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PubMed2007

Ethanol analysis from biological samples by dual rail robotic autosampler.

Detection, identification, and quantitation of ethanol and other low molecular weight volatile compounds in liquid matrices by headspace gas chromatography-flame ionization detection (HS-GC-FID) and headspace gas chromatography-mass spectrometry (HS-GC-MS) are becoming commonly used practices in forensic laboratories. Although it is one of the most frequently utilized procedures, sample preparation is usually done manually. Implementing the use of a dual-rail, programmable autosampler can minimize many of the manual steps in sample preparation. The autosampler is configured so that one rail is used for sample preparation and the other rail is used as a traditional autosampler for sample introduction into the gas chromatograph inlet. The sample preparation rail draws up and sequentially adds a saturated sodium chloride solution and internal standard (0.08%, w/v acetonitrile) to a headspace vial containing a biological sample, a calibrator, or a control. Then, the analytical rail moves the sample to the agitator for incubation, followed by sampling of the headspace for analysis. Using DB-624 capillary columns, the method was validated on a GC-FID and confirmed with a GC-MS. The analytes (ethanol, acetonitrile) and possible interferences (acetaldehyde, methanol, pentane, diethyl ether, acetone, isopropanol, methylene chloride, n-propanol, and isovaleraldehyde) were baseline resolved for both the GC-FID and GC-MS methods. This method demonstrated acceptable linearity from 0 to 1500 mg/dL. The lower limit of quantitation (LOQ) was determined to be 17 mg/dL and the limit of detection was 5 mg/dL.

Biomarkers↗
PubMed1998

Chromatographic removal of endotoxin from hemoglobin preparations. Effects of solution conditions on endotoxin removal efficiency and protein recovery.

In this work, affinity chromatography was used to remove endotoxin from human hemoglobin preparations with a Sterogene Acticlean Etox column. The effects of solution conditions on endotoxin removal efficiency and protein recovery have been investigated. It has been found that cations Na(+) or Ca(2+) reduced endotoxin removal efficiency from 73% (sample prepared with endotoxin-free water) to 31% (sample prepared with 0.15 M NaCl, ionic strength, I = 0.15 M), and from 73% sample prepared with endotoxin-free water) to 9% (sample prepared with 0.05 M CaCl2, ionic strength, I = 0.15 M). It has also been found that the protein recovery was increased from 90% to 99%, respectively.

Calcium Chloride↗
PubMed2005

Quantification of ACE inhibiting peptides in human plasma using high performance liquid chromatography-mass spectrometry.

An HPLC-MRM-MS method was developed for the quantification of 17 small ACE inhibiting (ACEI) peptides in plasma samples collected from human volunteers after the consumption of a peptide-enriched drink. The assay shows the high selectivity and sensitivity necessary to monitor small changes in the levels of the ACEI peptides after consumption of drinks developed to effect lowering of the blood pressure. Four different sample preparation methods were tested and evaluated. The final sample preparation method selected is simple and effective and consists mainly of the removal of proteins by acidification and heating, followed by a large volume injection. Additional sample preparation steps such as solid phase extraction and liquid/liquid partitioning were studied. Although they resulted in cleaner extracts, losses of specific peptides such as SAP were frequently seen. The isotope labeled form of one of the peptides to be quantified, [U(13)C]IPP, was used as an internal standard. The limit of detection of the assay is below 0.01 ng ml(-1). The limit of quantification is between 0.05 and 0.2 ng ml(-1), which is approximately 10% of the expected peptide concentration in plasma based on a normal diet. The intra- and inter-day relative standard deviations for all peptides have shown to be below 25% and the method has an accuracy of better than 75%. The long-term stability is good. At least 200 samples could be analysed before the system had to be cleaned. The assay has been successfully applied to blood samples collected from volunteers during a human trial.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2006

Automated target preparation for microarray-based gene expression analysis.

DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.

Animals↗
PubMed1995

Computer tool FUNSITE for analysis of eukaryotic regulatory genomic sequences.

