Determination of bile acids by hollow fibre liquid-phase microextraction coupled with gas chromatography.

A method based on hollow-fibre liquid phase microextraction combined with gas chromatography was developed for determination of specific bile acids in caecal materials of rats. Nine unconjugated bile acids, including the primary bile acids (cholic acid, chenodeoxycholic acid and α-muricholic acid) and the secondary bile acids (lithocholic acid, deoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, ß-muricholic acid and ω-muricholic acid) were quantified. Extraction conditions were evaluated, including: sample pH, type of organic solvent and amount of caecal material to be extracted. To compensate for sample matrix effects during extraction the method of standard addition was applied. The satisfactory linearity (r(2)>0.9840), high recovery (84.2-108.7%) and good intra-assay (6.3-10.6%) and inter-assay (6.9-11.1%) precision illustrated the good performance of the present method. The method is rapid, simple and capable of detecting and determining bile acids with limit of detection (LOD) ranged from 0.002 to 0.067µg/mL and limits of quantification (LOQ) varied from 0.006 to 0.224µg/mL. The results indicated that the concentration of some secondary bile acids, which usually are associated with health problems, were lower in rats fed with fermentable dietary fibre compared with a fibre free control diet, while the concentration of primary bile acids, usually connected with positive health effects, were higher in rats fed with diets containing dietary fibre. Of the dietary fibres, guar gum and to some extent the mixture of pectin+guar gum had the most positive effects. Thus, it was concluded that the composition of bile acids can be affected by the type of diet.

Analysis and quantification of parabens in cosmetic products by utilizing hollow fibre-supported liquid membrane and high performance liquid chromatography with ultraviolet detection.

A simple and direct method based on hollow fibre-supported liquid membrane (HFSLM) extraction and liquid chromatography equipped with a UV detector was developed for analysis and quantification of parabens in cosmetic products. The parabens analysed included methyl, ethyl, propyl, isobutyl and butyl paraben. The HFSLM extraction was carried out by employing di-n-hexyl ether as organic liquid that was immobilized in the hollow fibre membrane. The HFSLM extraction is simple, cheap, minimizes the use of solvents and uses disposable material. In an investigation of 11 paraben-containing cosmetic products, the levels of parabens (sum of all parabens in a product) ranged from 0.43% to 0.79% (w/w) for skin care products, 0.07-0.44% for hair fixing gels and 0.30-0.52% for soap solutions. The levels of individual parabens in individual cosmetic products ranged between 0.03% and 0.42% w/w for skin care products, 0.07% and 0.26% w/w for hair fixing gels and between 0.11% and 0.34% w/w for soap solutions. Parabens were found in the highest concentrations in skin care products followed by soap solutions and the least amounts were found in hair fixing gels. Of the paraben-containing products tested, all of them contained methyl paraben and about 90% contained propyl paraben in addition to methyl paraben. One product contained all the parabens analysed.

Determination of ivermectin and transformation products in environmental waters using hollow fibre-supported liquid membrane extraction and liquid chromatography-mass spectrometry/mass spectrometry.

A simple and easy-to-use extraction method for aqueous samples based on hollow fibre-supported liquid membrane (HF-SLM) followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was developed to determine ivermectin and transformation products, the monosaccharide (TP1) and the aglycon of ivermectin (TP2). The proposed method attained enrichment factors up to 80, after optimising parameters, such as fibre length, organic solvent, stirring speed, salt level, pH in samples/fibre, extraction time and fibre emptying technique. Method validation with tap and lake water samples provided good linearity and detection limits of 0.2, 1.6 and 0.9 microg/l for ivermectin, TP1 and TP2 in lake water with RSD below 15%.

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.

Routine analysis of alcohol and nonylphenol polyethoxylates in wastewater and sludge using liquid chromatography-electrospray mass spectrometry.

A wide range of alcohol and nonylphenol polyethoxylates was determined by separation on a reversed-phase liquid chromatographic column, followed by electrospray ionisation-mass spectral analysis. The compounds were separated chromatographically according to their aliphatic chain length. The mass spectral analysis functioned as a second separation step during which homologues of the ethoxylates were separated according to their polyethoxylate chain length. In this manner a truly orthogonal separation was obtained. The compounds were detected as ammonium complexes. The analysis presented is capable of qualitative and quantitative determination of a large number of ethoxylates as well as their metabolites in a single run. The method was applied to many different sample types, ranging from primary and treated wastewater to sludge. Batches of 50 real samples were routinely analysed without the need for cleaning the mass spectrometer or regeneration of the column. By utilising the extracted mass chromatograms, detection limits of 1 to 10 microg/l could be obtained for individual compounds in water samples, while the detection limits were around 100 microg/kg in sludge, depending on the degree of pollution.

