The properties of organelles within a cell have been shown to be highly heterogeneous. Until now, it has been unclear just how much of this heterogeneity is endemic to the organelle subpopulations themselves and how much is actually due to stochastic cellular noise. An attractive approach for investigating the origins of heterogeneity among the organelles of a single cell is CE with LIF detection (CE-LIF). As a proof of principle, in this report we optimize and use a single cell CE-LIF method to investigate the properties of endocytic (acidic) organelles. Our results show that the properties of individual acidic organelles containing Alexa Fluor 488 Dextran suggest that there are two groups of CCRF-CEM cells: a group with a high dextran content per cell, and a group with a low dextran content per cell. Furthermore, the individual organelle measurements of the single cells allow us to compare in each group the distributions of doxorubicin content per acidic organelle and electrophoretic mobilities of these organelles.

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

Individual nuclei isolated from the human leukemia CCRF-CEM and CEM-C2 cells treated with doxorubicin (DOX) were in-column lysed with a sodium dodecyl sulfate (SDS) containing buffer, their contents were then separated by micellar electrokinetic capillary chromatography using the same lysing buffer, and the DOX content was detected by laser-induced fluorescence. Use of a microscope for the selection of one nucleus from the nuclear preparation decreases the possibility of introduction of other subcellular components that are commonly found as impurities in subcellular fractions. The presence of SDS in the running buffer made negligible the DNA's quenching effect on DOX fluorescence, which often compromises quantification of DOX by direct imaging, making it possible to carry out the first direct measurement of the doxorubicin content of isolated nuclei. On average, nuclei from CCRF-CEM and CEM/C2 cell lines contained 85 +/-64 (n = 6) and 91 +/- 51 (n =7) amol of DOX, respectively. These values correspond to 74 and 65% of the average total cellular content as determined by single-cell analysis of the corresponding cell types. It is envisioned that this approach could become an important bioanalytical tool to investigate the effect of treatments with fluorescent drugs targeting the nucleus.

We report the doxorubicin content in individual organelles following their capillary electrophoretic separation and illustrate that chemical accumulation at the subcellular level is highly heterogeneous. In individual mitochondria from cultured human leukemia cells DOX amount is around 50 zmol, 2 orders of magnitude higher than expected from diffusion during drug treatment, and spans 2 orders of magnitude.

A sensitive capillary electrophoretic method was developed to detect the presence of alpha-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-alpha-tubulin primary mouse antibodies and then stained with a fluorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced fluorescence detection, a technique that allows for sensitive detection of fluorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing alpha-tubulin (SATs), estimate the number of alpha-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of alpha-tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not influenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the alpha-tubulin content. Given the zeptomolar sensitivity of laser-induced fluorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions.

A stepwise solid-phase microextraction (SPME) method was developed for on-site calibration of SPME for volatile organic compounds analysis. In this approach, a 75-μm Carboxen–polydimethylsiloxane coated fibre was loaded with a standard prior to exposure to samples of interest. Extraction time for the target analytes can be controlled independently from that of the standard, and the response factors for the target analytes can be adjusted accordingly. A good reproducibility of the response factors for BTEXs (benzene, toluene, ethylbenzene and xylenes) was obtained with stepwise SPME. Satisfactory results were obtained by using this method for quantitative analysis of BTEXs in the air of a gas station when tetrachloroethylene was used as a standard. The introduction of standard via the stepwise SPME procedure makes SPME more useful in field applications. It can be used to detect leaks, contaminations and losses from loading of a standard onto a fibre to introduction of the fibre to an analytical instrument. However, this method cannot be used for compensation of sample matrix effects.

Platelet-derived microparticles (PMPs) formed by vesiculation during platelet activation seem to play a role in blood coagulation and in pathological disease states. Flow cytometry is currently the gold standard to characterize platelets and PMPs. Using this technique we distinguished between platelets and PMPs based on size and the presence of phosphatidyl serine ( PS); PMPs were arbitrarily defined to be smaller than one micrometer and capable of forming a stable complex with fluorescently-labeled Annexin V, a protein that forms a calcium-dependent complex with PS. Further confirmation of PMP and platelet identity was done by use of fluorescently-labeled antibodies against CD41a, a glycoprotein found on the surface of both platelets and PMPs. In this report we also introduce the use of capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) for the analysis of fluorescently labeled platelets and PMPs. While both flow cytometry and CE-LIF can measure individual fluorescent events, only CE-LIF allowed us to calculate individual electrophoretic mobilities of activated platelets and PMPs that were then represented as distributions. A comparison between distributions suggests that PMPs have less negative mobilities. The fact that activated platelet preparations include PMPs partially obscure the interpretation of the data. While PMP and platelet number ml(-1) determined by flow cytometry is lower than the same parameter determined by CE-LIF, signal-to-noise ratio was 20 fold better for flow cytometry than for CE-LIF. This is the first time that a direct comparison between these two techniques is reported.