We present the computer tool FUNSITE for description and analysis of regulatory sequences of eukaryotic genomes. The tool consists of the following main parts: 1) An integrated database for genomic regulatory sequences. The integrated database was designed on the basis of the databases TRANSFAC (Wingender 1994) and TRRD (Kel et al. 1995) that are currently under development. The following functions are performed: i) linkage to the EMBL database; ii) preparing samples of definite types of functional sites with their flanking sequences; iii) preparing samples of promoter sequences; iv) preparing samples of transcription factors classified with regard to structural and functional features of DNA binding and activating domains, functional families of the factors, their tissue specificity and other functional features; v) access to data on mutual disposition of cis-elements within the regulatory regions. 2) The second component of FUNSITE tool is the set of programs for analysis of the structural organization of regulatory sequences: i) Program for revealing of potential transcription factors binding sites based on their consensi; ii) program for revealing of the potential binding sites using homology search with nucleotide sequences of real binding sites; iii) program for analysis of oligonucleotide context features which are characteristic of flank sequences of the binding sites; iv) program for design of recognition method for the functional sites based on generalized weight matrix; v) program for revealing potential composite elements. The results of analysis of the promoter sequences of eukaryotic genes with the FUNSITE are presented, too.

Amino Acid Sequence↗
PubMed2003

Improved sensitivity of the Chlamydia trachomatis Cobas Amplicor assay using an optimized procedure for preparation of specimens.

The aim of this study was to evaluate the performance of an improved sample preparation procedure that enhances the sensitivity of the Chlamydia trachomatis Cobas Amplicor assay (Roche, Switzerland). This procedure was developed after it was observed that, in some cases, endocervical swabs positive for Chlamydia trachomatis in a direct immunofluorescence assay were negative in the Chlamydia trachomatis Cobas Amplicor. For this procedure, the initial sample volume was increased over that recommended by the manufacturer, the sample was concentrated by centrifugation, and the yield of the extraction was increased by an additional enzymatic lysis. Five hundred sixteen endocervical swabs from women visiting a family planning clinic were tested in parallel by the Chlamydia trachomatis Cobas Amplicor using the extraction procedure recommended by the manufacturer and the modified in-house sample preparation procedure. Eight samples were positive with both procedures. Seven additional samples were positive with the in-house method only. The results show that the in-house sample preparation procedure markedly improves the test sensitivity of the Chlamydia trachomatis Cobas Amplicor assay. Consequently, the use of this improved screening protocol will facilitate the detection of even low-level infections with Chlamydia trachomatis, which in turn will lead to the introduction of earlier therapeutic interventions.

Adult↗
PubMed1998

High-performance liquid chromatographic separation and nanogram quantitation of bupivacaine enantiomers in blood.

Chiral separation of rac-bupivacaine extracted from blood was achieved with similar limits of detection but using a much simpler sample preparation than reported previously. The simple one-step sample preparation devised was highly robust and efficient and allowed a very high throughput of samples. The high-performance liquid chromatography (HPLC) conditions used gave baseline separation of the enantiomers with high sensitivity. R-(+)-bupivacaine and S-(-)-bupivacaine blood concentrations were determined using a chiral stationary phase (AGP, ChromTech) with diode array detection at 220 nm; this wavelength produced a stable baseline allowing semi-automated analysis. Sample preparation involved addition of internal standard (diphenhydramine), basification of blood, extraction with n-hexane, concentration of the extract to dryness and reconstitution in 0.002 M phosphoric acid. At rac-bupivacaine concentrations of 0.5, 5 and 50 microg/ml in blood, assay accuracy as estimated by coefficients of variation (C.V.s), were 3.3, 1.4, and 1.6%, respectively, for R-(+)-bupivacaine and 3.7, 2.0 and 1.5%, respectively, for S-(-)-bupivacaine. Using 0.6-ml samples, the estimated limits of detection for R-(+)-bupivacaine and S-(-)-bupivacaine were both 15 ng/ml of blood. Calibration curves (n=188) were linear from 0.1 to 50 microg/ml with all correlation coefficients being greater than 0.99. This semi-automated method was applied to studies involving whole body pharmacokinetics with intravenous doses ranging from 12.5 to 350 mg and regional myocardial pharmacokinetics with coronary arterial doses ranging from 2.5 to 12.5 mg. These studies generated approximately 12000 blood samples.

