 ng and limits of quantification 0.65-1.66 microg g(-1). Comparison of the results with those obtained by other techniques confirmed the good performance.

Determination of pesticide residues in red wines with microporous membrane liquid-liquid extraction and gas chromatography.

A sample pretreatment method based on microporous membrane liquid-liquid extraction (MMLLE) was developed for the subsequent gas chromatographic determination of pesticides in wine. MMLLE provided efficient and selective extraction with enrichment factors in the range 3-13. The gas chromatographic separation was carried out using on-column injection and flame ionization detection. The method was linear, repeatable and sensitive. The limits of quantification were better than 0.006 mg/L for all the analytes except for iprodione (0.37 mg/L). The method was applied to the determination of pesticides in several red wines of different origin.

Use of a partial filling technique and reverse migrating micelles in the study of N-methylcarbamate pesticides by micellar electrokinetic chromatography-electrospray ionization mass spectrometry.

This study describes three ways to couple micellar electrokinetic chromatography (MEKC) on-line with electrospray ionization mass spectrometry (ESI-MS) for the analysis of N-methylcarbamate pesticides. The methods involved the use of a partial filling (PF) technique under basic conditions and the use of reverse migrating micelles (RMMs) under acidic and basic conditions. The use of RMMs in basic electrolyte solutions required coated capillaries with low electroosmotic flows, and capillaries coated with anionic poly(sodium 2-acrylamide-2-methylpropanesulfonate) were selected for the purpose. Before the on-line MEKC-ESI-MS coupling, the MEKC and MS conditions were separately optimized under off-line conditions. The methods were compared in terms of detection limits and the stability of the electrospray process. The PF method offered good separation but poorer stability of the electrospray relative to the other methods. A more stable electrospray performance was obtained with use of RMMs in acidic electrolyte solutions, but some of the analytes were protonated and could not be detected due to the increase in their retention factors. However, with the use of anionic polymer-coated capillaries and RMMs at pH 8.5, all analytes were successfully separated. The high-salt stacking method was applied to improve the sensitivity of MEKC-ESI-MS and the detection limits were in the range of 0.04-2.0 microg/ml.

Analysis of pesticides in red wines by on-line coupled reversed-phase liquid chromatography-gas chromatography with vaporiser/precolumn solvent split/gas discharge interface.

Pesticides in red wines were analysed by on-line coupled reversed-phase liquid chromatography-gas chromatography where a vaporiser/precolumn solvent split/gas discharge interface enabled direct transfer of aqueous eluent to the GC system. The LC part of the system provided sample clean-up and re-concentration, and the GC the final analytical step. The method developed allowed automated and quantitative analysis of the wine samples, where the only manual step was filtration. The limits of quantification were clearly below the maximum residue limits established for grapes, being lower than 10 micrograms l-1 for all pesticides studied.

Liquid chromatographic sample cleanup coupled on-line with gas chromatography in the analysis of beta-blockers in human serum and urine.

An on-line coupled reversed-phase liquid chromatographic-gas chromatographic (LC-GC) method with minimal manual sample preparation is developed for the analysis of metoprolol, oxprenolol, propranolol, timolol, and codeine (as an internal standard) in human serum and urine. The method is based on a loop-type interface and concurrent eluent evaporation technique. On-line liquid-liquid extraction (LLE) is used to extract the analytes from aqueous eluent to organic solvent before injection onto the GC, and the two phases are separated with a sandwich-type phase separator. The LC is used for cleanup, and the GC is used for the final separation and detection of the analytes. Total analysis time is less than 45 min, which is much less than those of traditional analysis methods. Recoveries in LC cleanup and on-line LLE are excellent. A marked increase in the recoveries with on-line LLE is obtained by heating the aqueous eluent and the extraction coil. Linearity and repeatability of the method are good for both serum and urine, and the limits of quantitation for the analytes are 18-44 ng/mL.

Determination of the antileukemic drug mitoguazone and seven other closely related bis(amidinohydrazones) in human blood serum by high-performance liquid chromatography.

A reversed-phase (C18) HPLC method with diode-array detection was developed for the separation and determination of methylglyoxal bis(amidinohydrazone) (mitoguazone) and seven closely related aliphatic analogs thereof, namely the bis(amidinohydrazones) of glyoxal, dimethylglyoxal, ethylmethylglyoxal, methylpropylglyoxal, butylmethylglyoxal, diethylglyoxal and dipropylglyoxal. The mobile phase consisted of a non-linear binary gradient of methanol and 0.03 M aqueous sodium acetate buffer (pH 4.3). Good separation of the eight congeners was achieved. On increasing the methanol content of the eluent, the bis(amidinohydrazones) eluted in the order of increasing number of carbon atoms in the side-chains. The method was also applied to the quantitative analysis of the compounds in aqueous solution and, combined with ultrafiltration, for the separation of the eight congeners in spiked human blood serum. A separate simplified method for the quantitative determination of each of the compounds in spiked human blood serum samples was also developed. The methods developed made for the first time possible the simultaneous HPLC analysis of more than one bis(amidinohydrazones). The results obtained indicate that the bis(amidinohydrazones) studied obviously have a distinct tendency to form ion associates with acetate ions and probably also other carboxylate ions in aqueous solution. This aspect may be of biochemical significance, especially concerning the intracellular binding of the compounds. Each one of the compounds studied invariably gave rise to one peak only, this result supporting the theory that the conventional synthesis of each of the compounds gives rise to one geometrical isomer only. This result is completely in agreement with the results of previous proton and carbon NMR spectroscopic as well as X-ray diffraction studies.

