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PubMed2004

Enzymatic tissue digestion as an alternative sample preparation approach for quantitative analysis using liquid chromatography-tandem mass spectrometry.

Compound extraction from biological tissue often presents a challenge for the bioanalytical chemist. Labor-intensive homogenization or sonication of whole or powdered tissue is performed before compounds can be extracted and analyzed. Enzymatic digestion is commonly used for tissue dissociation and cell harvesting and offers the advantages of unattended sample preparation, potential automation, and low cost. The feasibility of enzymatic digestion as an alternate tissue preparation technique was evaluated for bioanalysis of drugs in conjunction with LC/MS/MS. Two different enzymes (collagenase and proteinase K) that are known to degrade connective tissues to allow tissue dissolution were chosen for evaluation, employing well-known antidepressants desipramine and fluoxetine as test compounds in dog and rat brain tissue. Comparison between enzymatic digestion and conventional homogenization tissue preparation was performed, including investigation of matrix ionization suppression of both methods using a postcolumn infusion system. Results showed that enzymatic digestion has extraction efficiency comparable to homogenization. Matrix ionization suppression was not observed for either the test compounds evaluated or the sample extraction method. Test compound levels of incurred tissue samples prepared by enzymatic digestion were in good agreement with the values obtained by the conventional homogenization tissue preparation, indicating that enzymatic digestion is an appropriate tissue sample preparation method.

Animals↗
PubMed2002

Optimized sample preparation for tandem capillary electrophoresis single-stranded conformational polymorphism/ heteroduplex analysis.

Here we describe DNA sample preparation methods that allow the rapid, simultaneous generation of both single-stranded conformational polymorphism (SSCP) and heteroduplex DNA elements from a single sample in a single tube, which are suitable for direct injection into a capillary electrophoresis (CE) instrument with excellent sensitivity of genetic mutation detection. The p53 gene was used as a model DNA region for this study, which was performed on a high-throughput MegaBACE 96-capillary array electrophoresis instrument. We found that, contrary to the practice common in slab-gel SSCP analysis, denaturants such as formamide are incompatible with this novel technique because they result in homo- and heteroduplex peak broadening in CE (possibly as a result of incomplete dsDNA re-hybridization) that reduces the peak resolution and hence the sensitivity of mutation detection. We also have found that PCR buffers, which are typically used to suspend samples for slab-gel heteroduplex analysis (HA), but which are less suitable for CE because of the presence of extra salt that reduces the efficiency of electrokinetic injection, may be substituted with a 10 mM Tris-HCI buffer (pH 8.5). The use of this Tris-HCl buffer for sample preparation provides both a high sensitivity of mutation detection by tandem SSCP/HA and high efficiency ofelectrokinetic injection by CE. In a related study (published elsewhere), we have applied this optimized protocol to the screening of a set of 32 mutant DNA samples from p53 exons 7 and 8 and recorded 100% sensitivity of mutation detection for tandem CE-SSCP/HA, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA).

DNA↗
PubMed2003

Sample preparation technique and white cell content influence the detectable levels of growth factors in platelet concentrates.

BACKGROUND AND OBJECTIVES: Autologous platelet concentrate (PC) is applied locally to improve wound healing and tissue repair. Previous measurements of the growth factor content of platelets have given conflicting results. To date, there is no information on the influence of different preanalytical sample-preparation methods on the detectable amount of growth factors. MATERIALS AND METHODS: We measured the level of growth factors in PCs obtained by plateletpheresis and by leukapheresis. We subjected aliquots of these components to six different preparation methods: freezing/thawing once or twice; dissolution in 0.5% Triton-X-100; and clot formation by the addition of calcium and thrombin with subsequent incubation for 1 h, for 24 h, or for 1 h followed by freezing and thawing. RESULTS: In samples dissolved in Triton-X-100, higher levels of growth factors were detected than in the other specimens. In comparison to clot formation, freezing and thawing platelets twice was equivalent with respect to the release of platelet-derived growth factor (PDGF) but superior with respect to the release of transforming growth factor-beta1 (TGF-beta1). Overall, mean levels of 4.77 x 10(-16) g of PDGF-AB, 2.2 x 10(-17) g of PDGF-BB, and 2.41 x 10(-16) g of TGF-beta1 were found per single human platelet in white blood cell (WBC)-poor samples dissolved in Triton-X-100. CONCLUSIONS: Dissolving PC in Triton-X-100 releases maximum quantities of growth factors from platelets. The release of each growth factor by any sample preparation method should be investigated and interpreted separately. The preanalytical sample-preparation method, as well as the platelet and WBC content, influence the measurable levels of growth factors in PCs. The results implicate the need to correct, considerably upwards, previous estimations of the PDGF content of platelets.