Anesthetics, Local↗
PubMed2004

Quantitative determination of heme for forensic characterization of bacillus spores using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A quantitative method was developed for the determination of heme (ferriprotoporphyrin IX) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method was designed for forensic characterization of the use of blood agar in preparation of Bacillus spores. An alkali wash of 0.3 M ammonium hydroxide was used to solubilize heme from spore samples. The wash was concentrated and analyzed by MALDI-TOFMS. Experimental parameters were optimized to obtain the best signal intensity, maximize signal reproducibility, and improve day-to-day repeatability of the measurement. Sinapinic acid was found to be the best matrix. A sandwich sample preparation protocol was determined to increase the shot-to-shot and point-to-point reproducibility of the measurement. Cobalt(III) protoporphyrin was used as an internal standard and the analyte/internal standard ratio responses from solutions of known concentrations were used to construct a calibration curve (R(2) = 0.993). Limits of detection and quantitation for heme were calculated to be approximately 0.4 (200 fmol) and 0.8 microM (400 fmol), respectively. Spore samples prepared on blood agar and nonblood agar were analyzed using the method. Heme was detected at a concentration of approximately 0.3 ng/mg of spore on samples prepared on blood agar and purified by extensive washing. Heme was not detected on spore samples prepared without blood.

Agar↗
PubMed1981

Mutagenicity studies on alcohol extracts from gamma-irradiated potatoes--preparation of samples and their chemical analysis.

The preparation of alcohol extracts from gamma-irradiated potatoes of the "Danshaku" variety and their chemical aspects were studied. The final concentrate of alcohol extracts from potatoes showed pH values of 3-4. The o-quinones or "radiotoxins" reported by Kuzin, et al. were not detected in the alcohol extracts from potatoes of this variety by high performance liquid chromatography (HPLC) analysis, paper chromatography and the model enzymatic experiment.

Alcohols↗
PubMed1989

Particulate matter test in small volume parenterals: critical aspects in sampling methodology.

The following critical steps of the particulate matter test sampling methodology for small volume parenteral products (SVPs), conduct by light blockage method, were considered: 1) reliability of the small volume aspirator sampler for different sample volumes; 2) particulate matter distribution inside each ampoule in liquid products (8 liquid SVPs tested); 3) influence of the sample preparation method on the evaluation of the final contamination of the sample. Nine liquid SVPs were tested by preparing samples following the three U.S.P. XXI methods: 1) unit as it is (direct analysis), II) unit diluted, III) sample obtained by combining several units. Particles counts were performed by a HIAC/ROYCO model 3000 counter fitted with a small volume sampler. The validation of the sampler shows that it should be improved. A more accurate and strict validation than the one stated by U.S.P. XXI is suggested. The particulate matter distribution in liquid products is found to be uniform inside the ampoule in the size range greater than or equal to 2 microns-greater than or equal to 10 microns; the analysis can be performed examining only a portion of the whole content. The three sample preparation methods lead to significantly different contamination results. The particulate control test should be conduct by direct analysis, as it is carried out under the same conditions as for product use. The combining method (III) is suggested for products of less than 2 ml volume that cannot be examined by direct analysis.

Drug Contamination↗
PubMed2000

[Detection of the genome of bovine viral diarrhea virus (BVDV) using the polymerase chain reaction after reverse transcription (RT-PCR): comparison of methods for the isolation of ribonucleic acid (RNA) from clinical samples].

