Evaluation of cartilage and bone degradation in a murine collagen antibody-induced arthritis model.

The purpose of this work was to validate collagen antibody-induced arthritis (CAIA) model in two mice strains (Balb/c and CD-1) using clinical, biochemical, microstructural and histological techniques. We induced arthritis in mice using a cocktail of collagen type II (CII) antibodies followed by an injection with lipopolysaccharide (LPS) in different doses in Balb/c and CD-1 mice strains. Serum CTX-II levels were measured at study termination and correlated with microscopic severity of joint lesions as determined by a validated scoring systems. Bone involvement was assessed by microcomputer tomography (micro-CT). Balb/c mice developed rapid (day 6) and robust (100%) arthritis, whereas CD-1 mice showed only temporary macroscopic signs of disease. Serum CTX-II levels in Balb/c mice showed a significant increase in cartilage degradation in diseased animals (43-64% compared with non-diseased mice) and was decreased in animals receiving dexamethasone. Correlation of serum CTX-II with the microscopic score was statistically significant (P < 0.01). Micro-CT analysis demonstrated structural damage in bone in the CAIA Balb/c mice, which was prevented by dexamethasone. The CAIA-LPS model provides a useful supplement to currently available animal models of arthritis. This is a rapid onset and robust model; however, the choice of mouse strain should be evaluated carefully.

Selective three-phase liquid phase microextraction of acidic compounds from foodstuff simulants.

A three-phase liquid phase microextraction (LPME) technique with high selectivity for five aromatic carboxylic acids and three phenolic compounds has been developed and optimized. The system consists of an acidified donor phase, a thin layer of solvent inside the wall pores of a hollow fiber, and an alkaline acceptor phase located inside the hollow fiber. The analysis of the compounds in the acceptor phase was carried out by capillary electrophoresis with UV detection. Eugenol, thymol, and carvacrol were efficiently extracted from the aqueous solution using chloropentane as organic phase, with recoveries from 73.8% to 93.8%. However, using 2-octanone as the organic phase, the recoveries for the aromatic carboxylic acid compounds ranged from 60.7% to 93.7% whereas the phenols were not extracted. 2,6-naphthalene-dicarboxylic acid was found to remain in the organic phase. The influence of 10% ethanol and 3% acetic acid in the donor phase was deeply studied as these solutions are commonly used as food simulants. AS4 silicone oil was found to be the best organic phase for the extraction of phenols both in 3% acetic acid and matrices with a high content of alcohol. The results obtained are shown and discussed.

Liquid-liquid extraction procedures for sample enrichment in capillary zone electrophoresis.

This review article presents an overview of applications of liquid-liquid extraction (LLE) for analyte enrichment and clean-up of samples prior to capillary zone electrophoresis (CZE). The basic principles of LLE are discussed with special emphasis on analyte enrichment. In addition, attention is focused on the requirements for the final extract to be compatible with CZE. The paper discusses selected examples from the literature with special emphasis on detection limits in drug analysis and in environmental chemistry. Finally, the paper focus on alternative liquid-phase extraction concepts based on electroextraction, supported liquid membranes, and liquid-phase microextraction.

Microemulsion electrokinetic chromatography in suppressed electroosmotic flow environment. Separation of fat-soluble vitamins.

Microemulsion electrokinetic chromatography (MEEKC) was carried out in a pH 2.5 phosphate buffer to effectively suppress the electroosmotic flow (EOF). With 66.6% (w/w) 25 mM phosphate buffer pH 2.5, 20.0% (w/w) 2-propanol, 6.6% (w/w) 1-butanol, 6.0% (w/w) sodium lauryl sulphate (SDS), and 0.8% (w/w) n-octane as the separation medium, the fat-soluble vitamins A palmitate, E acetate, and D3 were baseline separated within 11 min. With strongly suppressed EOF, the polarity of the separation voltage was reversed (positive electrode at the outlet); the n-octane micro droplets surrounded by negatively charged SDS molecules migrated towards the detector. The aqueous part of the microemulsion was modified with 20% (w/w) 2-propanol to improve partition between the n-octane phase and the surrounding aqueous medium. The fat-soluble vitamins were separated in order of decreasing hydrophobicity with a high migration time stability (repeatable within 0.1% RSD). Excellent accuracy and precision were obtained when the system was applied for the determination of vitamin E acetate in commercial vitamin tablets; quantitative data corresponded to 97.0% of label claim, intra-day results varied within 1.72% RSD (n=6), and inter-day results varied within 3.22% RSD (n=5).

Liquid-phase microextraction and capillary electrophoresis of citalopram, an antidepressant drug.

