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PubMed2003

In situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization MS analysis of polypeptide mixtures.

A novel liquid-liquid extraction (LLE) procedure was investigated for preparation of peptide and protein samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). LLE using ethyl acetate as the water-immiscible organic solvent enabled segregation of hydrophobic and hydrophilic polypeptides in mixtures, thereby reducing the complexity of mass spectra obtained by MALDI MS. The LLE technique was optimized for rapid and sensitive in situ (on-target) sample preparation for MALDI MS analysis of proteins and peptides at low-picomole and subpicomole levels. Addition of MALDI matrix to the organic solvent enhanced the efficiency of the LLE-MALDI MS method for analysis of hydrophobic peptides and proteins. LLE-MALDI MS enabled the detection of the hydrophobic membrane protein bacteriorhodopsin as a component in a simple protein mixture. Peptide mixtures containing phosphorylated, glycosylated, or acylated peptides were successfully separated and analyzed by the in situ LLE-MALDI MS technique and demonstrate the potential of this method for enhanced separation and structural analysis of posttranslationally modified peptides in proteomics research.

Amino Acid Sequence↗
PubMed1999

Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.

Coumaric Acids↗
PubMedDate not supplied

Sample preparation by ultrafiltration for direct gas chromatographic analysis of ethylene glycol in plasma.

The application of ultrafiltration to sample preparation in the detection and quantification of ethylene glycol in plasma by gas chromatography is described. After addition of the butylene glycol internal standard and ultrafiltration, the sample is directly injected, obviating the need for further dilution. This simple, efficient method of sample preparation is also suitable for more sensitive gas chromatographic measurement of other volatiles.

Chromatography, Gas↗
PubMed1987

Sample preparation and column regeneration in biopolymer separations.

Successful use of high-performance liquid chromatography (HPLC) for biopolymer separations requires adequate sample preparation procedures for prefractionation of complex mixtures, solubilization of the compounds of interest, or removal of contaminants which would interfere with chromatography or damage the HPLC column. Rapid techniques have been described for solid-phase and liquid-liquid extraction, desalting, detergent removal, concentration, and filtration. A number of commercial products are available for off-line batch sample preparation and for on-line automated sample processing. These include ultracentrifugation cartridges and a wide variety of sorbents sold in bulk form or packed in disposable cartridges or minicolumns. In cases where HPLC column performance has degenerated to unacceptable levels, procedures have been described to achieve partial or complete recovery of performance. These include addition of material to fill voids caused by compaction or dissolution of the packing, replacement or cleaning of contaminated frits, regeneration of lost stationary phase material, and stripping of strongly retained contaminants from the stationary phase with suitable strong solvents.

Chromatography, High Pressure Liquid↗
PubMed1994

A simple method of sample preparation for analysis of biphenyl residues in citrus fruit peels by gas chromatography.

A simple method of sample preparation for analysis of residues of the fungicide biphenyl in peels of citrus fruits by gas chromatography (GC) is described. The blended peels are macerated after addition of anhydrous sodium sulphate in dichloromethane for 2 h. After filtration the biphenyl is quantified directly without concentration of the solution by GC with a flame ionization detector (FID) or a mass selective detector (MSD) under single ion mass conditions. Due to the simple method of sample preparation and good average recoveries of 98-103% with standard deviations of 1-4% in the concentration range of 1-10 ppm, the method described is reliable for analysis of biphenyl in citrus fruit peels. In order to avoid loss of the relatively volatile biphenyl no evaporation of the extraction solution occurs.

Biphenyl Compounds↗
PubMed1998

Picoliter sample preparation in MALDI-TOF MS using a micromachined silicon flow-through dispenser.

This paper presents a picoliter sample preparation technique utilizing the flow-through principle, allowing on-line coupling of chromatographic systems to be made. The work was performed in order to investigate the characteristics and the physicochemical properties of the sample preparation using typical mobile phase conditions from mu-CLC (column liquid chromatography) separations. The device presented here is a pressure pulse-driven dispenser, formed by two silicon structures processed by conventional micromachining. The pressure pulse is generated in the flow-through channel by a piezoceramic element. Depending on the orifice size, the droplets ejected range between 30 and 200 pL. The maximum ejection frequency is 500 Hz, limited by resonances within the unit. A pyramid-shaped nozzle improves the directivity of the droplets since it reduces the wetting of the orifice front surface area. The risk of particles sticking close to the orifice is also minimized. The analyses of the deposited sample spots were carried out on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer with delayed extraction. It was possible to detect attomole amounts (159-248 amol) of various proteins (cytochrome c, ribonuclease A, lysozyme, and myoglobin) from a single droplet of matrix:analyte 1:1 (drop volume approximately 110 pL). Additionally, it was found that sample enrichment could be carried out using multiple depositions on the same spot; i.e., 31 nM of insulin was easily detected when more than four depositions were made on the same spot, while no detection was possible without sample enrichment. Size optimization of the MALDI sample spot gave target zones of 100-500-micron diameter that matched the size of the laser focal point and resulted in a considerably increased sample throughput.

