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PubMed1979

Sample preparation of some shelled treenuts and peanuts in a vertical cutter-mixer for mycotoxin analysis.

A procedure has been devised for more rapid preparation of large samples of nutmeats (10-80 lb) for mycotoxin analysis without the use of grinding aids, by combining the grinding and mixing steps into one operation. Ease of cleaning equipment facilitates the preparation of several samples in a short time. The procedure was tested using shelled raw and roasted peanuts, and raw almonds, walnuts, and pecans. The reduction of particle size to pass a 20 mesh screen was attained using a 25 qt Hobart vertical cutter-mixer (VCM) equipped with standard serrated blades and a 40 qt VCM using either a standard serrated blade or a smooth-edge blade modified with sharp-edge notches to increase its cutting ability. Portions of ground composite were removed at various time intervals and particle sizes were measured. Original and check analyses of 12 naturally contaminated samples over a 3 year period indicate that this procedure is practical and reproducible.

Arachis↗
PubMed1998

Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing.

A procedure for visualization and sensitive detection of protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent or visible tags for certain amino acids in protein molecules, e.g., lysines modified with dansyl/dabsyl chloride and cystines/cysteines or phosphorylated serines modified with iodoacetamidofluorescein (I-15) after proper sample pretreatments. Modifications are performed prior to SDS-PAGE, eliminating the need for fixing, staining, and destaining as required for the conventional procedures. After electrophoresis, the fluorescent or visible bands are excised from the gel, homogenized in microcentrifuge tubes, and soaked in an appropriate buffer to release the separated proteins into solution. Enzymatic digestion can then be carried out in solution for better efficiency of digestion and recovery. The subsequent HPLC mapping and collection of protein digests are performed on PE Applied Biosystems Model 173A MicroBlotter. The separated peptides containing tagged amino acids are visible on the PVDF membrane and can be excised for direct sequence analysis. This approach has been employed for selectively isolating the lysine, cysteine, or phosphorylated serine containing peptides using model proteins. The sequencing results of the peptides generated from premodified proteins demonstrate that this approach facilitates sample preparation for microsequence analysis at low picomole level. Overall recoveries of 20-30% by sequencing initial yields have been achieved using our model proteins.

Amino Acid Sequence↗
PubMed2002

Recent applications of sample preparation techniques in food analysis.

Even with the emergence of advanced techniques of separation and identification, it is rarely possible to analyse food without manipulation. The traditional techniques for sample preparation are time consuming and require large amount of reagents, which are expensive, generate considerable waste, contaminate the sample and can enrich it for analytes. The more analytical techniques have become highly developed, the more has sample clean-up become important in order to fully take advantage of them. Due to the multiplicity of food matrices, it is not possible to use one sample preparation technique, so many methods have been proposed for meeting all the requirements. The newest variations of wet digestion, solvent and sorbent extraction and membrane separation are summarised and their most recent applications to food analysis are provided.

Food Analysis↗
PubMed2006

Simultaneous derivatization/preconcentration of volatile aldehydes with a miniaturized fiber-packed sample preparation device designed for gas chromatographic analysis.

A novel in-needle sample preparation device has been developed for the determination of volatile aldehydes in gaseous samples. The needle device is designed for the gas chromatographic (GC) analysis of aldehydes and ketones commonly found in typical in-house environments. In order to prepare the extraction device, a bundle of polymer-coated filaments was longitudinally packed into a specially designed needle. Derivatization reactions were prompted by 2,4-dinitrophenylhydrazine (NDPH) included in the needle, and so the aldehydes and ketones were derivatized to the corresponding hydrazones and extracted with the extraction needle. A reproducible extraction needle preparation process was established, along with a repeatable derivatization/extraction process that ensures the successful determination of aldehydes. The storage performance of the extraction needle was also evaluated at room temperature for three days. The results demonstrate the successful application of the fiber-packed extraction device to the preparation of a gaseous sample of aldehydes, and the future possibility of applying the extraction device to the analysis of in-house environments.

Journal Article↗
PubMed1998

Simple and broadly applicable sample preparation by use of magnetic glass particles.

A key issue in nucleic acid-based assays related to infectious disease testing and genetic testing is the sample preparation. This has to be convenient, efficient and suitable for automation. We describe a new method based on the use of glass magnetic particles. The steps involved are the lysis of bacterial cells and viral particles by chaotropic salts, binding of released nucleic acids to the magnetic glass particles, the removal of inhibitors and the release of purified nucleic acids ready for PCR amplification. The method fulfills most of the requirements for sample preparation such as sensitivity, universality and suitability for automation.

DNA↗
PubMed2006

Chromatographic quantitation at losses of analyte during sample preparation. Application of the modified method of double internal standard.