Adult↗
PubMed1999

Automated sample preparation techniques for the determination of drug enantiomers in biological fluids using liquid chromatography with chiral stationary phases.

The determination of drug enantiomers has become of prime importance in the field of pharmaceutical and biomedical analysis. Liquid chromatography (LC) is one of the most frequently used techniques for achieving the separation and quantitation of the enantiomers of drug compounds. In the bioanalytical field, the integrated systems present an interesting alternative to time-consuming sample preparation techniques such as liquid-liquid extraction. Solid phase extraction (SPE) on disposable cartridges, dialysis or column switching are sample preparation techniques that can be fully automated and applied to enantioselective analysis in biological fluids. The selection of the most appropriate LC mode and chiral stationary phase for enantioseparations in bioanalysis is discussed and some aspects of these automated sample preparation procedures are compared, such as selectivity, detectability, elution of the analytes from the extraction sorbent, sample volume and analyte stability.

Buffers↗
PubMed2002

Proteomic study of a model causative agent of harmful red tide, Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis.

A comprehensive study to find the optimal sample preparation conditions for two-dimensional electrophoresis (2-DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2-DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre-electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2-DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2-DE but this treatment did yield 2-DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2-DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3-[(3-cholamidoprphyldimethylamino]-1-propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2-DE analysis on the soluble protein fractions extracted from light-starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2-DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.

Acetone↗
PubMed2002

Characterization of low molecular weight hydrocarbon oligomers by laser desorption/ionization time-of-flight mass spectrometry using a solvent-free sample preparation method.

A new solvent-free sample preparation method using silver trifluoroacetate (AgTFA) was developed for the analysis of low molecular weight paraffins and microcrystalline waxes by laser desorption/ionization time-of-flight mass spectrometry (LDI-TOFMS). Experiments show that spectral quality can be enhanced by dispersing AgTFA directly in liquid paraffins without the use of additional solvents. This preparation mixture is applied directly to the MALDI probe. Solid waxes could be examined by melting prior to analysis. The method also provides sufficiently reproducible spectra that peak area ratios between mono- and bicyclic alkane peaks indicated variations in the cycloalkane content of paraffin samples. Dehydrogenation of hydrocarbons observed during the desorption/ionization process was studied by analysis of alkane standards.

Journal Article↗
PubMed1998

Sample Preparation for Arsenic, Copper, Iron, and Lead Determination in Sugar.

Two sample preparation methods were modified and optimized for the determination of As, Cu, Fe, and Pb in white sugar using atomic absorption spectrometry (AAS). Analyses were carried out using hydride generation AAS (HG-AAS) for As and flame AAS (FAAS) for Cu, Fe, and Pb. White sugar samples were prepared by dry ashing of the samples with H(2)SO(4) and Mg(NO(3))(2). Recoveries for As, Cu, Fe, and Pb were 87.6, 92.8, 95.8, 91.1% respectively,in ashing with H(2)SO(4). They were 95.1, 92.4, 97.4, and 91.1%, respectively, in ashing with Mg(NO(3))(2).6H(2)O. When small quantities of spikes were used, 94.7 and 95.3% recoveries of arsenic were found for the procedures applying H(2)SO(4) and Mg(NO(3))(2), respectively.

Journal Article↗
PubMed2006

A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples.

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.

DNA↗
PubMed1991

Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine.

An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitive detection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction (PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows were used. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 10(7) to 10(8) Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelleted leptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation method enabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the need for dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by experimentally infected cows.