Supported liquid membrane extraction of peptides.

The application of supported liquid membrane (SLM) extraction for the enrichment of short peptides is presented. The extraction efficiency is dependent on the pH of donor phase and salt concentration in acceptor phase. Moreover, the extraction efficiency is also influenced by the peptide amino-acid sequence and hydrophobicity.

Determination of thiophanate-methyl and its metabolites at trace level in spiked natural water using the supported liquid membrane extraction and the microporous membrane liquid-liquid extraction techniques combined on-line with high-performance liquid chromatography.

On-line supported liquid membrane (SLM) extraction and microporous membrane liquid-liquid extraction (MMLLE) techniques for sample preparation of natural water samples have been developed for the determination of thiophanate-methyl (TM), carbendazim (MBC) and 2-aminobenzimidazole (2-AB) using reversed-phase HPLC. The combination of SLM extraction and MMLLE offers extraction conditions that makes it possible to determine a wide variety of compounds, i.e., permanently charged, ionisable and non-polar at sub ppb level. The detection limits obtained after extraction are about 0.1 microg/l for MBC and 2-AB using SLM, and 0.5 x Lg/l for TM using MMLLE and the precision is better than 5% for both systems. Typical enrichment rates are 0.6 and 2.7 times/min using SLM and MMLLE, respectively.

Automated liquid membrane extraction for high-performance liquid chromatography of Ropivacaine metabolites in urine.

An automatic method for the determination of metabolites of Ropivacaine in urine was set up. It utilizes supported liquid membrane extraction for sample clean-up and enrichment, followed by ion-pair chromatography determination using UV detection. The extraction was very selective with no observed interfering compounds from the urine matrix, permitting simple isocratic chromatographic analysis. The detection limits for spiked urine samples were 2-18 nM for the different compounds. The repeatability was 1-3% (RSD) with an internal standard that was also extracted, and about twice without this standard. A throughput of 3.3 samples per hour was achieved and the liquid membrane was stable for more than a week.

Microporous membrane liquid-liquid extraction coupled on-line with normal-phase liquid chromatography for the determination of cationic surfactants in river and waste water.

Membrane-based continuous liquid-liquid extraction combined on-line with normal-phase liquid chromatography is proposed for the determination of cationic surfactants in complex aqueous samples. The technique has the potential for complete automation. Selective enrichment of cationic surfactants from spiked river water and waste-water samples with simultaneous removal of matrix constituents, followed by a quantitative transfer of the extract onto a liquid chromatographic column and separation of the surfactant homologues yielding low detection limits, has been realised. The homologues of alkyldimethylbenzylammonium chloride (Dodigen 226) were chosen as model compounds in the method development. Dodigen homologues were ion-paired with heptanoic acid and extracted into chlorobutane by means of microporous membrane liquid-liquid extraction. It was thereby possible to attain an enrichment of over 250 times for one of the homologues, viz. the concentration in the organic liquid is 250 times higher than in the original sample. Detection limits for the three best-detected homologues of the mixture were in the range 0.7-5 microg/l in spiked river water samples. Ion-pair normal-phase liquid chromatography, again with heptanoic acid as counter-ion, gave the necessary separation of the surfactant homologues.

Membrane-based techniques for sample enrichment.

Sample preparation techniques based on non-porous membrane extraction generally offer a high degree of selectivity and enrichment power, together with convenient possibilities for direct and automated connections to chromatographic and other analytical instruments. In this review principles and applications for techniques as supported liquid membrane extraction, microporous membrane liquid-liquid extraction, polymeric membrane extraction and membrane extraction with a sorbent interface are described and compared.

Alpha1-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound.

Alpha1-microglobulin (alpha1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three-dimensional (3D) structure that forms an internal hydrophobic ligand-binding pocket. Alpha1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alpha1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of alpha1m. A 3D model of alpha1m, based on the structure of rat epididymal retinoic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of alpha1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha1m is complex bound to IgA. Only the free alpha1m carried colored groups, whereas alpha1m linked to IgA was uncolored.