A newly developed disposable device for liquid-phase microextraction (LPME) was evaluated for the capillary electrophoresis (CE) of the antidepressant drug citalopram (CIT) and its main metabolite N-desmethylcitalopram (DCIT) in human plasma. CIT and DCIT were extracted from 1 ml plasma samples through hexyl ether immobilised in the pores of a porous polypropylene hollow fibre and into 25 microl of 20 mM phosphate buffer (pH 2.75) present inside the hollow fibre (acceptor phase). Prior to extraction, the samples were made strongly alkaline in order to promote LPME of the basic drugs. Owing to the high ratio between the volumes of sample and acceptor phase, and owing to high partition coefficients, CIT and DCIT were enriched by a factor of 25 to 30. In addition, sample clean-up occurred during LPME since salts, proteins and the majority of endogenic substances were unable to penetrate the hexyl ether layer. Since the extracts were aqueous, they were injected directly into the CE instrument. Limits of quantification (S/N= 10) for CIT and DCIT in plasma were 16.5 ng/ml and 18 ng/ml respectively, while the limits of detection (S/N=3) were 5 ng/ml and 5.5 ng/ml respectively. This enabled CIT (and DCIT) to be analysed within the therapeutic range by LPME-CE and detection limits were comparable with previously reported HPLC methods.

Development of a simple in-vial liquid-phase microextraction device for drug analysis compatible with capillary gas chromatography, capillary electrophoresis and high-performance liquid chromatography.

A simple, inexpensive and disposable device for liquid-phase microextraction (LPME) is presented for use in combination with capillary gas chromatography (GC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). 1-4 ml samples of human urine or plasma were filled into conventional 4-ml vials, whereafter 15-25 microl of the extraction medium (acceptor solution) was filled into a short piece of a porous hollow fiber and placed into the sample vial. The drugs of interest were extracted from the sample solutions and into the small volumes of acceptor solution based on high partition coefficients and were preconcentrated by a factor of 30-125. For LPME in combination with GC, the porous hollow fiber was filled with 15 microl n-octanol as the acceptor solution. Following 30 min of extraction, the organic acceptor solution was injected directly into the GC system. For LPME in combination with CE and HPLC, n-octanol was immobilized within the pores of the hollow fiber, while the internal volume of the fiber was filled with either 25 microl of 0.1 M HCl (for extraction of basic compounds) or 25 microl 0.02 M NaOH (for acidic compounds). Following 45 min extraction, the aqueous acceptor solution was injected directly into the CE or HPLC system. Owing to the low cost, the extraction devices were disposed after a single extraction which eliminated the possibility of carry over effects. In addition, because no expensive instrumentation was required for LPME, 10-30 samples were extracted in parallel to provide a high number of samples per unit time capacity.

On-line dialysis, liquid chromatography and post-column reaction detection of oxytetracycline in salmon muscle extracts.

The development of a sensitive automated method for residue control of oxytetracycline (OTC) in salmon muscle is described. Tissue homogenate is dialysed and the dialysate enriched on a small on-line polystyrene column. OTC and the internal standard (tetracycline) are separated by HPLC on a polystyrene column using an ion-pair eluent system. The column effluent is mixed with sodium hydroxide and irradiated at 366 nm and the resulting derivatives monitored by means of a fluorescence detector (excitation: 358 nm, emission: 460 nm). By the method OTC is detected down to 5 ng g-1. The standard curve was linear (r = 0.9999) over the range 50-1000 ng g-1. Within-day and between-day relative standard deviations (n = 6) at 50 and 200 ng g-1 ranged from 1.0 to 1.7%.

Automated on-line dialysis for sample preparation and HPLC analysis of antidepressant drugs in human plasma. Inhibition of interaction with the dialysis membrane.

Antidepressant drugs interact with the dialysis membrane and were selected as model substances to study inhibition of analyte-membrane interactions. A chemometric approach based on response surface modelling was used for screening and optimisation of dialysis recoveries. Optimal dialysis recoveries (52-65%) were obtained for the model compounds (mianserine, amitriptyline, nortriptyline, imipramine and desimipramine) when a cationic surfactant added to the donor solution of the dialyser was used to inhibit analyte-membrane interactions. Automated analysis of antidepressants in plasma was performed by connecting the ASTED (Automated Sequential Trace Enrichment of Dialysates) system to high-performance liquid chromatography (HPLC). The drugs were detected by ultraviolet detection and fluorescence detection after post-column photochemical reaction. Validation of the method showed linear standard curves for all the drugs in the concentration range 50-2000 nmol 1-1. Within-and between day relative standard deviations ranged from 1.1 to 5.7%.

Automated determination of 'Ecstasy' and amphetamines in urine by SPME and capillary gas chromatography after propylchloroformate derivatisation.

