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.

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.

Reduction of extraction times in liquid-phase microextraction.

Recently, we introduced a simple and inexpensive disposable device for liquid-phase microextraction (LPME) based on porous polypropylene hollow fibres. In the present paper, extraction times were significantly reduced by an increase in the surface of the hollow fibres. The model compounds methamphetamine and citalopram, were extracted from 2.5 ml of urine, plasma, and whole blood after dilution with water and alkalisation with 125 microl of 2 M NaOH though a porous polypropylene hollow fibre impregnated with hexyl ether and into an aqueous acceptor phase consisting of 0.1 M HCl. Two commercially available hollow fibres, which differed in surface area, wall thickness and internal diameter, were compared. An increase in the contact area of the hollow fibre with the sample solution by a factor of approximately two resulted in reduction in equilibrium times by approximately the same factor. Thus, the model compounds were extracted to equilibrium within 15 min from both urine and plasma, and within 30 min from whole blood. For the first time LPME was utilised to extract drugs from whole blood, and the extracts were comparable with plasma both with regard to sample clean-up and extraction recoveries. Extraction recoveries for methamphetamine and citalopram varied from 60 to 100% using the two fibres and the different matrices.

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.

Liquid-phase microextraction as a sample preparation technique prior to capillary gas chromatographic-determination of benzodiazepines in biological matrices.

Liquid-phase microextraction (LPME) and gas chromatography were applied to determine diazepam and the main metabolite N-desmethyldiazepam in human urine and plasma. The analytes were extracted from 3.0-3.5 ml sample volumes directly into 25 microl of extraction solvent. The microextraction device consisted of a porous hollow fiber of polypropylene attached to two guiding needles inserted through a septum and a 4 ml vial. The hollow fiber filled with extraction solvent was immersed in sample solution. The extraction device was continuously vibrated at 600 rpm for 50 min. An aliquot (1 microl) of the extraction solvent with preconcentrated analytes was injected directly into the capillary gas chromatograph. Thirty samples were extracted simultaneously on the vibrator, providing a high sample capacity. The limits of detection were from 0.020 to 0.115 nmol/ml for diazepam and N-desmethyldiazepam in plasma and urine using a nitrogen-phosphorus detector (NPD).

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).

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.

Liquid-phase microextraction and capillary electrophoresis of acidic drugs.

Vial liquid-phase microextraction (LPME) combined with capillary electrophoresis (CE) was evaluated for the determination of the acidic drugs ibuprofen, naproxen, and ketoprofen present in water samples and in human urine. The 2.5 mL samples containing the drugs were filled into conventional vials and subsequently acidified by 250 microL of 1-10 M HCl. Porous hollow fibers of polypropylene containing 25 microL of an aqueous solution of 0.01-0.1 M NaOH (acceptor solution) and with dihexyl ether immobilized in the pores of the wall were placed into each of the samples. The acidic drugs were extracted from the acidified sample solutions into the dihexyl ether phase, in the pores of the hollow fiber, and further into the alkaline acceptor solution forced by high partition coefficients. The drugs were extracted almost quantitatively (75-100% extraction efficiency) from the 2.5 mL samples and into the 25 microL acceptor solutions, providing 75-100 times preconcentration. The acceptor solutions were collected for automated CE analysis, which enabled the drugs to be detected down to the 1 ng/mL level.

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.

Liquid-liquid-liquid microextraction for sample preparation of biological fluids prior to capillary electrophoresis.

