Pre-column switching techniques for the determination of drugs and metabolites in body fluids in research and routine analysis

The use of a column switching system for direct injection of samples and of a sample clean-up on reversed phase pre-columns is described. The pre-columns were filled with spherical C-18 silica gel of particle size 30 microns. Two applications are reported on: (1) the direct injection of serum samples for the simultaneous analysis of nine antiepileptic drugs and metabolites and (2) the determination of phenytoin and of carbamazepine in serum ultra-filtrates. The purge liquid for the sample clean-up was diluted phosphoric acid, and the eluent mixture for the chromatographic separation was water/acetonitrile. The analytical column (length 12.5 cm) was filled with C-18 silica gel of particle size 5 micron. A gradient elution was chosen for the first application, while the second application was carried out using isocratic chromatographic conditions.     

Pre-Column Switching Techniques for the Determination of Drugs and Metabolites in Body Fluids in Research and Routine Analysis

Abstract

The use of a column switching system for direct injection of samples and of a sample clean-up on reversed phase pre-columns is described. The pre-columns were filled with spherical C-18 silica gel of particle size 30 μm.

Two applications are reported on: (1) the direct injection of serum samples for the simultaneous analysis of nine antiepileptic drugs and metabolites and (2) the determination of phenytoin and of carbamazepine in serum ultra-filtrates.

The purge liquid for the sample clean-up was diluted phosphoric acid, and the eluent mixture for the chromatographic separation was water/acetonitrile. The analytical column (length 12.5 cm) was filled with C-18 silica gel of particle size 5 μm. A gradient elution was chosen for the first application, while the second application was carried out using isocratic chromatographic conditions.     

Evaluation of an automated clean-up system for the isotope-dilution high-resolution mass spectrometric analysis of PCB, PCDD, and PCDF in food

An automated clean-up system was evaluated for the simultaneous analysis of polychiorinated dibenzo-p-dioxins (PCDD), dibenzofurans (PCDF), and biphenyls (PCB) in different foods. In addition to the seventeen 2,3,7,8-substituted PCDDIPCDF and four non-ortho PCB, by use of the clean-up system studied, it was possible to collect the eight mono-ortho and two di-ortho PCB and the seven indicator PCB in two separate fractions during the same clean-up run. The study was first performed using standard mixtures containing PCDD, PCDF and PCB, and a certified reference material. The recoveries of the 13C-labeled compounds ranged from 51 to 90%, indicating that the PCDD, PCDF, and PCB clean-up worked satisfactorily. Next, the automated system for PCDD, PCDF, and PCB analysis was evaluated for foods such as milk, egg, meat (beef, chicken, and pork), mussel, and olive oil. The recoveries of the 13C-labeled compounds ranged from 40 to 120% for PCB and from 57 to 113% for PCDD/ PCDF, meeting the requirements of well accepted methods. Thus, the automated clean-up system studied is a suitable alternative to conventional clean-up methods.     

Quantitative Bestimmung von Benzo(a)pyren in Zigarettenrauchkondensaten durch Hochdruckflüssigkeits-Chromatographie (HPLC)

Following a clean-up procedure by liquid-liquid distribution and column chromatography on a layer-column silica gel/aluminium oxide, a benzo(a)pyrene concentrate from cigarette smoke condensate was separated by high pressure liquid chromatography. As selective separating system served a column with cross-linked cellulose acetate. Benzo(a)pyrene was quantitatively determined with a fluorescence detector at a detection limit of 10–30 ng/5 μl injection volume. A determination was carried out from the same benzo(a)pyrene concentrate on a reverse-phase system.     

GC-MS analysis of the PCB substitute Ugilec 141 and its occurrence in waste mineral oils

