Structural analysis of the reaction products of chitosan with o-, m- and p-phthalaldehydes

Chitosan, a $\text{(I}\text{a}\text{?}\text{4)-}\text{linked}$ 2-amino-2-deoxy-β-d-glucan, was allowed to react with o-, m- and p-phthalaldehydes in aqueous acetic acid-methanol at room temperature to give the corresponding Schiff's base derivative (d.s. ～ 1.0/hexosaminyl residue) in a gel form. The dry product was isolated in yields of 90–97%, and the fine structure was analysed. Both the crosslinking and N-formylbenzylidene structures were present in an almost equal molar ratio in the reaction product of chitosan with p-phthalaldehyde and at a molar ratio of 0.1:0.9 in the reaction product of chitosan with m-phthalaldehyde. However, both structures were absent in the reaction product of chitosan with o-phthalaldehyde, and an intramolecular cyclic structure was present.     

Biotransformation of p-, m-, and o-hydroxybenzoic acids by Panax ginseng hairy root cultures

The regioselective glycosylation of three isomers of hydroxybenzoic acids was observed in Panax ginseng hairy root cultures. p-Hydroxybenzoic acid (1) and m-hydroxybenzoic acid (2) were converted into their corresponding glycosides (1a and 2a) and glycosyl esters (1b and 2b) while no metabolite of o-hydroxybenzoic acid (3) was detected. A new compound, m-hydroxybenzoic acid β-d-xylopyranosyl (1 → 6)-β-d-glycopyranosyl ester (2c) was identified as a biotransformation product of 2. Further time-course studies of the biotransformation reactions showed that the glycosides were major products in the latter stage. The addition of carbohydrates or antioxidants increased glycosyl esters formation.     

Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection

Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.     

Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

Simultaneous determination of benzene, toluene, ethylbenzene, and xylenes in urine by thermal desorption–gas chromatography

The determination of metabolites of benzene, toluene, ethylbenzene, and xylenes in urine has been used to assess human exposure to these compounds. The analyses of urine samples for these metabolites are tedious and time consuming. The determination of unmetabolized individual compounds in urine has been studied previously with some success. A simultaneous determination of several unmetabolized VOC compounds in urine by thermal desorption–gas chromatography was conducted to assess the exposure of smokers and nonsmokers to these compounds. The method of thermal desorption–GC was sensitive enough to detect a significant difference in exposure levels due to the contribution of light smoking in the environmentally-exposed group.     

Oxidation of both termini of p- and m-xylene by Escherichia coli transformed with xylene monooxygenase gene

Xylene monooxygenase (XMO) from Pseudomonas putida mt-2 catalyzes oxidation of methyl group of toluene and xylenes. While it has been postulated that this enzyme oxidizes one methyl group of xylene, we observed that both methyl groups in p- and m-xylene were oxidized to alcohol and aldehyde when the relevant genes (xylM and xylA) were co-expressed in Escherichia coli C600 and MC4100. When p-xylene was used as a substrate, p-hydroxymethylbenzaldehyde and p-xylyleneglycol were identified, in addition to p-methylbenzylalcohol and p-tolualdehyde. When m-xylene was used as a substrate, m-hydroxymethylbenzaldehyde and m-xylyleneglycol were identified, in addition to m-methylbenzylalcohol and m-tolualdehyde. Ratio of the products varied significantly according to the reaction condition and host strain, presumably reflecting the relative activity of XMO and host-derived dehydrogenase(s). Using various oxidized compounds as substrates, it was indicated that dialcohol (p- or m-xylyleneglycol) was formed via p- or m-hydroxymethylbenzaldehyde, respectively, rather than directly from corresponding monoalcohol (p- or m-methybenzylalcohol).     

Determination of chloral hydrate and its metabolites in blood plasma by capillary gas chromatography with electron capture detection

A sensitive, accurate, and reliable method is described for the quantitative determination of chloral hydrate (CH) and its metabolites in blood plasma of mice and rats. Metabolites of CH include trichloroacetic acid (TCA), trichloroethanol (TCE), and trichloroethanol glucuronide (TCE-Glu). This new method uses capillary gas chromatography with electron-capture detection (GC/ECD). Procedures for improving sample stability and quality assurance are also described that were not mentioned in previous literature. Rat or mouse plasma (50 microl) is acidified (or treated enzymatically for TCE-Glu determination) and extracted with peroxide free methyl t-butyl ether. Distilled diazomethane (CH(2)N(2)) is added to derivatize TCA to its methyl ester. Detection limits were estimated at 0.2 microg/ml for CH and TCE, and 0.1 microg/ml for TCA. Detector response to TCA and TCE were shown to be linear in the range of 3.125-200 microg/ml (r> or =0.9996). For CH, the response fits a second-order equation in this same range (r=0.99994)     

Estimation of 18-hydroxycorticosterone concentration in human peripheral plasma by gas-liquid chromatography with electron capture detection

A gas-liquid chromatography method for determining the plasma concentration of plasma 18-hydroxycorticosterone based on the formation of the heptafluorobutyrate is described and evaluated. Detector contamination was avoided by using high column and detector temperatures and a high quench gas flow rate. The concentration in a group of normal subjects varied from 17.26 ± 7.66 (SD) ng 100 ml⁻¹ at 08.00 h to 11.27 ± 6.16 at 12.00 h and 5.97 ± 2.42 at 23.00 h, indicating some dependence on ACTH secretion. Coefficient of variation at a mean concentration of 10.39 ng 100ml⁻¹ was 15.7% and there was no detectable reagent blank.     