Animals↗
PubMed1995

Application of the fast-evaporation sample preparation method for improving quantification of angiotensin II by matrix-assisted laser desorption/ionization.

The fast-evaporation method of sample preparation has been applied for quantitative analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. An instrumental protocol focusing on improvement of shot-to-shot repeatability and compensation for signal degradation has been developed for quantification of angiotensin II using the fast-evaporation technique and an internal standard. The fast-evaporation method was compared to the standard method of sample preparation (using a multicomponent matrix) in the quantitative analysis of angiotensin II, and found to be superior in several respects. Improvement in sample homogeneity using the fast-evaporation method enhanced both point-to-point repeatibility and sample-to-sample reproducibility. The relative standard deviations of the analyte/internal standard ratios (point RSD) were decreased by a factor of three compared to those obtained using the multicomponent matrix method. The average point RSD was found to be ca. 5% for the fast-evaporation technique. Two internal standards were evaluated for quantification of angiotensin II. The better one, 1-SAR-8-Ile angiotensin II, yielded a relative standard deviation of the standard curve slope of ca. 2.2% over two orders of magnitude of concentration (45 nM to 3000 nM), an improvement by a factor of two over the standard preparation method. Renal microdialysate samples, spiked with angiotensin II and the internal standard 1-SAR-8-Ile angiotensin II, were also analyzed using the fast-evaporation technique. The detection limit was calculated to be in the high attomole range (675 amol). Furthermore, the accuracy for a single determination of angiotensin II concentration in these samples was found to be 13.9% with a relative error of 8.19%.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed2005

BIS repetita non placent -- a statistical analysis of the number of sample preparations and number of injections required for chromatographic analyses.

This article attempts to answer the question of how many replicate sample preparations and replicate chromatographic injections must be done to provide accurate results in chromatographic analyses of pharmaceuticals. Using a random selection of chromatographic runs obtained with 1-3 replicate preparations and duplicate injections, the variance associated with preparation-to-preparation and injection-to-injection variability were estimated by a mixed-model statistical analysis. The analysis also predicted the probability that two injections of the same sample preparation are not in agreement with each other. Results indicated that, with modern chromatographic equipment, duplicate injections do not improve the precision. The number of replicate preparations needed to provide accurate results for various types of analysis depends on the type of sample and the desired tightness of the specification limits.

Chromatography↗
PubMed1995

Sample preparation of human tumors prior to two-dimensional electrophoresis of proteins.

Sample preparation procedures are critical for obtaining high quality two-dimensional electrophoresis (2-DE) separations from clinical tumor materials. In this communication, we describe procedures which we have found satisfactory for preparing various tumor samples for 2-DE. By these procedures, tumor cells are enriched from serum proteins and contaminating stromal cells. Tumors can be prepared with good reproducibility to obtain high resolution using these techniques.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Sample preparation and determination of acetylcholine in corneal epithelium cells using liquid chromatography-tandem mass spectrometry.

A sample preparation method with subsequent liquid chromatography (LC)-mass spectrometry (MS)-MS analysis for acetylcholine in corneal epithelium is developed. The sample preparation is developed with a focus on compatibility with the LC-MS-MS system and the stability of acetylcholine because acetylcholine esterase is present in the tissue. It appears that both acetylcholine as well as the internal standard (IS) used (acetyl-beta-methylcholine) have fragments at m/z values in the tandem MS spectrum, which correspond with the m/z values of fragments of endogenous substances. Acetylcholine and (3-carboxypropyl)triethylammonium both have 146-->87 and 146-->60 transitions. Acetyl-beta-methylcholine and an unknown compound both have 160-->101 and 160-->60 transitions. This makes it necessary to use a chromatographic step, which has a baseline separation between these endogenous compounds, acetylcholine, and the IS. The analytical procedure has linearity from 1 ng/mL (30 pg/mg corneal epithelium tissue) to at least 250 ng/mL (7.55 ng/mg corneal epithelium tissue). The limits of detection and quantitation are 15 and 45 pg on column, respectively. Relative standard deviation and bias values are within the range of acceptance for all concentration levels.