On-line microporous membrane liquid-liquid extraction for sample pretreatment combined with capillary gas chromatography applied to local anaesthetics in blood plasma.

A new automated procedure for analyzing complex samples has been developed utilizing microporous membrane liquid-liquid extraction (MMLLE) combined with capillary gas chromatography. Some local anaesthetics were used as model compounds in aqueous solution as well as in blood plasma. The MMLLE procedure was performed in a flow system with the sample fed to the donor side of the hydrophobic microporous membrane and with an organic solvent (hexane) in the pores and as the acceptor solution. The analytes in a small volume of sample (< 1 mL) were extracted into the organic acceptor phase which was transferred into the gas chromatographic system by utilizing a loop-type interface compatible with large-volume (300 microL) injection. High selectivity and low carry-over effects were obtained with the system. The detection limits were 0.5-1 ng/mL using 0.5 mL of human plasma, and the precision was approximately 5%. The effects of pH, flow rates, and adsorption of the analytes were evaluated.

On-line supported liquid membrane-liquid chromatography with a phenol oxidase-based biosensor as a selective detection unit for the determination of phenols in blood plasma.

The potential of on-line combination of supported liquid membrane extraction and column liquid chromatography with a phenol oxidase-based biosensor as a selective detection unit has been investigated for the determination of phenols in human plasma. The phenols are selectively extracted into a porous PTFE (polytetraflouroethene) membrane impregnated with a water-immiscible organic solvent and further into an alkaline acceptor phase. Via an ion-exchange interface, the analytes are transferred to a reversed-phase column where they are separated and detected using the biosensor. No sample pretreatment before the extraction, except centrifugation, is made. Due to the high selectivity both in the extraction and in the detection steps and to the fact that the demands on the chromatographic separation are low, a quick separation using an eluent with a low concentration of organic modifier can be made, without affecting the biosensor response. Detection limits below the 50 microg/l level in blood plasma were obtained for the three model compounds, phenol, p-cresol and 4-chlorophenol.

Miniaturized supported liquid membrane device for selective on-line enrichment of basic drugs in plasma combined with capillary zone electrophoresis.

A hollow fiber miniaturized supported liquid membrane (SLM) device for sample preparation is connected on-line with capillary electrophoresis and used for determination of a basic drug, bambuterol, in human plasma. The analyte is extracted from the outside of the hollow fiber (donor) through the liquid membrane (pores of the fiber impregnated with organic solvent) into the acceptor solution in the fiber lumen. The process is driven by differences in pH between the donor and acceptor solution. The whole volume of the acceptor solution can then be injected into the CZE capillary by using the double-stacking procedure for large volume-injection. Very clean extracts of low ionic strength are obtained from the SLM treatment, making this sample pretreatment method compatible with the CZE double-stacking procedure, which in turn makes it possible to inject large volumes of sample onto the separation capillary. Good performance of the whole procedure is demonstrated, and detection limits in the low nanomolar range were obtained in spite of the relatively weak UV absorbance of bambuterol. Extractions through the miniaturized SLM unit can be performed for 5-6 h without regenerating the fiber. The regeneration procedure was tested, and no relevant changes in the performance of the extraction could be found after seven regenerations, allowing the same fiber to be used for a week.

Determination of a basic drug, bambuterol, in human plasma by capillary electrophoresis using double stacking for large volume injection and supported liquid membranes for sample pretreatment.

In this work we show the potential of using a double stacking procedure based on field enhancement as a means to increase the concentration sensitivity in CZE analysis of human plasma extracted by the supported liquid membrane (SLM) technique. A basic drug, bambuterol, was used as a model substance. The low ionic strength of the SLM extract makes this pretreatment technique compatible with the double stacking sequence. No significant loss of separation performance was observed when 3 microliters of SLM extract was concentrated by the CZE double stacking sequence. Almost no visible difference was seen between the electropherograms after enrichment of a plasma blank and an aqueous blank. Good performance of the whole procedure was demonstrated and detection limits in the low nM range were obtained in spite of the relatively weak UV absorbance of bambuterol. The developed procedure was evaluated for both achiral and chiral separation. In the latter approach chiral selectivity was obtained by adding cyclodextrin to the separation electrolyte.

Determination of Phenmetrazine in urine by gas chromatography-mass spectrometry.

