Comparative study of various extraction methods for the quantitative determination of free amino acids from brain tissue

Seven published extraction methods for the quantitative determination of the amino acids of biological fluids and tissues were applied to brain tissue and the experimental data analyzed statistically.     

新生隐球菌基因组DNA不同抽提方法的比较

目的 DNA是进行分子生物学研究的重要基础。在本研究中,我们建立了2种简单快速抽提基因组DNA的方法并可用作PCR扩增的模板。通过比较4种不同的DNA抽提方法以确定哪种更适合进行下一步的基因分析。方法这4种方法是:玻璃珠法,酶法,3%SDS法和氯化苄法。玻璃珠法是用玻璃珠在混漩器上剧烈振荡破碎细胞壁;3%SDS法是将细胞在含10mmol/LDTT的3%SDS溶液中加热,然后用5mmol/LKAc和异丙醇抽提,DNA的产量通过A260测定。结果 3%SDS溶解法、经典酶法、玻璃珠法和氯化苄法的DNA产量分别为0.4154±0.0367、0.8484±0.0756、1.2636±0.2040、0.4070±0.0339(g/L×108CFU/mL)。结论玻璃珠法是最敏感、重复性好、简单、费用合理的抽提方法 。     

High throughput method for the determination of organochlorine pesticides and polychlorinated biphenyls in human serum

An improved method for the determination of selected organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum was developed. The method requires low volume of serum (500 microl) and 48-96 samples per day can be prepared by one analyst without special automatic equipment. Initial extraction was performed using 96-well solid-phase extraction disk plates and was followed by a clean-up with silica gel/sulfuric acid. Different denaturation, elution and clean-up conditions were tested. Quantification was carried out by gas chromatography equipped with electron capture detector (GC-ECD) or mass spectrometer (GC-MS). Recoveries of PCB congeners 28, 52, 101, 118, 138, 153 and 180 and OCPs HCB, beta-HCH, p,p'-DDE and p,p'-DDT at two spiking levels (n=8) varied from 57 to 120%, and intra-day relative standard deviation from 1 to 11%, both depending on spiking level and compound. Inter-day relative standard deviation was <15% in all cases. Limit of quantification (LOQ) for these PCBs ranged from 0.08 to 0.13 ng/ml and for these OCPs from 0.16 to 0.40 ng/ml. The optimized method was applied to the analysis of 1000 serum samples from different places of Spain.     

A simple and fast extraction method for organochlorine pesticides and polychlorinated biphenyls in small volumes of avian serum

A solid-phase extraction (SPE) method was developed using 8M urea to desorb and extract organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs) from avian serum for analysis by capillary gas chromatography with electron capture detection (GC-ECD). The analytes were efficiently extracted from the denatured serum-lipoprotein-analyte complex by one passage through an Oasis((R)) hydrophilic-lipophilic-balanced (HLB) SPE cartridge. No further clean-up was necessary, the entire extraction procedure and GC-ECD analysis can be accomplished in less than 3h. Serum volumes ranged from 100 microL to 1 mL with absolute recoveries of 90-101% for PCBs and 74% to 101% for the OC pesticides.     

Current trends in solid-phase-based extraction techniques for the determination of pesticides in food and environment

Solid-phase extraction (SPE) procedures for pesticide residues in food and environment are reviewed and discussed. The use of these procedures, which include several approaches such as: matrix solid-phase dispersion (MSPD), solid-phase micro-extraction (SPME) and stir-bar sorptive extraction (SBSE), represents an opportunity to reduce analysis time, solvent consumption, and overall cost. SPE techniques differ from solvent extraction depending on the interactions between a sorbent and the pesticide. This interaction may be specific for a particular pesticide, as in the interaction with an immunosorbent, or non-specific, as in the way a number of different pesticides are adsorbed on apolar or polar materials. A variety of applications were classified according to the method applied: conventional SPE, SPME, hollow-fiber micro-extraction (HFME), MSPD and SBSE. Emphasis is placed on the multiresidue analysis of liquid and solid samples.     