Microwave-assisted headspace solid-phase microextraction (MA-HS-SPME) was developed as a simple and effective method for fast sampling of volatile organic compounds (VOCs) from Eucalyptus citriodora Hook (E. citriodora) leaves. During microwave heating, a simple shielding device made of aluminum foil was used to protect the SPME fiber from microwave irradiation while allowing the sample to be heated. A room temperature water bath was also used to allow microwave heating to be conducted in a more controlled manner. The inner heating caused by microwave irradiation dramatically accelerated the emission of VOCs from the sample, but no marked change in headspace temperature in the sample vial was found. Under optimum conditions, the extraction efficiencies obtained with microwave heating were much higher than those obtained without microwave heating for all fibers used, namely, 7-microm polydimethylsiloxane (PDMS), 100-microm polydimethylsiloxane (PDMS), 65-microm polydimethylsiloxane/divinylbenzene (PDMS/DVB), and 75-microm carboxen/polydimethylsiloxane (CAR/PDMS). The improvement of extraction efficiency using MA-HS-SPME allowed more VOC events to be detected, with more balanced extraction of VOCs of lower and higher molecular masses. Moreover, a good linear relationship was found between sample size and GC-FID response (total peak area of VOCs), indicating the usefulness of MA-HS-SPME for quantitative analysis of individual volatile compounds in E. citriodora leaves.

Microwave heating was employed for preparation of the standard gas of volatile organic compounds (VOCs) and semivolatile organic compounds (semi-VOCs) by using a 1000 W commercial domestic microwave oven and 1 L gas-sampling bulbs. The VOCs investigated were benzene, chloroform, 1,3-dichlorobenzene, tetrachloroethylene, toluene, and 1,1,2-trichloroethane, and the semi-VOCs used were the polychlorinated biphenyls (PCBs) PCB 1016 and PCB 1248. Since these weakly or nonpolar molecules are very poor absorbers of microwave energy, an appropriate amount of water was introduced to accept microwave radiation and act as the thermal source to accelerate their evaporation. The glass bulb may also contribute thermal energy to the VOCs/semi-VOCs by accepting microwave energy to a small degree. For 0.5 microL of liquid VOCs on 10 mg of glass wool, it was shown that 15 microL of H2O and 60 s of microwave heating yielded a very efficient evaporation [97.2-106.4%, compared with a classic method (Muller, L; Gorecki, T.; Pawliszyn, J. Fresenius' J. Anal. Chem. 1999, 364, 610-616)]. For 1 microL of PCB solution (1000 microg/mL in hexane), 15 microL of H2O and 90 s of microwave heating also provided a complete evaporation. The addition of water was particularly significant for microwave-assisted evaporation of PCBs because semi-VOCs are much more difficult to evaporate than VOCs. This developed microwave technique proved to be quite simple, powerful, rapid, accurate, and safe for the preparation of VOC/semi-VOC standard gas. Solid- phase microextraction combined with gas chromatography was used for the gas analysis.

A sample pretreatment by microwave-assisted extraction (MAE), microwave-assisted alkaline degradation (MAAD) and microwave-assisted saponification prior to the determination of PAHs, PCBs, triazines and cholesterol in diverse samples using GC, GC-MS and HPLC has been developed. When MAAD was coupled with MAE, the degradation of chlorinated pesticides and the extraction of PCBs analytes in the samples could be carried out simultaneously. Likewise, when MAAD and MAS were coupled with MAE, the degradation, saponification and extraction could be carried out simultaneously. In addition, a microwave-assisted derivatization (MAD) of fatty acids in edible oil, plasma and shark cartilage has been developed. The analytical results are satisfactory. The microwave-assisted sample pretreatment methods are rapid, labor and solvent saving, moreover, non-chlorinated organic solvents can be used.

