A high-throughput platform for low-volume high-temperature/pressure sealed vessel solvent extractions

A high-throughput platform for performing parallel solvent extractions in sealed HPLC/GC vials inside a microwave reactor is described. The system consist of a strongly microwave-absorbing silicon carbide plate with 20 cylindrical wells of appropriate dimensions to be fitted with standard HPLC/GC autosampler vials serving as extraction vessels. Due to the possibility of heating up to four heating platforms simultaneously (80 vials), efficient parallel analytical-scale solvent extractions can be performed using volumes of 0.5-1.5 mL at a maximum temperature/pressure limit of 200°C/20 bar. Since the extraction and subsequent analysis by either gas chromatography or liquid chromatography coupled with mass detection (GC-MS or LC-MS) is performed directly from the autosampler vial, errors caused by sample transfer can be minimized. The platform was evaluated for the extraction and quantification of caffeine from commercial coffee powders assessing different solvent types, extraction temperatures and times. For example, 141±11 μg caffeine (5 mg coffee powder) were extracted during a single extraction cycle using methanol as extraction solvent, whereas only 90±11 were obtained performing the extraction in methylene chloride, applying the same reaction conditions (90°C, 10 min). In multiple extraction experiments a total of ~150 μg caffeine was extracted from 5 mg commercial coffee powder. In addition to the quantitative caffeine determination, a comparative qualitative analysis of the liquid phase coffee extracts and the headspace volatiles was performed, placing special emphasis on headspace analysis using solid-phase microextraction (SPME) techniques. The miniaturized parallel extraction technique introduced herein allows solvent extractions to be performed at significantly expanded temperature/pressure limits and shortened extraction times, using standard HPLC autosampler vials as reaction vessels. Remarkable differences regarding peak pattern and main peaks were observed when low-temperature extraction (60°C) and high-temperature extraction (160°C) are compared prior to headspace-SPME-GC-MS performed in the same HPLC/GC vials.     

Polymeric nanocapsules containing an antiseptic agent obtained by controlled nanoprecipitation onto water-in-oil miniemulsion droplets

The modified nanoprecipitation of polymers onto stable nanodroplets has been successfully applied to prepare well-defined nanocapsules whose core is composing of an antiseptic agent, i.e., chlorhexidine digluconate aqueous solution. The stable nanodroplets were obtained by inverse miniemulsions with an aqueous antiseptic solution dispersed in an organic medium of solvent/nonsolvent mixture containing an oil-soluble surfactant and the polymer for the shell formation. The change of gradient of the solvent/nonsolvent mixture of dichloromethane/cyclohexane, obtained by heating at 50 degrees C, led to the precipitation of the polymer in the organic continuous phase and deposition onto the large interface of the aqueous miniemulsion droplets. The monodisperse polymer nanocapsules with the size range of 240-80 nm were achieved as a function of the amount of surfactant. Using various polymer contents, molecular weights and types, an encapsulation efficiency of 20-100% was obtained as detected by proton-nuclear magnetic resonance spectroscopy ((1)H NMR) measurements. The nanocapsules could be easily transferred into water as continuous phase resulting in aqueous dispersions with nanocapsules containing an aqueous core with the antiseptic agent. The encapsulated amount of the antiseptic agent was evaluated to indicate the durability of the nanocapsule's wall. In addition, the use of different types of polymers having glass transition temperatures (T(g)) ranging from 10 to 100 degrees C in this process has been also successful.     

High-performance liquid chromatography of aflatoxins in human urine

A method was developed for the extraction and determination of unconjugated aflatoxins in human urine by high-performance liquid chromatography. The analysis is based on the elimination of lipid-soluble constituents other than unconjugated aflatoxins in urine by light petroleum extraction. The unconjugated aflatoxins were subsequently extracted from the aqueous phase with chloroform-acetone. Chromatography was performed isocratically with a silica column at 40 degrees C. The resolved aflatoxins were detected and identified by ultraviolet and fluorometric detectors. The recoveries of aflatoxins B1 and G1 added prior to the extraction were 72% and 83%, respectively. This procedure is simple, sensitive and practically useful for epidemiological survey of unconjugated aflatoxins in human urine from areas with a high risk of aflatoxin consumption.     