The determination of amphetamines and their methylenedioxylated analogs in urine by propylchloroformate derivatisation and automated solid-phase microextraction is described. The urine sample was adjusted to pH 10.8 and added propylchloroformate reagent and an internal standard. Derivatisation resulted in water-stable carbamates which were automatically extracted by solid-phase microextraction. A fiber coated with polydimethylsiloxane was inserted into the urine matrix and agitated for 16 min. The fibre with the extracted carbamates was injected into the heated split-splitless injection port of the gas chromatograph where the analytes were evaporated at 300 degrees C, separated on a methylsilicone capillary column and detected by either a nitrogen phosphorous detector or by mass spectrometry. The method was shown to be highly reproducible and robust with respect to variations in the urine matrices. The detection limits were 5 ng/ml(-1) of methamphetamine, MDMA and MDEA and 15 ng/ml(-1) of amphetamine and MDA in urine. The method is a solvent free, automated alternative to traditional methods for determination of the amphetamine and their methylendioxylated analogs in urine.

Determination of benzodiazepines in human urine and plasma with solvent modified solid phase micro extraction and gas chromatography; rationalisation of method development using experimental design strategies.

Solid phase micro extraction (SPME) and gas chromatographic analysis was used for the analysis of several benzodiazepines (oxazepam, diazepam, nordiazepam, flunitrazepam and alprazolam) in human urine and plasma. Several factors likely to affect the analyte recovery were screened in a fractional factorial design in order to examine their effect on the extraction recovery. Parameters found significant in the screening were further investigated with the use of response surface methodology. The final conditions for extraction of benzodiazepines were as follows: Octanol was immobilised on a polyacrylate fibre for 4 min. The fibre was placed in the sample and extraction took place at pH 6.0 for 15 min. Urine samples were added to 0.3 g ml(-1) sodium chloride. In plasma, the extraction recovery was less than in urine and releasing the benzodiazepines from plasma proteins followed by protein precipitation was found necessary prior to sampling. The method was validated and found linear over the range of samples. The limits of detection in urine were determined to be in the range 0.01-0.45 micromol l(-1). The corresponding limits of detection in plasma were in the range 0.01-0.48 micromol l(-1). Finally, the method developed was applied to determine some benzodiazepines after administration of a single dose. This method offers sufficient enrichment for bioanalysis after a single dose of high dose benzodiazepines as diazepam, but for low dose benzodiazepines as flunitrazepam, further sensitivity is needed.

An all-purpose injection system for gas chromatographic analyses of biological samples.

A versatile injection system for the direct introduction of samples into the gas chromatograph (GC) is described. Several years of experience in biological and pharmaceutical chemistry have led to the construction and final modification of this device. No modification of the injection port of the GC is necessary.

Automated analysis of oxolinic acid and flumequine in salmon whole blood and plasma using dialysis combined with trace enrichment as on-line sample preparation for high-performance liquid chromatography.

The use of dialysis as sample clean-up for high-performance liquid chromatography makes fully automated determination of drugs in whole blood and plasma possible. High recoveries of the analytes oxolinic acid and flumequine and the internal standard nalidixic acid are obtained after a short time of dialysis (7.3 min). The dilute dialysates are enriched on a small column packed with polystyrene. When dialysis is discontinued, the analytes are eluted by mobile phase to the analytical column. With UV detection the limit of detection was 50 ng/ml for both oxolinic acid and flumequine. Validation showed good precision and accuracy and good correlation between determinations in plasma and whole blood.

Fully automated high-performance liquid chromatographic analysis of whole blood and plasma samples using on-line dialysis as sample preparation. Determination of oxytetracycline in bovine and salmon whole blood and plasma.

A fully automated technique for high-performance liquid chromatographic analysis of whole blood and plasma is described. Samples are automatically injected into a dialyser where proteins and blood cells are removed. The dialysates are concentrated on a small column prior to analysis. This technique is used for the determination of oxytetracycline in whole blood and plasma. After dialysis oxytetracycline and the internal standard, tetracycline, are retained on a polystyrene enrichment column and subsequently separated on a polystyrene analytical column by ion-pair chromatography. Using ultraviolet detection 50 ng/ml can be detected. Validation showed good within-day and between-day accuracy and precision. Different oxytetracycline concentrations were found in plasma and whole blood. This difference varied between the species.

Screening of hallucinogenic mushrooms with high-performance liquid chromatography and multiple detection.

A rapid, sensitive and specific method for the screening of hallucinogenic mushrooms has been developed. High-performance liquid chromatography with simultaneous use of ultraviolet, fluorescence and electrochemical detection was employed. Separation of the mushroom components was achieved on a silica column using an alkaline aqueous methanolic eluent. The use of detector response ratios for identification of hallucinogenic indole alkaloids has been evaluated.