Methamphetamine as a model compound was extracted from 2.5-mL aqueous samples adjusted to pH 13 (donor solution) through a thin phase of 1-octanol inside the pores of a polypropylene hollow fiber and finally into a 25-microL acidic acceptor solution inside the hollow fiber. Following this liquid-liquid-liquid microextraction (LLLME), the acceptor solutions were analyzed by capillary zone electrophoresis (CE). Extractions were performed in simple disposable devices each consisting of a conventional 4-mL sample vial, two needles for introduction and collection of the acceptor solution, and a 8-cm piece of a porous polypropylene hollow fiber. From 5 to 20 different samples were extracted in parallel for 45 min, providing a high sample capacity. Methamphetamine was preconcentrated by a factor of 75 from aqueous standard solutions, human urine, and human plasma utilizing 10(-1) M HCl as the acceptor phase and 10(-1) M NaOH in the donor solution. In addition to preconcentration, LLLME also served as a technique for sample cleanup since large molecules, acidic compounds, and neutral components were not extracted into the acceptor phase. Utilizing diphenhydramine hydrochloride as internal standard, repetitive extractions varied less than 5.2% RSD (n = 6), while the calibration curve for methamphetamine was linear within the range 20 ng/microL to 10 micrograms/mL (r = 0.9983). The detection limit of methamphetamine utilizing LLLME/CE was 5 ng/mL (S/N = 3) in both human urine and plasma.

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.

Analysis of vitamin formulations by electrokinetic chromatography utilizing tetradecylammonium ions as the pseudostationary phase.

A recently proposed method for the separation of fat-soluble vitamins by electrokinetic chromatography was further developed and investigated in the present study. The separation medium consisted of acetonitrile-water (80:20 v/v) and contained 80 mM tetradecylammonium bromide (TDA+); the content of acetonitrile served to maintain the hydrophobic vitamins dissolved during electrophoresis, while the TDA+ ions served as the pseudostationary phase. With the cathode placed at the outlet of the capillary, the fat-soluble vitamins were separated based on different hydrophobic interactions to the TDA+ ions and migrated in order of decreasing hydrophobicity prior to the electroosmotic flow. Migration time stability was significantly enhanced by the addition of 4 mM borate to the separation medium. The separation system was validated for the determination of vitamin E acetate in commercial tablets; quantitative results deviated by less than 3.5% from specified values, varying by less than 2.5% relative standard deviation (RSD) for within-day experiments, and by less than 6.5% RSD during between-day experiments. The separation system was compatible with injection solvents ranging in polarity from water to tetrahydrofuran, and was even capable of separating the water-soluble vitamins B1, B2, B12, and nicotinamide.

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%.

Strategies for the capillary electrophoretic separation of indole alkaloids in Psilocybe semilanceata.

While the hallucinogenic mushrooms Psilocybe semilanceata have previously been analyzed for the indole alkaloids psilocybin and baeocystin by capillary zone electrophoresis (CZE) at pH 11.5, the present work focused on the development of an alternative and complementary capillary electrophoretic method for their identification. Owing to their structural similarity and zwitterionic nature, the compounds were difficult to resolve based on different interactions with cationic or anionic micelles. However, while the attempts with micellar electrokinetic chromatography (MEKC) were unsuccessful, rapid derivatization with propyl chloroformate and reanalysis by CZE at pH 11.5 was effective to support identification of the two indole alkaloids. Psilocin was difficult to analyze by CZE at pH 11.5 owing to comigration with the electroosmotic flow. For this compound, the pH of the running buffer was reduced to 7.2 to effectively enhance the electrophoretic mobility.

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.

Aqueous alkylchloroformate derivatisation and solid-phase microextraction: determination of amphetamines in urine by capillary gas chromatography.

The alkylchloroformate derivatisation and solid-phase microextraction of amphetamine and methamphetamine directly in urine samples prior to capillary gas chromatographic analysis is described. The alkylchloroformate reagent was added to the urine sample, which was adjusted to pH 10.8, and an internal standard was added. The resulting products were water-stable carbamates that were extracted without organic solvent. The polydimethylsiloxane coated fibre was inserted into the modified sample and agitated for 14 min. The fibre with the extracted derivatisation products was injected into the capillary gas chromatograph. The extracted carbamates were evaporated at 300 degrees C in the split-splitless injection port of the gas chromatograph, separated on a methylsilicone capillary column and detected by either a nitrogen-phosphorus detector or by mass spectrometry. The method was shown to be reproducible with a detection limit of 50 ng/ml of amphetamine and methamphetamine in urine.