After the restrictions on production and use of PCBs, Ugilec 141, a technical mixture of dichlorobenzyldichlorotoluenes (DBDTs), has been used as a PCB substitute in hydraulic systems. In the Netherlands, the production, import and use of Ugilec 141 has been forbidden since 1988. A method has been developed for the analysis of Ugilec 141 in waste mineral oils to control waste oil import and processing. The method development was based on modification of procedures for the analysis of PCBs in oil and dioxins in fatty matrices and analysis was performed with gas chromatography with mass spectrometry. For sample clean-up, several combinations of column chromatography with activated carbon, alumina, and silica gel have been tested to obtain extracts free from matrix interferences. An internal standard of ¹³C-labeled DBDT was examined to check recoveries from the clean-up procedures. Quantification was performed by comparison with the six most abundant peaks of an external Ugilec 141 standard mixture. Validation experiments showed a linear range from 0.25 to 60 mg Ugilec 141 kg^?1 oil, a reproducibility of 3% and a limit of determination of 0.25 mg Ugilec 141 kg^?1 waste oil. The method was applied in a survey of 70 samples of waste mineral oils from producers all over the Netherlands; this revealed levels below the limit of determination and one positive sample with a concentration of 5.7±0.1 mg Ugilec 141 kg^?1 oil. In an inspection inquiry, one contaminated oil was found containing 116 ± 9 g Ugilec 141 kg^?1 oil.     

The international validation of bio- and chemical-analytical screening methods for dioxins and dioxin-like PCBs: the DIFFERENCE project rounds 1 and 2

The European research project DIFFERENCE is focussed on the development, optimisation and validation of screening methods for dioxin analysis, including bio-analytical and chemical techniques (CALUX, GC-LRMS/MS, GC×GC-ECD) and on the optimisation and validation of new extraction and clean-up procedures. The performance of these techniques is assessed in an international validation study and the results are compared with the reference technique GC-HRMS. This study is set up in three rounds and is in accordance with the International Harmonized Protocol for Proficiency Studies and the ISO 5725 standard. The results of the first two rounds are very promising in particular for GC-LRMS/MS. The results obtained with this technique were as accurate as the results reported by the labs using the GC-HRMS. The initial results reported for GC×GC-ECD overestimate the dioxin concentration in the samples. The results reported by the labs using the CALUX technique underestimate the total TEQ concentrations in the samples, compared to the GC-HRMS reference method. The repeatability of the CALUX is significantly higher than the other screening techniques. It was shown that accelerated solvent extraction (ASE) is a valid alternative extraction and clean-up procedure for fish oil and vegetable oil. The results obtained with CALUX and GC-HRMS after ASE are equivalent to the results obtained with the classical extraction and purification procedures.     

Oil powders and gels from particle-stabilized emulsions

We report on the use of silica particle-stabilized oil-in-water emulsions as a template for the preparation of oil powders and gels obtained in a single step by either slow or rapid evaporation of water using freeze-drying or spray-drying, respectively. Using olive oil and partially hydrophobic fumed silica nanoparticles, an oil powder containing nearly 90 wt % oil can be formed by spray-drying, which shows no sign of oil leakage over several months. Upon slow evaporation of water by freeze-drying, a transparent oil gel with oil content as high as 98 wt % can be formed, in which the silica particles form a space-filling network thickening the oil. Comparisons are made throughout with a typical surfactant-stabilized emulsion which does not produce oil powders or gels.     

An improved clean-up strategy for simultaneous analysis of polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and polychlorinated biphenyls (PCB) in fatty food samples

The study and extension of a simple automated clean-up method for polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) to a broad range of polychlorinated biphenyls (PCB) is described. The isolation of seven PCDD, ten PCDF, and three coplanar PCB (cPCB) is extended to eight monoortho substituted PCB and seven so-called "marker PCB" (Aroclor 1260) for fatty food samples. This enables quantification of 35 compounds - including all congeners with a WHO toxic equivalent factor (TEF)--in a single extraction and single purification step. The chromatographic behaviour of mono-ortho PCB and marker PCB on a variety of adsorbents, including basic alumina, has been studied. Partitioning of analytes through multi-column sequences is described and correlated with their structural and electronic properties, by use of molecular modelling calculations. The fractionation process available with the Power-Prep automated clean-up system enables rapid independent analysis of the different groups of compounds. Gas chromatography with high resolution mass spectrometry (GC-HRMS) is used for the PCDD/F and cPCB fraction and quadrupole ion-storage tandem in time mass spectrometry (GC-QISTMS) for analysis of the remaining PCB. A comparison study was performed on quality-control samples and real fatty food samples to evaluate the robustness of the new strategy compared with a reference method. On the basis of this simultaneous clean-up, a rapid simplified strategy for PCDD/F and selected PCB analysis determination is proposed for fatty food samples.     