Known methods of quantitative chromatographic analysis (calibration, external standard, internal standard and standard addition) require the application of sample preparation techniques without significant losses of analytes. If this condition cannot be satisfied, the compensation of these losses should be provided. The modification of known method of quantitative chromatographic analysis (double internal standard), implying the addition of two homologues (previous and following) of target analytes as internal standards into initial samples is considered. This approach permits us to compensate significant losses both analytes and standards at all stages of sample preparation. The advantages of this method are demonstrated on the examples of liquid-liquid extraction, head space analysis (HSA), distillation of volatile compounds with volatile solvents (concentration in condensates) and evaporation of volatile solvents (concentrating in the residues of solvents). In all cases the application of two homologues as internal standards provides accurate results (the typical relative errors are within 1-6%) at the values of a factor of composition distortion of initial samples (K', the definition is suggested) from 0.2 up to 4. These results are in accordance with general relationships between variations in any physicochemical properties of organic compounds within homologous series. The single found exception was the evaporation of volatile solvents (the open phase transition process) when to get the results with relative errors not more then +10% requires the minimal changes in the composition of initial samples (K' values should not be more then approximately 1.5).

Calibration↗
PubMed2006

The essence of DNA sample preparation for MALDI mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important analytical technique in nucleic acid research. MALDI is used for quality control of oligonucleotides as well as for analyzing DNA markers. Sample preparation of nucleic acids is crucial for obtaining high-quality mass spectra. Sample purity, solvent content, suitable matrices, and substrate surfaces, as well as laboratory conditions affect spectra quality. This review presents essential information with regard to sample preparation, DNA modification chemistry, and DNA purification, along with a discussion of instrumental advances, which facilitate and extend the applicability of MALDI in genomics.

Chromatography↗
PubMed2002

High-speed gradient parallel liquid chromatography/tandem mass spectrometry with fully automated sample preparation for bioanalysis: 30 seconds per sample from plasma.

In this work, a high-throughput and high-performance bioanalytical system is described that is capable of extracting and analyzing 1152 plasma samples within 10 hours. A Zymark track robot system interfaced with a Tecan Genesis liquid handler was used for simultaneous solid-phase extraction of four 96-well plates in a fully automated fashion. The extracted plasma samples were injected onto four parallel monolithic columns for separation via a four-injector autosampler. The use of monolithic columns allowed for fast and well-resolved separations at a considerably higher flow rate without generating significant column backpressure. This resulted in a total chromatographic run cycle time of 2 min on each 4.6 x 100 mm column using gradient elution. The effluent from the four columns was directed to a triple quadrupole mass spectrometer equipped with an indexed four-probe electrospray ionization source (Micromass MUX interface). Hence, sample extraction, separation, and detection were all performed in a four-channel parallel format that resulted in an overall throughput of about 30 s per sample from plasma. The performance of this system was evaluated by extracting and by analyzing twelve 96-well plates (1152) of human plasma samples spiked with oxazepam at different concentrations. The relative standard deviation (RSD) of analyte sensitivity (slope of calibration curve) across the four channels and across the 12 plates was 5.2 and 6.8%, respectively. An average extraction recovery of 77.6% with a RSD of 7.7% and an average matrix effect of 0.95 with a RSD of 5.2% were achieved using these generic extraction and separation conditions. The good separation efficiency provided by this system allowed for rapid method development of an assay quantifying the drug candidate and its close structural analog metabolite. The method was cross-validated with a conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay.

Animals↗
PubMed2005

Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis.

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.

Albumins↗
PubMed2001

Comparison of various sample preparation methods for PCR diagnosis of visceral leishmaniasis using peripheral blood.

We have compared various sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and tested the influence of these protocols upon sensitivity. Four methods of lysis-DNA extraction were used with two types of blood samples: whole blood (WB) and buffy coat (BC). Comparisons were first carried out with seeded samples at various parasite concentrations. At high concentrations (> or = 1,000 parasites/ml), there were no significant differences in PCR sensitivity among the methods tested. At concentrations of < or = 100 parasites/ml, proteinase K (PK)-based methods proved clearly superior to guanidine-EDTA-based methods. Moreover, a 10-fold increase in sensitivity was observed for BC over that for WB. Thus, the best sensitivity was obtained with the BC prepared with PK-based methods. With this combination, the PCR reliably detected 10 parasites/ml but was inconsistent when the sample contained 1 parasite/ml of blood. The methods that yielded the highest sensitivities were evaluated with seven dogs and four human VL patients. Again, the utilization of the BC prepared with PK-based methods gave the best results. The optimization of each step of the assay (sample preparation, DNA extraction, and PCR conditions) yielded a highly sensitive tool for the diagnosis of VL using patient blood, thus avoiding more invasive diagnostic procedures and allowing the detection of low parasitemia during posttherapeutic follow-up.

