Determination of catechins and catechin gallates in tissues by liquid chromatography with coulometric array detection and selective solid phase extraction

Catechins levels in organ tissues, particularly liver, determined by published methods are unexpectedly low, probably due to the release of oxidative enzymes, metal ions and reactive metabolites from tissue cells during homogenization and to the pro-oxidant effects of ascorbic acid during sample processing in the presence of metal ions. We describe a new method for simultaneous analysis of eight catechins in tissue: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG) and (-)-epigallocatechin gallate (EGCG) (Fig. 1). The new extraction procedure utilized a methanol/ethylacetate/dithionite (2:1:3) mixture during homogenization for simultaneous enzyme precipitation and antioxidant protection. Selective solid phase extraction was used to remove most interfering bio-matrices. Reversed phase HPLC with CoulArray detection was used to determine the eight catechins simultaneously within 25 min. Good linearity (>0.9922) was obtained in the range 20-4000 ng/g. The coefficients of variance (CV) were less than 5%. Absolute recovery ranged from 62 to 96%, accuracy 92.5 +/- 4.5 to 104.9 +/- 6%. The detection limit was 5 ng/g. This method is capable for determining catechins in rat tissues of liver, brain, spleen, and kidney. The method is robust, reproducible, with high recovery, and has been validated for both in vitro and in vivo sample analysis.     

Comparison of homogenization with the GentleMacs Dissociator vs conventional methods for routine tissue processing

Tissue specimens are valuable materials for microbiological diagnosis. The method of tissue processing can have a significant effect on sensitivity. This study aimed to compare different biopsy processing methods in terms of efficacy and standardization. Pork tissue artificially inoculated with Staphylococcus aureus and Escherichia coli, and samples of infected human tissue were processed by different methods before culture, and the results compared. Bacterial recovery from artificially inoculated pork tissue was significantly higher by homogenization with GentleMacs Dissociator than with sonication. No significant difference was observed between the GentleMacs Dissociator and manual treatment with a scalpel and vortexing. The microbial yield from homogenized human tissues was significantly higher after homogenization with GentleMacs Dissociator than with the conventional method. Homogenization with the GentleMacs Dissociator retrieves bacteria from tissue effectively. Tissue homogenization with the Dissociator is easy and fast to perform and allows for a high degree of standardization.     

Protein Extraction From Leaves of Aloe vera L., A Succulent and Recalcitrant Plant, for Proteomic Analysis

An improved method for extracting proteins from leaf tissues of Aloe vera L., a recalcitrant plant species, for proteomic analysis is presented. In this protocol, the following critical components are included. A washing step is added prior to homogenization of the tissue to eliminate contaminants, and a concentrated 2× extraction buffer (pH 7.5) is used to increase protein yield. Compared to classical trichloroacetic acid–acetone and phenol extraction methods, this novel protocol has yielded two-dimensional electrophoresis gels with minimal (if any) streaking and provided high-quality protein samples. This protocol is expected to be applicable to other recalcitrant plant tissues.     

A new method for isolation of polyamines from animal tissues

A new method for isolation of polyamines from tissues was developed and compared with the butanol extraction method which has been widely used for quantitative determination of polyamines. In the new method protein-free tissue extracts are applied to a small Dowex-50 column. The column is washed with appropriate buffers to remove ninhydrin-positive contaminants and the polyamines are eluted. With this method, the overall recovery of polyamines, after separation by paper electrophoresis and subsequent colorimetric determination with ninhydrin, is always over 90% (average 95%). This method is much better than the butanol method, which gives variable recoveries of 70–90%. The new method also has the advantages over the butanol method that the isolated polyamines are purer and the procedure is simpler.     

A simple and efficient method for isolation of DNA in high mucilaginous plant tissues

A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was used with healthy Bixa orellana and virus-infected Malvaceae plants.     

Quantitative analysis of polychlorinated biphenyls, organochlorine insecticides, polycyclic aromatic hydrocarbons, polychlorinated hydrocarbons and polynitrohydrocarbons in spiked samples of soil, water and plasma by selected-ion monitoring gas chromatography–mass spectrometry

A broad range of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated hydrocarbons (PCHs), polynitrohydrocarbons (PNHs), polychlorinated biphenyls (PCBs) and organochlorine (OCs) insecticides were simultaneously analyzed in spiked soil, water or plasma samples by using gas chromatography–mass spectrometry (GC–MS). Water and plasma samples containing the pollutants were extracted by a solid-phase extraction (SPE) method using florisil columns. The soil samples, fortified with the toxicants, were extracted with water, methanol or dichloromethane (DCM). The water extract was processed by the SPE method. The methanol and DCM samples were dried, dissolved in acetonitrile and subjected to the SPE extraction. The extracted samples were analyzed by GC–MS programmed to monitor selected ions. The deuterium labelled compounds were used as the internal standards. The chromatographic profile of total ions indicated complete separation of some compounds such as isophorone, naphthalene, all PCBs, most OC insecticides and PNHs; high M_r PAHs and some PCHs were partially or incompletely separated. The chromatographic profile of individual ion indicated good separation of each ion. The minimum detection limit ranged from 1 to 4 pg injected when 1 or 2 ions were monitored or from 20 to 200 pg injected when 20 ions were monitored. The SPE method that provided 60–105% recovery of pollutants from water samples, provided only 2–60% recovery from plasma samples. This may be due to the binding of pollutants to plasma proteins. Water recovered 1–30%, while methanol or DCM recovered 65–100% of the pollutants added to the soil samples. The use of internal standards corrected for the loss of pollutants from plasma or soil.     

Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography–electrospray tandem mass spectrometry

We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC–MS–MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C₁₈ column (125×3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate–acetonitrile as the mobile phase at a flow-rate of 0.7 ml min⁻¹. Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC–MS–MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL–2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.     

Method for recovery of intact DNA for community analysis of marine intertidal microbial biofilms

A protocol is described for rapid DNA isolation from marine biofilm microorganisms embedded in large amounts of exopolysaccharides. The method is a modification of the hot phenol protocol used for plants tissues, where nonexpensive and easily available enzymes were used. The method is based on the incubation of biofilm biomass samples in an extraction buffer mixed with phenol preheated at 65°C. The procedure can be completed in 2 h and up to 20 samples can be processed simultaneously with ease and DNA of excellent quality, as shown by successfully amplification of polymerase chain reaction (PCR) products. DNA was recovered from a range of intertidal marine biofilms with varying amounts of exopolysaccharides.     

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.     

Determination of lamivudine in plasma, amniotic fluid, and rat tissues by liquid chromatography

An HPLC method for the quantification of lamivudine (3TC) in rat plasma, amniotic fluid, placental and fetal tissues has been developed, validated and applied to the study of the placental transport of this drug in the pregnant rat. Placental and fetal tissues were processed using liquid-liquid extraction enhanced by salting out the sample using a saturated solution of ammonium sulfate. Plasma and amniotic fluid samples were processed by protein precipitation using 2 M perchloric acid. Reverse phase chromatography was performed using a phenyl column (5 microm, 150 mm x 2 mm i.d.) under a flow rate of 0.2 ml/min. The mobile phase consisted of 5% methanol in 20 mM dibasic phosphate buffer (pH 6). The method was validated over the range from 0.1 to 50 microg/ml for plasma and amniotic fluid and 0.2-50 microg/ml for the placental and fetal tissues.     

An Improved Method for the Isolation of Total RNA from Malva pusilla Tissues Infected with Colletotrichum gloeosporioides

A modified RNA extraction method was developed for fungal and plant tissues that has several advantages compared with existing, published methods for materials with high contents of ribonuclease, phenolics and polysaccharides. The method uses guanidinium isothiocyanate in the homogenization buffer, and then hexadecyltrimethylammonium bromide (CTAB). soluble polyvinylpyrrolidone and insoluble polyvinylpolypyrrolidone in the extraction buffer. The procedure consistently produced a high yield of relatively high purity, undegraded RNA from a variety of biological materials and should be useful for other tissues where RNases, phenolics and sticky polysaccharides are a problem.     

A Rapid and Simple Method for Estimating Sulfate Reduction Activity and Quantifying Inorganic Sulfides

A simplified passive extraction procedure for quantifying reduced inorganic sulfur compounds from sediments and water is presented. This method may also be used for the estimation of sulfate reduction rates. Efficient extraction of FeS, FeS(inf2), and S(sup2-) was obtained with this procedure; however, the efficiency for S(sup0) depended on the form that was tested. Passive extraction can be used with samples containing up to 20 mg of reduced sulfur. We demonstrated the utility of this technique in a determination of both sulfate reduction rates and reduced inorganic sulfur pools in marine and freshwater sediments. A side-by-side comparison of the passive extraction method with the established single-step distillation technique yielded comparable results with a fraction of the effort.     

An improved method for determining phytoplankton chlorophyll a concentration without filtration

The common and routine procedure for the quantification of chlorophyll a (chl a) in aquatic studies has a series of steps. Here, we sought to find optimal conditions for phytoplankton cell harvesting, chl a extraction, and chl a measurement and calculation, to find an effective, cost-saving, safe, and environment-friendly procedure for determining phytoplankton chl a concentration. We replaced the traditional GF/C filters with inorganic polymer flocculants (IPFs) and clay for phytoplankton harvesting, and then various solvents (acetone, ethanol, DMF, and DMSO), IPFs (PAC, PFS, and PAFS) and clay were tested for their suitability for chl a extraction, with or without homogenization at different temperatures for different extraction durations. About 0.3–1.0 g l^?1 of PAC or PFSA combined with 1.0–2.5 g l^?1 clay were found to provide optimal conditions in terms of yield and cost for phytoplankton cell harvesting from water samples. Based on our results, we recommend flocculation and centrifugation instead of glass-fiber membrane filtration for harvesting phytoplankton cells from environmental water samples, 95% ethanol for chl a extraction without homogenization and heating, and spectrophotometry to determine chl a concentration.     