A simple novel configuration for in-vial microporous membrane liquid–liquid extraction

A novel arrangement for microporous membrane liquid–liquid extraction from the aqueous donor phase to the organic acceptor phase within a micro-vial, which is compatible with the chromatograph autosampler is presented. The device consisted of a stoppered glass micro-vial containing the organic solvent where the septum of the screw stopper was replaced by a sized piece of membrane which is hermetically assembled to the volumetric flask containing the aqueous donor solution. The placement of the membrane in alternative contact with the solutions was achieved by orbital agitation. As a preliminary study, 2-ethylhexyl 4-(dimethylamino)benzoate has been determined (limit of quantification 0.11 μg L⁻¹, precision 7.4%). The small quantity of organic solvent used, the achieved sample cleanup, and the minimal handling and risk of cross-contamination are significant operational advantages.     

Effect of the medium on the ionization constants of some triazole compounds

The deprotonation and acid ionization constants of some triazole derivatives in various aqueous-organic solvent mixtures were determined potentiometrically at 20 degrees C. The organic solvents used were methanol, ethanol, DMF, DMSO, acetonitrile, acetone and dioxane. The high stabilization of both the non-protonated form by dispersion forces and of the proton by its interaction with the organic solvent are the main factors influencing the deprotonation constant in aqueous mixtures of methanol, ethanol, DMF or DMSO. On the other hand, the hydrogen bonding interactions and the solvent basicity, in addition to the electrostatic effect, contribute to the major effects in the deprotonation process (in solutions enriched with acetonitrile, acetone or dioxane) and the acid ionization process in different aqueous-organic solvent mixtures. Some thermodynamic parameters (delta H, delta G, delta S) of the ionization processes in a pure aqueous medium are also determined and discussed.     

液相色谱-三重串联四极杆质谱测定粮油中的黄曲霉毒素

建立了超声提取-液相色谱-电喷雾三重串联四极杆质谱测定玉米、大米、大豆等粮油固体样品中黄曲霉毒素B1、B2、G1和G2(AFB1、AFB2、AFG1和AFG2)的方法。分析前对样品进行超声提取,优化得到最佳超声提取条件: 溶剂为甲醇-水(含40 g/L NaCl) (80:20, v/v)溶液、料液比为1:3(g:mL)、温度为50 ℃、时间为3 min。然后对提取的样品进行免疫亲和特异性净化。最后与液相色谱-电喷雾三重串联四极杆质谱联用,使用C18反相色谱柱,流动相为甲醇-10 mmol/L乙酸铵水溶液梯度洗脱,以黄曲霉毒素M1(AFM1)作为内标进行定量测定。结果表明,AFB1、AFB2、AFG1和AFG2的检出限分别为0.002、0.004、0.004和0.012 μg/kg。方法的加标回收率为87%～111%,日内相对标准偏差(RSD)和日间RSD分别不大于6.7%和5.6%。实验结果表明该方法可以有效地降低基质效应的影响,相比于外标法能极大地提高方法的准确度。     

MEPS as a rapid sample preparation method to handle unstable compounds in a complex matrix: determination of AZD3409 in plasma samples utilizing MEPS-LC-MS-MS