A sensitive and simple gas chromatographic--mass spectrometric method is described for the determination of the central nervous system (CNS) stimulant phenmetrazine in urine. The extraction and derivatization were combined into a single step with isooctane and methyl chloroformate. The limit of quantitation was 0.05 micrograms/mliters urine, and the method was linear up to 100 micrograms/mliters. The coefficients of variation (CV) for within-day runs were 1.2% and 2.4% (n = 5) for two controls containing 1.0 micrograms/mliters and 50 micrograms/mliters, respectively. During a six-month period, the same controls showed CVs of 9.1% and 8.7%, respectively (n = 40), indicating a somewhat lower between-run precision. Phenmetrazine was present in 83 out of 3000 urine samples that were screened for CNS stimulants during this period, and the concentrations ranged from 0.5-370 micrograms/mliters.

Micro-CLC as an interface between SLM extraction and CZE for enhancement of sensitivity and selectivity in bioanalysis of drugs.

This work demonstrates the high selectivity and sensitivity obtainable in bioanalysis using the supported liquid membrane (SLM) technique coupled on line with capillary zone electrophoresis (CZE) through a micro-column liquid chromatography (CLC) interface. The system utilizes two selective, sequential enrichment steps before the third analyte focusing and separation step with double-stacking CZE. The enantiomers of bambuterol in human plasma can be concentrated about 40,000 times (approx. 6 times by the SLM treatment, approx. 17 times by micro-CLC focusing, and approx. 400 times by double-stacking CZE) on their way through the system, and extremely high selectivity is obtained. Determinations in the subnanomolar region are achievable for the enantiomers despite relatively weak UV absorbance. Good performance of the entire procedure is demonstrated and a method to increase the sample throughput is presented.

Sample preparation using a miniaturized supported liquid membrane device connected on-line to packed capillary liquid chromatography.

A miniaturized supported liquid membrane device has been developed for sample preparation and connected on-line to a packed capillary liquid chromatograph. The device consists of hydrophobic polypropylene hollow fiber, inserted and fastened in a cylindrical channel in a Kel-F piece. The pores of the fiber are filled with an organic solvent, in this study 6-undecanone, thus forming a liquid membrane. The sample is pumped on the outside of the hollow fiber (donor), and the analytes are selectively enriched and trapped in the fiber lumen (acceptor). With this approach, the volume of the acceptor solution can be kept as low as 1-2 µL. This stagnant acceptor solution is then transferred through capillaries attached to the fiber ends to the LC system. The system was tested with a secondary amine (bambuterol), as a model substance in aqueous standard solutions as well as in plasma. The best extraction efficiency in aqueous solution, with an acceptor volume of 1.9 µL, was 32.5% at a donor flow rate of 2.5 µL/min. At flow rates above 20 µL/min, the concentration enrichment per time unit was approximately constant, at 0.9 times/min, i.e., 9 times enrichment in about 10 min. The overall repeatability (RSD) for spiked plasma samples was ∼4% (n = 12). Linear calibration curves of peak area versus bambuterol concentration were obtained for both aqueous standard solutions and spiked plasma samples. The detection limit for bambuterol in plasma, after 10 min of extraction at a flow rate of 24 µL/min, was 80 nM.

Supported liquid membrane technique for selective sample workup of basic drugs in plasma prior to capillary zone electrophoresis.

The potential of using the supported liquid membrane (SLM) technique for pretreatment of plasma samples before analysis with capillary zone electrophoresis (CZE) has been investigated. A basic drug, bambuterol, was used as a model substance in a system, where either 6-undecanone or a mixture of di-n-hexyl ether (DHE) and tri-n-octyl phosphine oxide (TOPO) was used as membrane liquids. It was found that the electropherograms obtained after SLM enrichment of bambuterol in plasma samples were as clean as when aqueous samples containing the same substance were processed in the same way. The low ionic strength of the SLM treated blood plasma samples permitted subsequent sample stacking in the CZE step. The linearity of the detector signal for different concentrations of bambuterol in plasma was satisfactory from 50 to 1000 nmol/L with regression co-efficients of 0.997 using 6-undecanone as membrane liquid and 0.999 with the other liquid. In both systems, the confidence interval of the intercept included the origin. The detection limit was about 50 nmol/L. The long-term stability of the two membrane liquids proved adequate as a membrane lasted at least through the working day.