The RT-PCR is an in vitro technique that is increasingly being used for diagnosis of viral animal pathogens. Due to its high sensitivity it is considered as an alternative to current standard methods for detecting BVDV especially in pooled samples, e.g. from bulk tank milk. A prerequisite for the performance of RT-PCR is an efficient and simple method for sample preparation. The aim of this work was to compare the efficiency of three commercially available kits for RNA extraction, and their suitability for sample preparation for the detection of the BVDV genome by RT-PCR in blood, milk and tissue samples. The kits were based on different methods for extraction of RNA and differed in costs, labour and time consumption. The most sensitive RT-PCRs (exception: heparinised blood) were obtained when sample preparation was performed by acidic guanidinium-isothiocyanate-phenol-chloroform extraction with the Trizol (Gibco) reagent. Using a kit based on the binding of RNA to silica membrane in a spin column, positive results in RT-PCR were obtained from all samples, but with lower sensitivity. The advantage of the column-based kits is that they are less time-consuming, easier to handle and suitable for automatisation of sample preparation. A kit using salt precipitation of the desoxribose nucleic acid (DNA) and proteins was unsuitable for the isolation of viral RNA from the samples.

Animals↗
PubMed2006

Determination of drugs in biological fluids by direct injection of samples for liquid-chromatographic analysis.

The analysis of drugs in various biological fluids is an important criterion for the determination of the physiological performance of a drug. After sampling of the biological fluid, the next step in the analytical process is sample preparation. The complexity of biological fluids adds to the challenge of direct determination of the drug by chromatographic analysis, therefore demanding a sample preparation step that is often time-consuming, tedious, and frequently overlooked. However, direct on-line injection methods offer the advantage of reducing sample preparation steps and enabling effective pre-concentration and clean-up of biological fluids. These procedures can be automated and therefore reduce the requirements for handling potentially infectious biomaterial, improve reproducibility, and minimize sample manipulations and potential contamination. The objective of this review is to present an overview of the existing literature with emphasis on advances in automated sample preparation methods for liquid-chromatographic methods. More specifically, this review concentrates on the use of direct injection techniques, such as restricted-access materials, turbulent-flow chromatography and other automated on-line solid-phase extraction (SPE) procedures. It also includes short overviews of emerging automated extraction-phase technologies, such as molecularly imprinted polymers, in-tube solid-phase micro-extraction, and micro-extraction in a packed syringe for a more selective extraction of analytes from complex samples, providing further improvements in the analysis of biological materials. Lastly, the outlook for these methods and potential new applications for these technologies are briefly discussed.

Body Fluids↗
PubMed2005

The structure of gas-phase bradykinin fragment 1-5 (RPPGF) ions: an ion mobility spectrometry and H/D exchange ion-molecule reaction chemistry study.

Ion mobility-mass spectrometry (IM-MS) data is interpreted as evidence that gas-phase bradykinin fragment 1-5 (BK1-5, RPPGF) [M + H](+) ions exist as three distinct structural forms, and the relative abundances of the structural forms depend on the solvent used to prepare the matrix-assisted laser desorption ionization (MALDI) samples. Samples prepared from organic rich solvents (90% methanol/10% water) yield ions having an ion mobility arrival-time distribution (ATD) that is dominated by a single peak; conversely, samples prepared using mostly aqueous solvents (10% methanol/90% water) yield an ATD composed of three distinct peaks. The BK1-5 [M + H](+) ions were also studied by gas-phase hydrogen/deuterium (H/D) exchange ion-molecule reactions and this data supports our interpretation of the IM-MS data. Plausible structures for BK1-5 ions were generated by molecular dynamics (MD). Candidate MD-generated structures correlated to measured cross-sections suggest a compact conformer containing a beta-turn whereas a more extended, open form does not contain such an interaction. This study illustrates the importance of intra-molecular interactions in the stabilization of the gas-phase ions, and these results clearly illustrate that solution-phase parameters (i.e., MALDI sample preparation) greatly influence the structures of gas-phase ions.

Bradykinin↗
PubMed2005

Optimization and validation of a fully automated silica-coated magnetic beads purification technology in forensics.