Detection of beta-blockers in urine by solid-phase extraction-supercritical fluid extraction and gas chromatography-mass spectrometry.

The most convenient way to perform supercritical fluid extraction (SFE) of liquid sample matrices is to combine it with solid-phase extraction (SPE). beta-Blockers from urine were collected on an Empore disc, which was then placed into an extraction cell for derivatization and SFE. SPE recovery was best at pH 10. Effects of temperature, pressure and volume of pyridine on the acetylation and SFE processes were studied. Without acetylation the beta-blockers were not significantly soluble in CO2. SFE temperatures of 70 degree C and 150 degree C together with 200 microliters of acetic anhydride and 400 microliters pyridine gave the best results. With the SPE-SFE-GC-MS method developed here, beta-blockers like oxprenolol, metoprolol and propranolol could easily be detected in urine samples, and the limit of detection (LOD) for these compounds was found to be 20 ng/ml, 30 ng/ml and 40 ng/ml, respectively.

Screening of beta-blockers in human serum by ion-pair chromatography and their identification as methyl or acetyl derivatives by gas chromatography-mass spectrometry.

A simultaneous screening method for atenolol, acebutolol, metoprolol, oxprenolol, alprenolol and propranolol by ion-pair chromatography with a column-switching technique was developed. The serum samples were purified using either liquid-liquid extraction or solid-phase extraction methods. The pretreatment of the samples consisted of hydrolysis and protein precipitation. The drug separation was on either octadecylsilica or polymer-based alkyl column material. Binary eluent mixtures containing methanol and a buffer solution with a quaternary ammonium salt as an ion-pair former were used. Detection of the compounds in liquid chromatographic analysis was based on ultraviolet spectra. The effects of methanol, two buffers and the ion-pair former on the retention of the compounds were studied. The determination limits ranged from nanograms to micrograms in the ion-pair chromatographic method, depending on the drug studied. Identification was based on the mass spectra or, if necessary, on selected-ion monitoring spectra of either the methylated or the acetylated compounds obtained by means of gas chromatography-electron impact or negative chemical ionization mass spectrometry. The detection limits for the identified compounds were in the picogram range. The matrix effect was strong, and this resulted in determination limits in the nanogram range with the scan method.

Remarkable differences between the species distributions of various bis(guanylhydrazones) at physiological conditions, and their possible involvement in the strict structural requirements for antileukemic activity.

The first systematic study on the acid-base properties of the antileukemic agents glyoxal bis(guanylhydrazone) (GBG) and methylglyoxal bis(guanylhydrazone) (MGBG) and of their non-antileukemic monoalkyl- and dialkylglyoxal analogs is reported. At physiological conditions (pH 7.4, 37 degrees C), the species distribution of GBG and MGBG differs remarkably from that of their inactive congeners, a noteworthy proportion of GBG (10.2%) and MGBG (4.0%) existing in the form of the free base while the corresponding proportion of their non-antiproliferative analogs is only 0.5% or less. Ethylglyoxal bis(guanylhydrazone) (EGBG), which has antiproliferative properties in vitro but is devoid of antileukemic activity in vivo, is intermediate between the two groups, 2.6% of it existing in the free base form. In contrast to what has been generally assumed, at physiological conditions, the predominant species of GBG, MGBG, and EGBG is the monocation form and not the dication. Considerable proportions of other congeners also exist in the monocation form. At slightly higher pH values that are of interest because of the known antimitochondrial effects of GBG and MGBG (and, in high concentrations, EGBG), the species distribution of GBG and MGBG differs even more remarkably from that of the dialkylglyoxal analogs. Thus, at pH 8.0 and 37 degrees C, as much as 36% of GBG and 19% of MGBG exist in the free base form, the corresponding proportion of EGBG being 14% and that of the other congeners studies only ca. 3-4%. On the basis of the results, it appears possible that the unusually strict structure-activity relationships of this class of antineoplastic agents may be based on the remarkable differences between the species distributions of the various congeners. The hypothesis is presented that the actual antiproliferative and antimitochondrial species of the compounds is the free base form. A compilation of pKa1 and pKa2 values, measured by potentiometric methods in 0.1 M NaCl (aq) at 25 degrees C and 37 degrees C, is given for six bis(guanylhydrazones). The species distribution curves of the compounds (at 37 degrees C) are given for the pH range 6-10.

Simultaneous determination of benzene and toluene in the blood using head-space gas chromatography.

A head-space method for the simultaneous determination of benzene and toluene in blood using a gas chromatograph equipped with a photoionization detector was developed. Internal standards for benzene and toluene were fluorobenzene and o-xylene, respectively, and the detection limit was 5 nmol/l for both solvents. This method is sensitive enough for needs of biological monitoring of benzene and toluene in exposed workers. With automation it offers a possibility for routine measurements. An application of the method in monitoring exposed workers in the industry is presented.