Animals↗
PubMed2006

Fast gas chromatography-negative-ion chemical ionization mass spectrometry with microscale volume sample preparation for the determination of benzodiazepines and alpha-hydroxy metabolites, zaleplon and zopiclone in whole blood.

Fast gas chromatography/negative-ion chemical ionization mass spectrometric (GC/NICI-MS) assay combined with rapid and nonlaborious sample preparation is presented for the simultaneous determination of benzodiazepines and alpha-hydroxy metabolites, zaleplon and zopiclone in whole blood. The compounds were extracted from 100 microl of whole blood by simultaneous multitube, microscale liquid-liquid extraction (LLE) and derivatized by N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), without the need for the time-consuming concentration stage. In the analytical separation, various parameters of fast GC/NICI-MS were applied, e.g. the use of hydrogen as a GC carrier gas, a high carrier gas velocity, a small film thickness of the analytical column, fast MS data acquisition, fast temperature ramping, and high initial and final temperatures of GC column. Sensitive identification, screening and quantitation of 18 compounds of interest were achieved in chromatographic separation in only 4.40 min. Accurate and reproducible results were obtained by using five different and carefully selected deuterated analogues on the basis of the chemical properties of the target analytes. Nevertheless, for alpha-OH-midazolam, and for bromazepam and flunitrazepam at low concentrations, the results can be considered only semiquantitative on the basis of the validation data. The extraction efficiencies ranged from 74.3 to 105.7% and the limits of quantitation (LOQ) from 1 to 100 ng ml(-1). Rapid sample preparation and fast chromatographic separation allowed cost-efficient, reliable and high sample-throughput analyses with a low amount of manual work. The method was fully validated and accredited according to EN ISO/IEC 17025 standards and is applicable for sensitive, reliable and quantitative determination of benzodiazepines, zaleplon and zopiclone, e.g. in clinical and forensic toxicology.

Acetamides↗
PubMed2004

Comparison of sample preparation procedures for the determination of trace heavy metals in house dust, tobacco and tea samples by atomic absorption spectrometry.

The effect of wet ashing, dry ashing and microwave procedure for the determination of trace metal levels was investigated in house dust, tobacco and tea samples by atomic absorption spectrometry. The study of sample preparation procedures showed that the microwave method was the best. The recovery of trace metals was very good and precision and accuracy were compatible with standard reference material. The relative standard deviations for all measured metal concentrations were lower than 10%. The digestions of HNO3/H2SO4/HClO4 (4: 1: 1) mixture for house dust, HNO3/H2SO4/H2O2 (2: 2: 2) mixture for tea and HNO3/H2O2 (4: 2) mixture for tobacco were very efficient.

Dust↗
PubMed1998

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching.

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 microm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5+/-2.8% with a linear range of 0.1-100 microg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.

Acetonitriles↗
PubMed2003

Comparison of methods for sample preparation of individual rat cerebrospinal fluid samples prior to two-dimensional polyacrylamide gel electrophoresis.

Different pre-treatment methods have been compared for two-dimensional mapping of individual rat cerebrospinal fluid samples based on acetone, trichloroacetic acid/acetone and methanol/acetone precipitation of proteins. Acetone precipitation following incubation with DTT gave the highest protein recovery (72%) and the largest number of protein spots (92 +/- 4) as well as minimizing the time taken.

Acetone↗
PubMed1993

Image analysis versus flow cytometry for DNA ploidy quantitation of solid tumors: a comparison of six methods of sample preparation.

With the availability of user-friendly interactive image analysis instruments for DNA analysis, there is a growing need for comparison with the established methodology of flow cytometry. We have compared the results of DNA ploidy quantitation in 12 solid tumors prepared by six different techniques of sample preparation: flow cytometry of fresh cell suspensions and of nuclei isolated from formalin-fixed, paraffin-embedded tissue; and image analysis of touch preparations, of disaggregated cells from paraffin-embedded tissue as well as of 3- and 7-microns-thick tissue sections. Complete agreement in DNA ploidy results obtained by the six methods was found in six out of 12 solid tumors. Image analysis of touch preparations detected most tetraploid and multiple aneuploid peaks. Sections of 7-microns-thick tissue gave better histogram quality than 3-microns-thick sections, however tetraploid peaks were not resolved in one case. Image analysis of disaggregated paraffin-embedded tumor showed comparable ploidy to fresh touch preparations in seven out of 12 cases, the discrepancies being due to loss of tetraploid or multiple aneuploid peaks. Flow cytometry gave good histograms, but tetraploid and multiple aneuploid peaks were occasionally not detected. Each method presents advantages and disadvantages. Flow cytometry and image analysis are complementary methods for DNA quantitation, and more than one method may be necessary to confirm the DNA content of solid tumors.