The aim of the present investigation is to develop a simple, fast, and sensitive method for the determination of a new candidate drug, AZD3409, in rat, dog, and human plasma samples. AZD3409 is stable in aqueous solutions at low pH (< 4) but not in whole blood or in plasma. In rat plasma at 25 degrees C, more than 90% of the compound is degraded within 40 min. When 20 mg of NaF and 50 microL of protease inhibitor cocktail are added to 1.0 mL of rat blood, AZD3409 is stable for up to about 90 min. Due to the instability of AZD3409, microextraction in packed syringe (MEPS) is used as an online and fast sample-preparation method, followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the quantitation of this compound in plasma samples. In MEPS, the sampling sorbent is 1 mg of polystyrene polymer packed in a 250-microL syringe. When the plasma sample (50-250 microL) is withdrawn through the syringe by an autosampler, the analyte is adsorbed to the solid phase. The analyte is then eluted with an organic solvent such as methanol or the LC mobile phase (20-50 microL) directly into the instrument's injector. MEPS is rapid and easy to use. The lower limit of quantitation for AZD3409 is established to be 0.024 microM. The accuracy of the quality-control samples ranged from 89% to 102%, and the precision (C.V.%) had a value of 11-16% for the plasma samples. The calibration curve in plasma is obtained in the concentration range 0.022-9.0 microM. The coefficients of determination (R2) for plasma samples were > or = 0.998 for all runs. The present method is used for the analysis of rat and dog plasma samples.     

Extraction of aflatoxins B1 and G1 from maize by using aqueous sodium dodecyl sulfate

Aflatoxins are potent carcinogens produced by certain Aspergillus fungi. The aflatoxins were first discovered in the 1960s, and since then have been found to be distributed worldwide in a variety of commodities, foods, and feeds. Many of the early techniques for detecting aflatoxins involved extraction with halogenated solvents. With the increased availability and use of reversed-phase solid-phase extraction cartridges and the availability of immunoaffinity columns, aqueous mixtures of nonhalogenated solvents have been frequently used. To further reduce the need for solvents, we examined the effects of eliminating solvents during the extraction of maize, using aqueous mixtures of the detergent sodium dodecyl sulfate. After extraction and filtration, aflatoxins B1 (AFB1) and G1 (AFG1) were isolated by using commercially available immunoaffinity columns. The isolated AFB1 and AFG1 were derivatized with trifluoroacetic acid before separation by liquid chromatography with fluorescence detection. In spiked maize, the limits of detection were 0.5 and 1 ng/g for AFB1 and AFG1, respectively. Recoveries of AFB1 from maize spiked at 1-20 ng/g averaged 87.5% (range, 76.3-99.0%), with an average repeatability relative standard deviation (RSDr) of 4.0%. Recoveries of AFG1 from maize spiked at 2-20 ng/g averaged 80.4% (range, 70.3-85.8%), with an average RSDr of 3.5%. This is the first reported demonstration of an effective solvent-free extraction of aflatoxins from maize at ambient pressure, and this extraction procedure may serve to help reduce solvent consumption during aflatoxin analysis.     

Factors influencing performance in the rapid separation of aflatoxins by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) is applied to the high-speed analysis of aflatoxins. Baseline separation of the four common aflatoxins G(1), G(2), B(1) and B(2), is accomplished in less than 30 sec. Small (25 mum) internal diameter capillaries are found to be critical in maintaining high efficiency under rapid MECC separation conditions. Van Deemter-like plots are generated in order to study the effects of capillary diameter and organic solvent on efficiency under high electric field conditions. Other experimental parameters affecting efficiency are investigated, including buffer concentration, surfactant concentration, and detector time constant. Simple on-column laser-based fluorescence detection, employing helium-cadmium laser radiation at 325 nm for excitation, allows for limits of detection in the range of 0.05-0.9 femtomoles injected for underivatized aflatoxins. Considerations important in the analysis of aflatoxins in real matrices are presented.     

Combining ultrasound‐assisted extraction and vortex‐assisted liquid–liquid microextraction for the sensitive assessment of aflatoxins in aquaculture fish species