Determination of m- and p-O-methylated products of -3,4-dihydroxyphenylalanine using high-performance liquid chromatography and electrochemical detection

In a study of in vitro and in vivo metabolism of -3,4-dihydroxyphenylalanine ( _Dopa), two methods of high-performance liquid chromatography (HPLC) were used to separate the m- and p-O-methylated products. A reversed-phase column and an aqueous mobile phase by gradient elution were used; the elute was analyzed electrochemically with a single amperometric and dual coulometric electrode. The -Dopa and its O-methylated products could be detected individually in the enzymatic methylation of rat liver homogenate and in patients with Parkinson's disease. Meta/para ratios of O-methylation are easily obtained by this method.     

Work in progress: Analysis of the prostaglandins E by glass capillary gas chromatography with electron capture detection

Using a wide bore capillary column coated with D.E.G.S. in combination with a micro volume ⁶³Ni electron capture detector a simple and rapid method with high sensitivity was developed for the analysis of E prostaglandins.For electron capture detection the naturally occurring PGE's have to be transformed to the PGB configuration, esterified and silylated. This method with sensitivity in the low picogram range, tested with prostaglandin standards, will be used for the analysis of prostaglandins and prostaglandin metabolites in biological fluids.     

Conformation and quantification of chloramphenicol in cow's urine, muscle and eggs by electron capture negative ion chemical ionization gas chromatography/mass spectrometry.

A method is described for the determination of residues of the antibiotic chloramphenicol in biological samples. The method is based on gas chromatography/negative ion chemical ionization mass spectrometry and uses (37Cl2)chloramphenicol as internal standard. Selective ion monitoring of four analyte-specific ions enables the determination of chloramphenicol levels in urine of 3 micrograms l-1 with a coefficient of variation of 8%. The limit of detection of the method is 0.1 p.p.b. for urine, muscle and egg.     

Co-polymerization of MTPC (methylene tri p-cresol) and m-cresol using CiP (Coprinus cinereus peroxidase) to improve the dissolution characteristics of the enzyme-catalyzed polymer

MTPC (Methylene tri p-cresol) and m-cresol were copolymerized by Coprinus cinereus peroxidase in aqueous acetone. Although MTPC did not dissolve completely in the aqueous acetone, copolymerization was achieved owing to the radical transfer between solute and solid surface. Various polymerized products with different molecular weights and hydroxyl values were synthesized depending upon reaction compositions (ratio of MTPC to m-cresol and buffer to acetone). Poly(MTPC–m-cresol), a copolymer of MTPC and m-cresol, was mixed with a diazonaphthoquinone derivative to form a new type of photoresist, a thin film of which was formed on a silicon wafer and immersed in alkaline solution (tetramethylammonium hydroxide) to measure speed of dissolution. Poly(MTPC–m-cresol), with higher hydroxyl value (over 80%), showed remarkably improved dissolution characteristics (dark loss in alkaline solution decreased by almost half), which is prerequisite for sensitive photoresist polymer.     

Sample preparation for gas chromatography with electron capture detection: determination of total and free thyroxin in serum

Relatively clean gas chromatography with electron capture detection (GC-ECD) chromatograms are obtained for both total and free thyroxin (T4) in serum by improving sample preparation. This is based on establishing a sequence of steps that cumulatively overcome two classes of interference: those present in the initial sample and those introduced by the procedure. The main source for the latter contaminants is the derivatization step, a problem that was largely overcome by employing HPLC after this step. Also it is helpful to use ion-exchange columns early in the procedure under fast-flow conditions with intermediate flows of air to speed up and enhance their reliability. The work establishes some guidelines for future applications of GC-ECD to the determination of sub-nanogram analytes requiring derivatization, an area in which GC-ECD has been remiss in the past. As a side benefit, total T4 in serum is determined by HPLC for the first time with uv detection.     

Preparation of biological fluids for simultaneous analysis of prostaglandin cyclo-oxygenase synthesized compounds by gas chromatography with electron capture detection

A method for isolating climax products of the arachidonic acid cascade from biological fluids is described which allows simultaneous measurement of PGs (PGE2, PGD2, 6-keto-PGF1 alpha, TXB2, 6-keto-PGE1, 6, 15-diketo-13,14-dihydro-PGF1 alpha) by electron capture detection of pentafluorobenzyloxime methyl ester trimethylsilyl (TMS) ether derivatives. PGs are adsorbed onto Amberlite XAD-2 from acidified solution and nonadhering substances removed by sequential administration of water, then petroleum ether. PGs are extracted into methanol. Following evaporation and reconstitution in water, the PGs are extracted into ethyl acetate at pH 3 and the ethyl acetate extracts are purified by lipidex column chromatography. Derivatization to pentafluorobenzyloxime methyl ester TMS ethers of PGs in the sample is followed by separation on either glass packed-columns or SCOT capillary columns, and detection by an electron capture detector. PGA2, added to the unpurified sample, is used as an internal standard for quantification. The methods have performed well on all biological fluids thus far examined. Examples of chromatographs from urine, Krebs-perfused lung effluents, and blood are shown.     

Determination of total dopamine, R- and S-salsolinol in human plasma by cyclodextrin bonded-phase liquid chromatography with electrochemical detection

A reliable and sensitive high-performance liquid chromatographic (HPLC) method is presented for the determination of total (free and conjugated) plasma dopamine and the enantiomers R- and S-salsolinol. Plasma is purified on two cartridges, containing primary and secondary amines and phenylboronic acid. Dopamine, R- and S-salsolinol are then separated by HPLC using a β-cyclodextrin-OH phase column. The eluate is monitored electrochemically, without further purification nor derivatization. The method is suited for routine analysis. It allows the detection of total (free and conjugated) dopamine and R- and S-salsolinol in human plasma in concentrations as low as 0.02 ng/ml plasma. The sensitivity is sufficient to measure the naturally occurring levels of salsolinol.