Evaluation of automated analysis of 15N and total N in plant material and soil

Simultaneous determination of ¹⁵N and total N using an automated nitrogen analyser interfaced to a continuous-flow isotope ratio mass spectrometer (ANA-MS method) was evaluated. The coefficient of variation (CV) of repeated analyses of homogeneous standards and samples at natural abundance was lower than 0.1%. The CV of repeated analyses of ¹⁵N-labelled plant material and soil samples varied between 0.3% and 1.1%. The reproductibility of repeated total N analyses using the automated method was comparable to results obtained with a semi-micro Kjeldahl procedure. However, the automated method gave results which were 3% to 5% higher than those obtained with the Kjeldahl procedure. Since only small samples can be analysed, careful sample homogenization and fine grinding are very important. Evaluation of a diffusion method for preparing nitrate and ammonium in solution for automated ¹⁵N analysis showed that the recovery of inorganic N in the NH₃ trap was lower when the N was diffused from water than from 2 M KCl. The results also indicated that different proportions of the NO₃ ^- and the NH₄ ⁺ in aqueous solution were recovered in the trap after combined diffusion. The method is most suited for diffusing either NO₃ ^- or NH₄ ⁺ alone, but can be used for combined diffusion of the two ions.     

Emerging Lyngbya wollei toxins: A new high resolution mass spectrometry method to elucidate a potential environmental threat

Mass spectrometric methods for the quantitative and qualitative analyses of algal biotoxins are often complicated by co-eluting compounds that present analytically as interferences. This issue is particularly critical for organic polyamines, where co-eluting materials can suppress the formation of cations during electrospray ionization. Here we present an extraction procedure designed specifically to overcome matrix-derived ion suppression of algal toxins in samples of Lyngbya wollei, a filamentous benthic algae known to produce several saxitoxin analogues. Lyngbya wollei samples were collected from a large, persistent harmful algal bloom in Lake Wateree, SC. Six known Lyngbya wollei-specific toxins (LWT1–6) were successfully resolved and quantified against saxitoxin using hydrophilic interaction liquid chromatography coupled with triple quadrupole and quadrupole time-of-flight mass spectrometry. The parent ions [M²⁺ – H⁺]⁺ were observed for LWTs 1–6 and the [M]²⁺ ion was observed for LWT5. High resolution mass spectra and unique fragmentation ions were obtained for LWTs 1–6. A dilution factor of 50 resulted in a linear calibration of saxitoxin in the algae matrix. Ion suppression was resolved by sample dilution, which led to linear, positive correlations between peak area and mass of the extracted sample (R² > 0.96). Optimized sample extraction method and instrument parameters are presented.     

Procedure for isolation of gangliosides in high yield and purity: simultaneous isolation of neutral glycosphingolipids

While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.     

Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

Protein estimation in tissues containing high levels of lipid: modifications to Lowry's method of protein determination

A method to estimate protein in detergent-solubilized homogenates of lipid-rich biological samples (e.g., adipose tissue, myelin-enriched fractions of sheep brain) is described. The method is also suitable for samples in which protein is present as a protein-detergent complex. The method involves homogenization of tissue in the presence of a suitable detergent and KCl. Protein is then estimated in an aliquot of this homogenate by Lowry's method in the presence of excess sodium dodecyl sulfate, the solutions being clarified by extraction with ethyl acetate. Protein solubilization by Triton X-100 from adipose tissue was biphasic, extracting two to three times more protein under optimum conditions [1.7 +/- 0.1% (v/v) Triton X-100 and 0.75 M KCl], compared with homogenization without salt and detergent. Unlike adipose tissue, protein solubilization from myelin-enriched fractions of sheep brain peaked at 1% (v/v) Triton X-100, resulting in the extraction of approximately three times more protein than homogenization in the absence of detergent and salt.     

A simple and general plant tissue extraction procedure for two-dimensional gel electrophoresis

A fast and simple extraction procedure of plant tissue for two-dimensional gel electrophoresis is presented. The procedure is especially useful for the extraction of plant cell suspension cultures, callus and other plant tissues having a high content of phenol oxidases, polysaccharides, polynucleotides, terpenoids and other substances interfering with isoelectric focusing. Due to the speed of the extraction procedure (about 20 min), large numbers of samples containing only milligram amounts of tissue can be easily processed. The simplicity of the method makes it particularly suitable for the extraction of radiolabeled tissues (³⁵S, ³²P). This method is perfectly compatible with silver staining, autoradiography and Western blotting analysis.Abbreviations DTT dithiothreitol - IEF isoelectric focusing - SDS sodium dodecyl sulfate - TEMED N,N,N,N-tetramethylethylenediamine     

An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,<Emphasis Type="Italic">Theobroma cacao</Emphasis> L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.