Automated procedures for forensic DNA analyses are essential not only for large-throughput sample preparation, but are also needed to avoid errors during routine sample preparation. The most critical stage in PCR-based forensic analysis is DNA isolation, which should yield as much highly purified DNA as possible. The extraction method used consists of pre-treatment of stains and samples, cell lysis using chaotropic reagents, binding of the DNA to silica-coated magnetic particles, followed by elution of the DNA. Our work focuses mainly on sample preparation, obtaining the maximum possible amount of biological material from forensic samples, and the following cell lysis, to create a simple standardized lysis protocol suitable for nearly all forensic material. After optimization and validation, the M-48 BioRobot((R)) workstation has been used for more than 20,000 routine lab samples. There has been no evidence of cross contamination. Resulting DNA from as small as three nuclear cells yield reliable complete STR amplification profiles. The DNA remains stable after 2 years of storage.

Bone and Bones↗
PubMedDate not supplied

Recommendations for preparing test samples for AOAC collaborative studies of microbiological procedures for foods.

Preparation of test samples for microbial collaborative studies poses problems not encountered in studies on chemical analytes. For Associate Referees who are considering a collaborative study of a microbiological procedure for food analysis, these problems have not been adequately addressed. Types of contamination (natural or artificial), number of test samples required, analyte selection, proper controls, and container selection are addressed herein. The discussion is a supplement to the guidelines contained in the Handbook for AOAC Members.

Food Contamination↗
PubMedDate not supplied

Testing shelled corn for aflatoxin, Part I: estimation of variance components.

The variability associated with testing lots of shelled corn for aflatoxin was investigated. Eighteen lots of shelled corn were tested for aflatoxin contamination. The total variance associated with testing shelled corn was estimated and partitioned into sampling, sample preparation, and analytical variances. All variances increased as aflatoxin concentration increased. With the use of regression analysis, mathematical expressions were developed to model the relationship between aflatoxin concentration and the total, sampling, sample preparation, and analytical variances. The expressions for these relationships were used to estimate the variance for any sample size, subsample size, and number of analyses for a specific aflatoxin concentration. Test results on a lot with 20 parts per billion aflatoxin using a 1.13 kg sample, a Romer mill, 50 g subsamples, and liquid chromatographic analysis showed that the total, sampling, sample preparation, and analytical variances were 274.9 (CV = 82.9%), 214.0 (CV = 73.1 %), 56.3 (CV = 37.5%), and 4.6 (CV = 10.7%), respectively. The percentage of the total variance for sampling, sample preparation, and analytical was 77.8, 20.5, and 1.7, respectively.

Aflatoxins↗
PubMedDate not supplied

Testing green coffee for ochratoxin A, part I: estimation of variance components.

The variability associated with testing lots of green coffee beans for ochratoxin A (OTA) was investigated. Twenty-five lots of green coffee were tested for OTA contamination. The total variance associated with testing green coffee was estimated and partitioned into sampling, sample preparation, and analytical variances. All variances increased with an increase in OTA concentration. Using regression analysis, mathematical expressions were developed to model the relationship between OTA concentration and the total, sampling, sample preparation, and analytical variances. The expressions for these relationships were used to estimate the variance for any sample size, subsample size, and number of analyses for a specific OTA concentration. Testing a lot with 5 microg/kg OTA using a 1 kg sample, Romer RAS mill, 25 g subsamples, and liquid chromatography analysis, the total, sampling, sample preparation, and analytical variances were 10.75 (coefficient of variation [CV] = 65.6%), 7.80 (CV = 55.8%), 2.84 (CV = 33.7%), and 0.11 (CV = 6.6%), respectively. The total variance for sampling, sample preparation, and analytical were 73, 26, and 1%, respectively.

Algorithms↗
PubMed1997

A desalting approach for MALDI-MS using on-probe hydrophobic self-assembled monolayers.

One of the problems encountered in preparing samples for matrix-assisted laser desorption/ionization (MALDI) analysis is the presence of nonvolatile salts in the sample. This difficulty is often exacerbated by the necessity to prepare the sample in the appropriate sample-to-matrix ratio. This paper reports a probe surface derivatization method that greatly simplifies this sample preparation process. By constructing self-assembled monolayers of octadecyl mercaptan (C18) on the MALDI probe surface, we were able to generate a surface capable of reversibly binding polypeptides via hydrophobic interactions, which in turn, permits the analyte to be easily concentrated and desalted directly on the probe tip.

Chelating Agents↗