Determination of psilocybin in Psilocybe semilanceata by capillary zone electrophoresis.

A capillary zone electrophoretic (CZE) method was developed for the rapid determination of psilocybin in Psilocybe semilanceata. Following a simple two step extraction with 3.0+2.0 ml methanol, the hallucinogenic compound was effectively separated from matrix components by CZE utilizing a 10 mM borate-phosphate running buffer adjusted to pH 11.5. The identity of psilocybin was confirmed by migration time information and by UV spectra, while quantitation was accomplished utilizing barbital as internal standard. The calibration curve for psilocybin was linear within 0.01-1 mg/ml, while intra-day and inter-day variations of quantitative data were 0.5 and 2.5% R.S.D., respectively. In addition to psilocybin, the method was also suitable for the determination of the structurally related compound baeocystin.

Automated on-line dialysis, trace enrichment and high-performance liquid chromatography. Inhibition of interaction with the dialysis membrane and disruption of protein binding in the determination of clozapine in human plasma.

Problems related to interaction of drugs with the dialysis membrane and to protein binding must be overcome in order to develop automated methods for drug analysis based on on-line dialysis, trace enrichment and HPLC. In order to study these problems, clozapine and its active metabolite N-desmethylclozapine were chosen as model compounds because they were found to interact with the dialysis membrane, and clozapine is highly protein bound. Addition of a cationic surfactant, dodecylethyldimethyl ammonium bromide, to the donor solution and to the plasma samples was found to inhibit interaction of the drugs with surfaces. The protein binding in plasma was disrupted prior to dialysis by lowering the pH with hydrochloric acid and the plasma proteins were solubilised with glycerol. The results obtained were used to develop a fully automated method for the determination of clozapine and N-desmethylclozapine in human plasma. More than 100 samples could be analysed within 24 h. The limit of detection in human plasma was 0.050 mumol/l for clozapine and 0.055 mumol/l for N-desmethylclozapine. Linearity was found for drug concentrations between 0.25-3 mumol/l. The relative standard deviations were between 1.2-6.7% and the method was applicable for therapeutic drug monitoring.

Solvent-modified solid-phase microextraction for the determination of diazepam in human plasma samples by capillary gas chromatography.

This paper describes microextraction and gas chromatographic analysis of diazepam from human plasma. The method was based on immobilisation of 1.5 microliters of 1-octanol on a polyacrylate-coated fiber designed for solid-phase microextraction. The solvent-modified fibre was used to extract diazepam from the samples. The plasma sample was pre-treated to release diazepam from the protein binding. The fibre was inserted into the modified plasma sample, adjusted to pH 5.5 an internal standard was added and the mixture was carefully stirred for 4 min. The fibre with the immobilised solvent and the enriched analytes was injected into the capillary gas chromatograph. The solvent and the extracted analytes were evaporated at 300 degrees C in the split-splitless injection port of the gas chromatograph, separated on a methylsilicon capillary column and detected with a nitrogen-phosphorus detector. The method was shown to be reproducible with a detection limit of 0.10 nmol/ml in human plasma.

Solid-phase microextraction for the determination of the free concentration of valproic acid in human plasma by capillary gas chromatography.

The potential of solid-phase microextraction in the bioanalysis of drugs is demonstrated. The free concentration of valproic acid in human plasma was determined by equilibrium dialysis at room temperature. To the dialysate was added an internal standard and the pH was adjusted to 2.5. The polymethylsiloxane-coated fused-silica fibre of the solid-phase microextraction device was inserted into the dialysate for 3 min. The sorbed analytes were then thermally desorbed at 210 degrees C in the split-splitless injection port of the gas chromatograph, separated on a Nukol capillary column and detected with a flame ionization detector. The method was shown to be highly reproducible with a detection limit of 1 microgram/ml of free valproic acid in human plasma.