Serum organochlorine pesticides residues and risk of cancer: A case-control study

Organochlorine pesticides (OCPs) are frequently used worldwide as insecticides, fungicides, herbicides and termiticides and have been associated with a variety of cancers in animal and human studies. In the present study, we examined residues of fourteen OCPs in the serum samples of diagnosed cancer patients and healthy residents of Karachi, Pakistan. A random collection of fasting blood samples was carried out from the donors with informed consent. Serum was separated within 2?h of blood collection and was then subjected to extraction with organic solvents followed by purification with florisil column. The final organic extract of each serum sample was processed with Gas Chromatograph coupled with Electron Capture Detector (GC-ECD). OCPs were detected in 97.59% of the cancer cases and 93.75% of the healthy subjects. Mean concentrations of total OCPs (ΣOCPs) was found elevated in the cancer group (0.606?mg/kg) compared with the control group (0.322?mg/kg). Endosulfan was the highest prevalent OCP with a mean concentration of 0.214?mg/kg in the cancer group and 0.166?mg/kg in the control group. The second most prevalent OCP was 4,4-DDE with a mean concentration of 0.131?mg/kg in the cancer group and 0.019?mg/kg in the control group. Highest level of ΣOCPs was detected in the breast cancer cases (20.411?mg/kg) with a mean level of (2.041?mg/kg). In light of the obtained results and available literature on the subject, it has been concluded that OCPs are positively associated with the risk of various cancers in humans.     

Technical note: evaluation of extraction methodologies for the determination of an organochlorine pesticide residue in vegetation

Numerous extraction methodologies are used to quantify pesticide levels in vegetation. Sample availability, resource use, efficiency, time consumption, space allocation, and cost vary considerably among the commonly employed techniques. A study was conducted to compare the efficiency of microwave assisted extraction (MAE), blender homogenized extraction (BE), Soxhlet extraction (SE), the QuEChERS ("Quick, Easy, Cheap, Effective, Rugged, and Safe") method, and a simple oven assisted extraction (OAE), to recover p,p'-DDE from the tissues of Cucurbita pepo. A hot-solvent soak of stem or root tissues in a 2-propanol/hexane mixture, OAE yields recoveries that are statistically equivalent to the other procedures. The method recovered 1800 +/- 190 ng g(-1) and 8100 +/- 900 ng g(-1) (BCF = 87 +/- 9.7) p,p'-DDE from stem and root tissue, respectively. Recoveries for the other methods ranged from 1400-2200 ng g(-1) for the stems and 3600-7200 ng g(-1) for the roots. Statistical analyses for stem and root extraction indicate that there is no significant difference among the variances of each method. Given its simplicity, precision, and efficiency, OAE appears to be suitable for the extraction of an organic pollutant such as p,p'-DDE from plant tissues and for use in phytotechnology development and risk assessment.     

Biosorption of organochlorine pesticides using fungal biomass

Cladosporium strain AJR³18,501 was tested for its ability to sorb the organochlorine pesticide (OCP) p,p′-DDT from aqueous media. When p,p′-DDT was added to distilled water, ethanol or 1-propanol solutions in excess of its solubility, p,p′-DDT was sorbed onto the fungal biomass. Increasing the amount of p,p′-DDT in solution by changing the medium composition increased sorbent uptake: p,p′-DDT uptake by the fungal biomass was 2.5 times greater in 25% 1-propanol (17 mg of p,p′-DDT g⁻¹ dry weight fungal biomass) than in distilled water. When p,p′-DDT was dissolved in 25% 1-propanol (12 mg l⁻¹), rapid p,p′-DDT sorption occurred during the first 60 min of incubation. p,p′-DDT in solution was reduced to 2.5 mg l⁻¹ with the remaining p,p′-DDT recovered from the fungal biomass. A number of environmental parameters were tested to determine their effect on p,p′-DDT biosorption. As arsenic (As) is prevalent at DDT-contaminated cattle dip sites, its effect on p,p′-DDT uptake was determined. The presence of As [As(III) or As(V) up to 50 mg l⁻¹] did not inhibit p,p′-DDT uptake and neither As species could be sorbed by the fungal biomass. Changing the pH of the medium from pH 3 to 10 had a small effect on p,p′-DDT sorption at low pH indicating that an ion exchange process is not the major mechanism for p,p′-DDT sorption. Other mechanisms such as Van der Waals forces, chemical binding, hydrogen bonding or ligand exchange may be involved in p,p′-DDT uptake by Cladosporium strain AJR³18,501. Journal of Industrial Microbiology & Biotechnology (2002) 29, 163–169 doi:10.1038/sj.jim.7000280 Received 15 January 2002/ Accepted in revised form 18 May 2002     