Although aflatoxins contamination in feedstuff is a well‐known problem, and hence these residues are controlled in poultry products, there is scarce information regarding the presence of these toxic substances in aquaculture fish, facilities that use several feedstuff for fish breeding. A simple, rapid, and sensitive method has been therefore developed for aflatoxins (B1, B2, G1, and G2) assessment in aquaculture products by combining ultrasound probe‐assisted extraction and vortex‐assisted liquid–liquid microextraction as a sample pretreatment, and high‐performance liquid chromatography‐tandem mass spectrometry as a separation/detection system. Aflatoxins were extracted from fish flesh/liver with a 60:40 acetonitrile/aqueous phosphate buffer (pH 7.0) mixture before preconcentration and clean‐up by vortex‐assisted liquid–liquid microextraction under the following optimized conditions: 5.0 mL of fish extract at pH 7.0 and NaCl at 0.5% (w/v), 400 μL of chloroform as extracting solvent, and vortex shaking at 2000 rpm for 1 min. The proposed method is shown to be precise and accurate, and the limit of quantitations (from 0.20 to 1.10 μg kg^?1) were lower than the value established by the European Commission Regulation for aflatoxins in foodstuff. Results have shown that fish flesh is free of aflatoxins, but aflatoxins B2 and G1 were quantified in fish liver.     

Rapid extraction of aflatoxin from creamy and crunchy peanut butter

A rapid extraction technique was developed for the isolation and subsequent liquid chromatographic determination of aflatoxins B1, B2, G1, and G2 in creamy and crunchy peanut butter. Peanut buftter samples were extracted with a methanol 15% sodium chloride (7 + 3) solution followed by a second extraction with methanol. The extract was subjected to a cleanup using a Vicam Aflatest immunoaffinity column. Control samples for both smooth and crunchy peanut butter were fortified at 4 different levels for aflatoxin B1, B2, G1, and G2. The average aflatoxin B1, B2, G1, and G2 recoveries from smooth peanut buffer were 95.2, 89.9, 94.1, and 62.4%, respectively, and 92.4, 84.3, 85.5, and 53.7%, respectively, from crunchy peanut butter. This extraction method and the official AOAC Method 991.31 produced comparable results for peanut butter samples. This method provides a rapid, specific, and easily controlled assay for the analysis of aflatoxins in peanut butter with minimal solvent usage. Organic solvent consumption was decreased by 85% and hazardous waste production was decreased by 80% in comparison with the AOAC method. Along with the decreased solvent consumption, significant savings in time were observed.     

Long term stability of organic selenium species in aqueous solutions

Long term stability of organic selenium compounds (selenocystine, selenomethionine, trimethylselenonium ion) has been studied over a one year period for 2 analyte concentrations: 25 and 150 μg/L Se, at pH 4.5 in the dark, under different storage conditions: temperature of –20°C, 4°C, 20°C, 40°C; in Pyrex, Teflon, or polyethylene containers; in an aqueous matrix or in the presence of a chromatographic counter ion (pentyl sulfonate at 10^–4 mol/L concentration). Light effects have also been tested. The stability of the selenium species was monitored by HPLC-ICP/MS. Storage conditions can drastically alter the stability of organic selenium species. Organoselenium compounds were shown to be stable in the dark over a one year period in an aqueous matrix at pH 4.5 in Pyrex containers at both 4°C and 20°C. Pyrex vials exposed to natural sunlight at room temperature resulted in a steady decrease of the selenoamino acid concentration. Teflon containers caused losses of less than 25% at both 4° C and 20° C in the dark. However, polyethylene vials presented, at all temperatures tested, a rapid decrease of the TMSe⁺ concentration. The stability of the Se species studied did not show significant differences between 4° C and 20° C in any container material used. Storage of solutions at 40° C led to slight differences between the Pyrex and Teflon containers. However, polyethylene presented a drastic decrease of the three species over time at this higher temperature. Solutions frozen at –20° C in polyethylene vials did not stabilize the TMSe⁺ signal. Finally, concentrations and matrices of the samples did not significantly affect the stability of the species. Received: 15 July 1996 / Revised: 14 July 1997 / Accepted: 18 July 1997     