Acetylcholine↗
PubMed1993

Sample preparation for HPLC by Centricon ultrafiltration.

Peptides and other bioactive materials can be purified from complex biological sources by reverse-phase high-performance liquid chromatography (RP-HPLC), provided the mixture is suitably prepared before injection onto an HPLC system. Ultrafiltration offers a convenient and rapid sample preparation technique with numerous advantages over alternative methods such as conventional gel filtration chromatography. We demonstrate the use of ultrafiltration as an HPLC sample preparation step in the purification of peptides bound to class I major histocompatibility complex (MHC-I) membrane proteins. When ultrafiltration was performed with a Centricon-10 ultrafiltration device, peptides were efficiently separated from the alpha (45 kDa) and beta 2m (12 kDa) chains of MHC-I proteins and could be subjected to HPLC without further treatment. Furthermore, even samples as crude as whole cell lysates or supernatants could be prepared for HPLC in a single ultrafiltration step, affording a remarkably straightforward route to the purification of biologically important peptides.

Biotechnology↗
PubMed2002

Centrifugal concentrator for the substitution of nitrogen blow-down micro-concentration in dioxin/polychlorinated biphenyl sample preparation.

A centrifugal concentrator was applied for the substitution of nitrogen blow-down micro-concentration in dioxin/PCB sample preparation. Automation was achieved through this apparatus and resulted in significant improvements in sample preparation productivity, along with excellent reproducibility and recovery rate of dioxin/PCB. Mean recoveries of dioxin congeners were 90-106%, respectively.

Centrifugation↗
PubMedDate not supplied

Methods of sample preparation for detecting alkaline phosphatase in casein: collaborative study.

A collaborative study was conducted in which 2 different sample preparation techniques were used to determine alkaline phosphatase in casein by the rapid colorimetric test. Seven collaborators tested 10 unknown casein products containing different amounts of residual phosphatase. Results indicated that the phosphatase contents of casein prepared by the 2 methods were not significantly different. The collaborators correctly analyzed 100% of the test samples that were ground and 98% of the test samples that were unground. The alternative rapid sample preparation method has been adopted official first action.

Alkaline Phosphatase↗
PubMed1993

Selected area polishing for precision TEM sample preparation.

A selected area mechanical polishing technique has been developed to improve the precision of cross-sectional TEM sample preparation, based upon the early work of Benedict and colleagues [Benedict et al. (1990) MRS Symp. Proc. Vol. 199, p. 189]. TEM samples were made from a pre-selected section through the middle of a 1 micron wide band of transistors extending laterally for more than 1 mm by precise control over the plane of polish with a corresponding reduction in sample preparation time. To illustrate the application of this technique, a uniformly thin, electron transparent TEM sample of a single, specific, failed transistor is obtained from a 4 mm by 10 mm device array.

Microscopy, Electron↗
PubMed1996

A total sample preparation method for the measurement of airborne asbestos and other fibers by optical and electron microscopy.

A total sample preparation method utilizing one membrane filter for all or some of the measurements by phase contrast optical microscopy (PCM), optical microscopy (OM), dispersion staining polarized microscopy (DS/PLM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), was developed in order to evaluate airborne asbestos and other mineral fibers comprehensively, including fiber number, sizes and types, in various environments. This method consists of two alternate procedures: parallel and serial preparation. The former uses different portions of a filter for each measurement of PCM, OM, DS/PLM, SEM and TEM. The latter uses a single filter portion for all OM, SEM and TEM measurements by which the same area and same fibers on the filter can be observed. This allows fiber numbers to be directly compared using these measurements on the same filter area, and the fibers observed by OM can be also examined by SEM and/or TEM equipped with an energy dispersive X-ray spectrometer (EDX) to determine the types and precise sizes. Using this method, it was found that OM measurements developed in this study can visualize thinner fibers than PCM measurements and make it easier to count fibers. We also found that currently used PCM measurement can detect fibers thicker than about 0.4-0.5 microns in diameter. This total sample preparation method can be used not only for air samples, but also for liquid and biological tissue samples.

Air Pollutants, Occupational↗