Cell Nucleus↗
PubMed2004

Sub-sampling and preparing forensic samples for pollen analysis.

The main forensic application of palynology is in providing associative evidence, assisting to prove or disprove a link between people and objects with places or with other people. Although identification and interpretation of pollen is a specialist job, sub-sampling and preparing pollen samples for analysis may be carried out by non-specialists. As few forensic laboratories have residing palynologists, laboratories may wish to reduce the cost of analysis or risk of contamination by doing their own sub-sampling and preparation. Presented is a practical guide for sub-sampling and preparing forensic samples for pollen analysis, providing a complete standard procedure for both the palynologist and non-specialist. Procedures for sub-sampling include a wide variety of materials commonly collected for forensic analysis (soil, clothing and other fabrics, footwear, twine and rope, firearms, granulated materials, plant and animal material, and illicit drugs), many of which palynologists will not be familiar with. Procedures for preparation of samples (pollen concentration) are presented as a detailed, step-by-step method. Minimizing the risks of laboratory and cross-sample contamination during sub-sampling and preparation is emphasized.

Animals↗
PubMed1990

[Human papillomavirus typing by molecular hybridization in situ with biotinylated probes. Optimization of sample preparation and hybridization time].

In situ hybridization with non radioactive probes is more and more used to detect viral infections, especially human papillomavirus (HPV) infections. The quality of the reaction depends on several factors, such as sample preparation (including fixation and pretreatments). Their importance was evaluated on a model with cell lines including CaSki cells (harboring about 600 copies of HPV 16 DNA per cell) and Hela cells (containing 10-50 copies of HPV 18 DNA per cell). These cell lines were chosen in order to evaluate cytological and histological difficulties. Several parameters were studied: preparation of samples, fixation and hybridization duration. DNAs of biotinylated probes of HPV types 16 and 18 and cells were simultaneously denatured 10 min at 95 degrees C. Hybridization was carried out at 37 degrees C for various periods of time; it was followed by a 3-step reaction for detection of biotinylated DNA-DNA hybrids with immunoenzymatic staining using streptavidin-alkaline phosphatase complex. Typical intranuclear granulations were seen either in cell deposits fixed with acetone, methanol-acetic acid, paraformaldehyde, formaline, Bouin's, Baker's or Carnoy's fixatives; or in cytocentrifuged cells fixed with formaline, Bouin's or Baker's fixatives. The detergent pretreatment was unnecessary. On the contrary, the protease pretreatment was required with formaline, Bouin's or Baker's fixatives. In order to detect constantly HPV 16 in CaSki cells and HPV 18 in HeLa cells, hybridization should be performed for more than 4 h. The sensitivity of the technique could therefore be evaluated to few copies of HPV DNA per cell. This technique is reliable, sensitive and rapid; et can be applied to biopsy specimens fixed with Bouin's or Baker's fixatives and paraffin-embedded; it allows routine detection of HPV infections.

Biotin↗
PubMed1999

PCR assay for detection of the E. coli O157:H7 eae-gene and effect of the sample preparation method on PCR detection of heat-killed E. coli O157:H7 in ground beef.

A PCR assay targeting the 3' end of the eae-gene of E. coli O157:H7 has been developed. It was shown to be specific for the E. coli O157:H7 eae-gene. Sensitivity of the PCR assay was 1 pg DNA or 10(3) cfu per PCR reaction. Subsequently, a study was conducted to examine the effect of the food matrix and the sample preparation method on PCR detection of non-viable cells using heat-killed E. coli O157:H7 in ground beef as a model system. Inoculated ground beef samples were subjected to either selective enrichment or immediately prepared for PCR analysis. Four sample preparation methods were applied: centrifugation, buoyant density centrifugation (BDC), immunomagnetic separation (IMS), and chelex extraction. Production of false-positive results due to amplification of the DNA of dead cells occurred if the number of heat-killed cells exceeded 10(8) cfu/g. For inocula <10(8) cfu/g, PCR results for heat- killed E. coli O157:H7 cells depended upon the sample preparation method. It was shown that the inclusion of two washings steps of the bacterial pellet before DNA extraction was the critical factor for elimination of false-positive results due to heat-killed cells in the centrifugation and BDC procedure. IMS did not produce false-positive results due to heat-killed cells (two washing steps were included in the IMS procedure). With the chelex-extraction method, false-positive results due to heat-killed cells were obtained. An effect of the ground beef food matrix on the presence of amplifiable DNA was noted.

Adhesins, Bacterial↗