利用聚乙烯醇结晶辅助制备多重交联的高强度聚丙烯酰胺水凝胶

在表面带有C=C双键的乙烯基杂化二氧化硅纳米颗粒(vinyl hybrid silica nanoparticle,VSNP)上接枝丙烯酰胺(AM),所得到的纳米刷状凝胶因子通过聚丙烯酰胺(PAM)间的氢键形成物理交联点,则多官能化的VSNP可作为拟共价交联点构筑双重交联的单一网络纳米复合物理水凝胶(nanocomposite physical hydrogel,NCP gel),表现出较高的强度和超拉伸性.为了进一步提高凝胶的强度和韧性,将少量PVA和PAM/VSNP纳米刷混合制成凝胶,通过冷冻-融化处理,使与PAM分子链相互缠绕并形成氢键作用的PVA结晶,形成新的交联点进一步交联PAM NCP gel,得到多交联的PAM NCP gel体系.通过拉曼光谱和示差扫描量热分析,证明凝胶中的PVA通过氢键既可以与PAM相互作用,又形成微晶为新交联点,大大增强了NCP gel的力学性能,与PAM NCP gel相比,凝胶的拉伸强度和断裂能分别从313 k Pa和1.41×104 J/m~2提高到了557k Pa和4.65×104 J/m~2.     

Electrogenerated Chemiluminescence of Tris(2,2′‐bipyridyl)ruthenium(II) Immobilized in Humic Acid‐Silica‐Poly(vinyl alcohol) Composite Films

In this paper, we report the first attempt to use humic acid (HA) as modifiers to prepare the organic‐inorganic hybrid modified glassy carbon electrodes based on HA‐silica‐PVA (poly(vinyl alcohol)) sol‐gel composite. Electroactive species of tris(2,2′‐bipyridyl)ruthenium(II) (Ru(bpy) ) can easily incorporate into the HA‐silica‐PVA films to form Ru(bpy) modified electrodes. The amount of Ru(bpy) incorporated in the composite films strongly depends on the amount of HA in the hybrid sol. Electrochemical and electrogenerated chemiluminescence (ECL) of Ru(bpy) immobilized in HA‐silica composite films coated on a glassy carbon electrode have been studied with tripropylamine (TPA) as the coreactant. The analytical performance of this modified electrode was evaluated in a flow injection analysis (FIA) system with a homemade flow cell. The as‐prepared electrode showed good stability and high sensitivity. The detection limits (S/N=3) were 0.050 μmol L^?1 for TPA and 0.20 μmol L^?1 for oxalate, and the linear ranges were from 0.10 μmol L^?1 to 1.0 mmol L^?1 for TPA and from 1.0 μmol L^?1 to 1.0 mmol L^?1 for oxalate, respectively. The resulting electrodes were stable over two months.     

Fast clean-up for polychlorinated dibenzo-p-dioxins, dibenzofurans and coplanar polychlorinated biphenyls analysis of high-fat-content biological samples

A fast clean-up procedure for the low level analysis of polychlorinated dibenzo-p-dioxins. polychlorinated dibenzofurans and coplanar polychlorinated biphenyls in highly fatty biological matrices using high capacity disposable multi-layer silica columns is presented. Results were compared with gel permeation chromatography for removal of lipids. Analytical criteria such as recovery rates, repeatability, reproducibility and robustness are evaluated through a broad range of biological matrices and reference materials analysis. The final proposed procedure for the complete analysis, including pressurized liquid extraction, Power-Prep system clean-up and GC-high-resolution MS analysis requires only 48 h, and allows the simultaneous preparation of up to 10 samples.     

Liquid-liquid equilibria of polyrotaxane and poly(vinyl alcohol)

Liquid–liquid equillibria (LLE) of the tertiary system of hydroxypropylated polyrotaxane (HPPR)–poly(vinyl alcohol) (PVA)–solvent have been investigated by focusing on the internal structures of HPPR–PVA blend gels. The phase diagrams of the HPPR–PVA aqueous systems displayed two liquid phases at a high concentration and molecular weight of PVA. This result was consistent with the prediction of the Flory–Huggins lattice model. On the contrary, the HPPR–PVA–DMSO system exhibited only a single phase. The HPPR–PVA blend gels crosslinked in dimethylsulfoxide (DMSO) were highly transparent over a wide concentration range, while the gels prepared in water were opaque at high polymer concentrations. Spherical domains were observed in the opaque gels by laser scanning confocal microscopy, and the sizes of the domains were significantly dependent on the amount of cross-linking reagent utilized. These results indicated that the transparency of the HPPR–PVA blend gels was strongly affected by the competition between the liquid–liquid two-phase separation and the crosslinking HPPR and PVA polymers during the preparation of the blend gels.     