Comparative study of various methods for the extraction of free creatine and phosphocreatine from mouse skeletal muscle

The efficacy of various media regarding the extraction of free creatine and phosphocreatine of mouse skeletal muscle was evaluated. In anesthetized animals tissue was quick-frozen in situ and removed by means of a modified Rongeur forceps cooled in liquid N₂. Homogenization of muscle tissue in 1 m EDTA in 50% (v/v) ethanol at −20°C, which was gradually diluted with ice-cold 0.4 perchloric acid to a final concentration of 0.3 perchloric acid in 12.5% ethanol proved to be the most suitable procedure regarding rapid handling of tissue samples, recovery of total creatine, and the ratio of phosphocreatine to total creatine. Phosphocreatine values as high as 78% of total creatine of skeletal muscle were thus obtained. Extraction of free creatine and phosphocreatine with concentrated ethanolic solutions (50–80%, v/v) was found to be incomplete apparently due to irreversible binding of creatine and phosphocreatine to protein precipitates.     

Comparative characteristics of methods for the immunoenzyme determination of summary immunoglobulins

Two modifications of ELISA for summary immunoglobulin determination (with and without the use of the antibody-enzyme conjugate) are described with special attention to their sensitivity, reproducibility and the influence of different conditions on assay characteristics. The sensitivity of the assay carried out with the use of the conjugate lies in the nanogram range of concentration. The modification of the assay, not requiring the use and, therefore, the synthesis of the conjugate, may be used with antisera of different specificity.     

Comparative study of different extraction methods and assays of tannins in some pteridophytes]

Various processes of extraction and quantitative analysis of a condensed tannin in a plant extract, which also includes some chlorogenic acids, have been examined. 60% methanol, at 50 degrees C, proved the most efficient extraction solvent. Several methods of analysis have been tried. The measure of the colour intensity obtained by the action of sulphuric vanilline on flavanols cannot be used because it depends on the tannin condensation stage. It is impossible to separate tannin from chlorogenic acids using the methods of adsorption by skin or nylon powders, or precipitation by polyvinylpyrrolidone. Only paper chromatography, followed by the distinct elution of the various phenolic compounds, allows the tannin evaluation by subtraction; but owing to the variability of the results, many more experiments are necessary. Some other processes are being studied.     

Extraction and recovery of organochlorine pesticides from fungal mycelia

An extraction method was developed to recover organochlorine pesticides (OCPs) associated with live and mercuric chloride-treated fungal mycelia. A Cladosporium sp. was inoculated into potato dextrose broth, DDT (90 mg/l) added and incubated for seven days. The combination of a microextraction procedure for aqueous-phase-associated DDT and a sonication extraction for mycelia-bound DDT, using dichloromethane as the extracting solvent, resulted in the recovery of 31-51% of added DDT. DDT recovery was increased to 62-65% by grinding the fungal mycelia before sonication. Alkali and nitric acid pretreatments were tested to increase the recovery of DDT associated with fungal mycelia, however, these treatments resulted in the production of unidentified DDT transformation products. Pretreating mycelia containing DDT in concentrated hydrochloric acid at 60 degrees C for 2, 4 and 6 h resulted in DDT recoveries of 90-91%, 99% and 101-102% respectively without the production of transformation products. When an OCP mixture (DDT, DDD and DDE) was added to fungal mycelia, between 89% and 96% of DDT, DDD and DDE were recovered from live cultures compared to 85-91% in mercuric chloride-treated cultures using the microextraction/hydrochloric acid pretreatment (60 degrees C/6 h) sonication extraction method.