Thermal gelation of cellulose in a NaOH/thiourea aqueous solution

Utilizing a novel solvent of cellulose, 6 wt % NaOH/5 wt % thiourea aqueous solution, for the first time, we prepared the thermally induced cellulose gel. We investigated the thermal gelation of cellulose solutions with rheometry and the structure of the gel with 13C NMR, wide-angle X-ray diffraction, environmental scanning electron microscopy, and atomic force microscopy. The cellulose solutions revealed an increase in both the storage modulus (G') and the loss modulus (G") with an increase in the temperature during gelation. The temperature at the turning point, where G' overrides G" because of the onset of gelation, decreased from 38.6 to 20.1 degrees C with an increase of cellulose concentration from 4 to 6 wt %. Given enough time, G' of all solutions can exceed G" at a certain temperature slightly lower than the gelation temperature, indicating that the occurrence of the gelation is also a function of time. Each of the assigned peaks of NMR of the cellulose gel is similar to that of the cellulose solution, suggesting that the gelation resulted from a physical cross-linking. The gels were composed of relatively stable network units with an average diameter of about 47 nm. At either a higher temperature (at 60 degrees C for 30 s) or a longer gelation time (at 30 degrees C for 157 s), the gel in the 5 wt % cellulose solution could form. A schematic gelation process was proposed to illustrate the sol-gel transition: the random self-association of the cellulose chains having the exposed hydroxyl in the aqueous solution promotes the physical cross-linking networks.     

Molar absorptivities of aflatoxins B1, B2, G1, and G2 in acetonitrile, methanol, and toluene-acetonitrile (9 + 1) (modification of AOAC Official Method 971.22): collaborative study

Four laboratories participated in a mini-collaborative study of AOAC Official Method 971.22, Standards for Aflatoxins, Thin-Layer Chromatographic Method, to extend the method to 3 replacement solvents for benzene for calibration of standard aflatoxin solutions. Triplicate test sample vials, each containing 25 micrograms of the respective aflatoxin for each of the 4 aflatoxins and for each of the solvents, were prepared and sent to each collaborator. The collaborators dissolved the aflatoxin in each vial in 2 mL solvent, measured the UV spectrum, and reported the absorptivity maxima near 350 nm. The concentrations of the aflatoxins in the test samples were determined by dissolving identical test samples in benzene-acetonitrile (98 + 2) and following the procedure described in AOAC Official Method 971.22. These concentrations were, in turn, used to determine the molar absorptivities in the other 3 solvents (see Table 1). AOAC Official Method 971.22 has been modified to extend its applicability to 3 replacement solvents for benzene for calibration of standard aflatoxin solutions.     

Nickel and strontium nitrates as modifiers for the determination of selenium in wine by Zeeman electrothermal atomic absorption spectrometry

A mixed matrix modifier of nickel and strontium nitrates was used as a chemical modifier for the determination of selenium in wines by Zeeman electrothermal atomic absorption spectrometry. Wine samples were heated on a boiling water bath with small amounts of nitric acid and hydrogen peroxide. For complete elimination of interference, especially from sulfates and phosphates, selenium is complexed with ammonium pyrolidinedithiocarbamate (APDTC), extracted into methyl isobutyl ketone (MIBK), and measured by ETAAS. The graphite furnace temperature program was optimized for both aqueous and organic solutions. Pyrolysis temperatures of 1300 degrees C and 800 degrees C were chosen for aqueous and organic solutions, respectively; 2700 degrees C and 2100 degrees C were used as optimum atomization temperatures for aqueous and organic solutions, respectively. The optimum modifier mass established is markedly lower than those presented in the literature. The platform atomization ensures pretreatment stabilization up to 1100 degrees C and 1600 degrees C, respectively, for organic and aqueous selenium solutions. The procedure was verified by the method of standard addition. The investigated wine samples originated from the different regions of the Republic of Macedonia. The selenium concentration varied from not detectable to 0.93 microg L(-1).     

The acidity constants of some pyrimidine bases in various water-organic solvent media

The acid ionization constants of some pyrimidine bases of nucleic acids were determined pH-metrically at 25 degrees C and at the constant ionic strength I = 0.10 mol l(-1) (KNO3) in pure water as well as in aqueous media containing variable mole percentages (5-30%) of organic solvents. The organic solvents used were methanol, ethanol, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, acetone and dioxane. The results obtained indicated that the acidity constants are generally decreased as the content of an organic solvent in the medium is increased. It was deduced that the hydrogen bonding interactions and the solvent basicity in addition to the electrostatic effect are the major effects influencing significantly the acid ionization process of pyrimidine bases in the different water-organic solvent media. Some thermodynamic parameters (deltaH, deltaG degrees, deltaS degrees) of the ionization process over the temperature range 5-45 degrees C in pure water were also determined and discussed.     