Humic acid-bonded silica as a novel sorbent for solid-phase extraction of benzo[a]pyrene in edible oils

A novel solid-phase extraction (SPE) sorbent, humic acid-bonded silica (HAS), was prepared. Humic acids (HAs) were grafted onto silica matrices via an amide linkage between humyl chloride and the amido terminus of 3-aminopropyltrimethoxysilane (APTS)-silica gel. The resulting material was characterized by Fourier transform infrared spectrometer, elemental analysis, and nitrogen adsorption analysis. This sorbent exhibits an excellent adsorption capacity for some electron-abundant analytes owing to its peculiar structure. In this paper, we choose benzo[a]pyrene (BaP) in oil as a probe to validate the adsorption capacity of the material. Thus a fast, cheap and simple SPE method with humic acid-bonded silica cartridge for edible oil clean-up, followed by high-performance liquid chromatography (HPLC) with fluorescence detection was established. The effects of experimental variables, such as washing and elution solvents, and the amount of sorbents have been studied. The recoveries of BaP in edible oils spiked at 0.2-100 μg kg⁻¹ were in the range of 78.8-102.7% with relative standard deviations ranging between 1.3 and 9.3%; the limit of detection was -0.06 μg kg⁻¹.     

Miniaturized Salting-Out Assisted Liquid-Liquid Extraction Combined with Disposable Pipette Extraction for Fast Sample Preparation of Neonicotinoid Pesticides in Bee Pollen

As the main source of nutrients for the important pollinator honeybee, bee pollen is crucial for the health of the honeybee and the agro-ecosystem. In the present study, a new sample preparation procedure has been developed for the determination of neonicotinoid pesticides in bee pollen. The neonicotinoid pesticides were extracted using miniaturized salting-out assisted liquid-liquid extraction (mini-SALLE), followed by disposable pipette extraction (DPX) for the clean-up of analytes. Effects of DPX parameters on the clean-up performance were systematically investigated, including sorbent types (PSA, C18, and silica gel), mass of sorbent, loading modes, and elution conditions. In addition, the clean-up effect of classical dispersive solid-phase extraction (d-SPE) was compared with that of the DPX method. Results indicated that PSA-based DPX showed excellent clean-up ability for the high performance liquid chromatography (HPLC) analysis of neonicotinoid pesticides in bee pollen. The proposed DPX method was fully validated and demonstrated to provide the advantage of simple and rapid clean-up with low consumption of solvent. This is the first report of DPX method applied in bee pollen matrix, and would be valuable for the development of a fast sample preparation method for this challenging and important matrix.     

Structure formation of the mixed gels of zirconium oxyhydrate and silicic acid produced with different sequencing of reagents

The physicochemical characteristics of single and mixed zirconia and silica gels produced by the sol-gel technique at different synthesis pH and sequence of introducing the reagents in the mother liquid are presented. As a result of comparing the data obtained by different research methods, it is found that in the mixed gels, irrespective of the synthesis technique, Si-O-Zr bonds are present. The introduction of a zirconium salt in the mother liquid containing a silicate salt leads to the preferred formation of zirconia gel granules of 20–30 nm in diameter enclosed in the matrix of silica gel. The inverse sequence of introducing the gel-forming components in the reaction mixture promotes the formation of large gel aggregates containing the particles of less that 10 nm and having a high degree of polymerization. Mixed gels of zirconium oxyhydrate and silicic acid have an order higher sorptive capacity for yttrium (III) and calcium cations, as compared to single silica gels and zirconia gels.     

Chemical Modification of Activated Carbons

Mesoporous activated carbons and silica gel (Kieselgel 100, Merck) were modified as follows: (i) treatment with vinyltrimethoxysilane; or (ii)chlorination with CCl₄ followed by the reaction with a Grignard reagent. Modification of silica gel was proved by methods FTIR, NMR-¹³C and element analysis on carbon. The chemical modification of coals by alkans and olefins was supported by comparison of results of thermogravimetry for modified coals and modified silica gels. Polymerization of vinyl groups on carbon surface is shown by methods thermogravimetric analysis and differential scanning calorimetry. This revised version was published online in August 2006 with corrections to the Cover Date.     