The extraction of organic pollutants from solid environmental matrices has traditionally been done using non-instrumental approaches, e.g. Soxhlet extraction. Recent developments have seen the possibility of extraction using instrumental techniques, namely, supercritical fluid extraction, microwave-assisted extraction and pressurised fluid extraction. This review considers the applications of instrumental techniques for the extraction of polycyclic aromatic hydrocarbons, polychlorinated biphenyls and pesticides from soils/sediments. Then, in order to assist the analyst to select the most appropriate technique (traditional versus instrumental techniques), a comparison of their analytical figures of merit is considered.

有机氯农药（Ops）严重干扰多氯联苯（PCBs）的测定，本文采用微波加热技术将Ops碱解，在优化的实验条件下能完全消除六六六（HCH）、双对氯苯基三氯乙烷（DDT）、二氯二苯二氯乙烷（DDD）的干扰，二氯二苯二氯乙烯（DDE）、艾试剂（Aldrin）、狄试剂（Dieldrin）也有一定数量的减少，狄试剂经浓硫酸处理后其干扰完全消除。通过对合成土壤样品的处理，表明该法能有效地碱解Ops，消除其对测定PCBs的干扰；并能在碱解Ops的同时萃取PCBs，PCBS回收率为84．1％，相对标准偏差为2．7％。本方法快速、高效，减少了有机溶剂的使用量。

Three microwave-assisted techniques (MAT), namely microwave-assisted extraction (MAE), microwave-assisted saponification (MAS) and microwave-assisted decomposition (MAD), were studied and combined to be used in the samples pretreatment to determine polychlorinated biphenyls (PCBs) in soil, sediment and mussels. MAE was employed for the isolation of PCBs, MAS for the saponification of a mussel matrix, and MAD for the elimination of some organochlorine pesticides (OCPs) such as hexachlorocyclohexane (BHC) and DDT, which usually coexisted with PCBs in environmental samples. It was found that acetone-n-hexane (1:1, v/v), methanol and methanolic 1M KOH solution were all good extractants for MAE of PCBs (recovery >83%), and 15 g mussel could be completely saponified with 30 ml of methanolic 1M KOH or ethanolic KOH solution by 2 min microwave heating at 100% power output of 600 W. Moreover, 30 mi methanolic 1 M KOH or ethanolic 1 M KOH solution and 2 min microwave heating brought about a complete decomposition for each of 2 similar to 4 mu g alpha-BHC, beta-BHC, gamma-BHC, delta-BHC, o-p-DDT, p-p-DDT and p-p-DDD, while p-p-DDE, aldrin and dieldrin could only be partly degraded. Furthermore, compared with that by MAD in methanolic 1 M KOH solution, the degradation products of OCPs by MAD in methanolic 1 M KOH solution yielded more peaks in the chromatogram obtained by gas chromatography equipped with electron capture detector (GC-ECD), which may interfere with the GC-ECD detection of PCBs. Therefore, methanolic 1M KOK solution was chosen as the extractant for the pretreatment of soil or sediment. As a result, the MAD of OCPs could occur along with the MAE of PCBs. Similarly, the MAD of OCPs could well coincide with the MAS of mussels when methanolic 1 M KOH solution was employed as the saponifier for the pretreatment of mussels. Thus, isolating the analytical targets and eliminating the coexisting interferents could be simultaneously carried out using the combined MAT. In addition, it was found that PCBs are quite stable under the optimized conditions during both the MAE-MAD and MAS-MAD procedures. (C) 2000 Elsevier Science B.V. ALI rights reserved.

A new and simple microwave-assisted alkaline degradation (MAAD) method for the elimination of organochlorine-pesticides interference on the determination of polychlorinebiphenyls (PCBs) in soil by GC is presented. Under optimized conditions, alpha-HCH, beta-HCH, gamma-HCH, delta-HCH, o, p'-DDT, p, p'-DDD and p, p'-DDT could he degraded completely, and p,p'-DDE, Aldrin and Dieldrin partly, but Dieldrin could be completely degraded by using concentrated sulphuric acid. The method was evaluated by analysing the spiked soil sample. The mean recovery obtained was 84.1% and the RSD was 2.7%. Experimental results also indicate that the degradation of the interferences and the extraction of the target analytes, PCBs could be carried out simultaneously. Compared with the traditional methods, the MAAD method is a rapid, efficient, and solvent-saving.

Ternary vapor-liquid equilibrium (VLE) data for the system dimethyl carbonate (1) -Methanol (2) -Chlorobenzene (3) were calculated using binary VLE data by Wilson equation, and these data can be used for building the mathematical model of extractive separating DMC and methanol.