Improved gas chromatographic determination of nicotine in environmental tobacco smoke

A rapid gas chromatographic procedure with an analysis time of 5 min was developed for the determination of environmental nicotine collected on sorbent tubes containing XAD-4 resin. In validating this procedure, severe temporal losses of nicotine were observed for solutions in glass sample vials waiting in a queue in an autosampler tray for analysis. These losses were traced to adsorptive interactions of nicotine with the glass surface of the vials. The use of N-ethylnornicotine as the internal standard or the addition of triethylamine to all solutions were both successful in producing constant response ratios of nicotine to internal standard. Owing to the limited availability and expense of N-ethylnornicotine, our current procedure calls for the addition of triethylamine to all nicotine solutions at the 0.01% V/V level and the use of quinoline as internal standard.     

Effects of Organic Solvents on Immobilized Lipase in Pectin Microspheres

Lipase from Brevibacillus agri 52 was found stable up to 90% diethylenglycol (DEG), glycerol (GLY), and 1,2 propanediol (1,2 PRO) at 37 degrees C for 1 h and the stability was reduced only approximately 20% after 12 h incubation, but in 40% dimethylsulfoxide (DMSO), lipase activity was stable only for 1 h. Inhibition of the biocatalysts with dimethylformamide (DMF) was detected at 20% solvent concentration. In water immiscible systems, the stability of lipase in n-hexane, n-tetradecane and n-heptane resembles the water activity, but in the presence of isobutanol, 1-hexanol, and butylbutirate, the stability was significantly reduced. Lipase 52 precipitates in the presence of 50% acetone or ethanol/water mixtures, but enzymatic activity was partially recovered by adding 20% GLY, DEG, 1,2 PRO, or DMSO to the reaction mixture. Furthermore, by increasing DEG in 70% DMF/DEG mixtures, the lipase activity was protected. Encapsulation of lipase in pectin gels cross-linked with calcium ions brings three to four times more enzymatic activity in 70% water miscible organic solvents compared to aqueous systems.     

液相色谱-串联质谱法测定动物肝脏中黄曲霉毒素

建立了动物肝脏中黄曲霉毒素G2、G1、B2、B1的高效液相色谱-串联质谱检测方法。样品经体积比为84∶16的乙腈-水溶液提取,离心后通过真菌毒素多功能净化柱,净化液氮气吹干,用流动相定容,采用C18柱分离,10mmol/L的甲酸铵溶液和甲醇作为流动相,以50∶50比例等度洗脱,在多重反应监测(MRM)正离子模式下进行分析。各组分在9min内完全分离,方法线性关系良好,黄曲霉毒素G2、G1、B2、B1的检出限分别为0.030、0.026、0.016、0.027μg/kg,三个加标水平下平均回收率在81%～98%之间,相对标准偏差小于2%。该方法简便快速,准确可靠,可用于动物肝脏中黄曲霉毒素的测定。     

Crystal-face dependences of surface band edges and hole reactivity, revealed by preparation of essentially atomically smooth and stable (110) and (100) n-TiO(2) (rutile) surfaces

Essentially atomically smooth (100) and (110) n-TiO(2) (rutile) surfaces were prepared by immersion of commercially available single-crystal wafers in 20% HF, followed by annealing at 600 degrees C in air. The obtained surfaces were stable in aqueous solutions of pH 1-13, showing no change in the surface morphology on an atomic level, contrary to atomically flat surfaces prepared by ion sputtering and annealing under UHV. The success in preparation of the atomically smooth and stable n-TiO(2) surfaces enabled us to reveal clear crystal-face dependences of the surface band edges and hole reactivity in aqueous solutions.