A simple clean-up procedure for the quantitative determination of PCBs in complex materials

Summary A simple, rapid, low cost and uniform single-step clean-up procedure with a column containing two silica gel packings of different mesh size for the quantitative determination of PCBs by capillary gas chromatography (ECD) is reported. Its application to 9 complex matrices, among them a transformer oil, a waste motor oil, and an oil from a tempering bath, shows that in contradiction to other clean-Up methods nearly all accompanying substances can be separated. The reproducibility of 4.7% and the recovery rate of 92.7% for the PCBs are comparable with data found in literature.

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Einfaches Clean-up-Verfahren zur quantitativen Bestimmung von PCBs in komplexen Materialien

     

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model. III. Polar stationary phases

In this third paper, varied types of polar stationary phases, namely silica gel (SI), cyano (CN)- and amino-propyl (NH2)-bonded silica, propanediol-bonded silica (DIOL), poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), were investigated in subcritical fluid mobile phase. This study was performed to provide a greater knowledge of the properties of these phases in SFC, and to allow a more rapid and efficient choice of polar stationary phase in regard of the chemical nature of the solutes to be separated. The effect of the nature of the stationary phase on interactions between solute and stationary phases and between solute and carbon dioxide-modifier mobile phases was studied by the use of a linear solvation energy relationship (LSER), the solvation parameter model. The retention behaviour observed with sub/supercritical fluid with carbon dioxide-methanol is close to the one reported in normal-phase liquid chromatography with hexane. The hydrogen bond acidity and basicity, and the polarity/polarizability favour the solute retention when the molar volume of the solute reduces it. As with non-polar phases, the absence of water in the subcritical fluid allows the solute/stationary phase interactions to play a greater part in the retention behaviour. As expected, the DIOL phase and the bare silica display a similar behaviour towards acidic and basic solutes, when interactions with basic compounds are lower with the NH2 phase. On the CN phase, all interactions (hydrogen bonding, dipole-dipole and charge transfer) have a nearly equivalent weight on the retention. The polymeric phases, PEG and PVA, provide the most accurate models, possibly due to their better surface homogeneity.     

Optimised chromatographic method for the measurement of selected polychlorinated biphenyls in ambient air

The focus of this study was to optimise selected polychlorinated biphenyl analysis in air by gas chromatography. Sample preparation was improved by using an automatic solid/liquid extraction system and columns with 1.5 g of Florisil for cleaning extracts. Better precision on clean-up results was obtained from Florisil cartridges, when compared to those obtained with silica gel. Analytical procedure was checked by participating in an inter-comparison exercise. Finally, we have considered usefulness of evaluating uncertainties associated to our method because of the large handling samples and the extremely low concentrations of PCBs in ambient air.     

Development of an automated method for determining oil in water by direct aqueous supercritical fluid extraction coupled on-line with infrared spectroscopy

A direct aqueous supercritical fluid extraction (SFE) system was developed which can be directly interfaced to an infrared spectrometer for the determination of oil in water. The technique is designed to provide an environmentally clean, automated alternative to established IR methods for oil in water analysis which require the use of restricted organic solvents. The SFE-FTIR method involves minimum sample handling stages, with on-line analysis of a 500 ml water sample being complete within 15 min. Method accuracy for determining water samples spiked with gasoline, white spirit, kerosene, diesel or engine oil was 81-100% with precision (RSD) ranging from 3 to 17%. An independent evaluation determined a 2 ppm limit of quantification for diesel in industrial effluents. The results of a comparative study involving an established IR method and the SFE-FTIR method indicate that oil levels calculated using an accepted equation which includes coefficients derived from reference hydrocarbon standards may result in significant errors. A new approach permitted the derivation of quantification coefficients for the SFE-FTIR analyses which provided improved results. In situations where the identity of the oil to be analysed is known, a rapid off-line SFE-FTIR system calibration procedure was developed and successfully applied to various oils. An optional in-line silica gel clean-up procedure incorporated within the SFE-FTIR system enables the same water sample to be analysed for total oil content including vegetable oils and selectively for petroleum oil content within a total of 20 min. At the end of an analysis the SFE system is cleaned using an in situ 3 min clean cycle.