Cell Line↗
PubMed2000

Sample preparation for high throughput accurate mass analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An automated sample preparation for high throughput accurate mass determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed. Sample preparation was performed with an automated workstation and automated mass analyses were performed with a commercial MALDI-TOF mass spectrometer. The method was tested with a 41-sample library. MALDI-TOFMS was found to give the needed sensitivity, accurate mass measurement, and soft ionization necessary for structure confirmation, even of mixtures. A mass accuracy of 5 ppm or less was obtained in over 80% of known compound measurements. A mass accuracy better than 10 ppm was obtained for all measurements of known compounds. Analyses of parallel synthesis products resulted in 77% of the measurements with a mass accuracy of 5 ppm or better.

Automation↗
PubMed2005

Sample preparation of organic liquid for off-site analysis of chemical weapons convention related compounds.

Off-site analysis of chemical warfare agents (CWAs) and related compounds plays a key role in the verification program of the Chemical Weapons Convention (CWC). The analysis results, aiming toward unambiguous identication of compounds, depend on the type of sample preparation method. Development of milder sample preparation methods, which offer good recoveries and do not alter the structure of analytes, is highly desirable. Organic liquid with high hydrocarbon background is a frequently encountered challenge in off-site analysis and in official proficiency tests conducted by OPCW. Sample cleanup procedures, namely, solvent exchange followed by cooling and liquid-liquid extraction were studied to eliminate the hydrocarbons from organic liquid. Acetonitrile, a polar aprotic solvent, was effectively used to remove the background in both methods, and recoveries of spiked CWAs by the two techniques were between 69 and 99%.

Chemical Warfare Agents↗
PubMed2004

Glass-chip-based sample preparation and on-chip trypic digestion for matrix-assisted laser desorption/ionization mass spectrometric analysis using a sol-gel/2,5-dihydroxybenzoic acid hybrid matrix.

A glass-chip-based sample preparation method for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of tryptic digests of proteins and intact cells is described. A MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), was hybridized with sol-gels to generate a sol-gel-derived material. Taking advantage of the characteristics of sol-gels, the sol-gel-derived material readily adhered to the surface of a glass chip through covalent bonding. Only one step of sample preparation, deposition of the sample solution on the glass chip, was required before MALDI-MS analysis. Because 2,5-DHB was homogeneously dispersed on the sol-gel network structure, good spot-to-spot reproducibility was obtained in MALDI analysis using this approach and the analyte signals were uniform throughout the chip. The modified glass chips were robust and effective for at least 1 week. This glass-chip-based matrix preparation method provides a straightforward approach to developing techniques for analyzing the on-chip enzymatic digestion of proteins and intact cells of microorganisms. Cytochrome C and Escherichia coli were used as analytes to demonstrate the feasibility of this approach. The products of the on-chip enzymatic digests were identified through protein database searches.

Amino Acid Sequence↗
PubMed2002

Sample preparation of Gram-positive bacteria for identification by matrix assisted laser desorption/ionization time-of-flight.

A new sample preparation method was developed for fresh, whole-cell Gram-positive bacteria to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). With fresh, whole-cell Gram-negative bacteria of the Enterobacteriaceae family, we had previously achieved spectra consisting of >50 peaks and mass ranges of 2-25 kDa. Because similar spectral quantity could not be achieved for Gram-positive bacteria, using this same protocol, we investigated an alternative approach that focuses on the thick peptidoglycan layer of the cell wall. Gram-positive bacteria were incubated with 0.05-0.5 mg/ml lysozyme for 30 min prior to being analyzed by MALDI ToF MS. Lysozyme is an enzymatically stable, 14-kDa protein that specifically cleaves between peptidoglycan disaccharide subunits. A significant increase in overall number of peaks (>50) in the 2-14 kDa range was observed without interference from the presence of lysozyme. We show that for four different species (Staphylococcus aureus, S. haemolyticus, Streptococcus pyogenes, and S. agalactiae) reproducible subset of peaks were found within spectra from a reference strain and two unrelated clinical isolates. The data suggests that this sample preparation may be useful for increasing the overall number of peaks within spectra for subsequent development of bacterial identification strategies.

Biomarkers↗
PubMed2006

Sample preparation for MALDI mass spectrometry using an elastomeric device reversibly sealed on the MALDI target.

A new method for improving low-concentration sample recovery and reducing sample preparation steps in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is presented. In the conventional approach, samples are typically desalted and/or concentrated with various techniques and deposited on the MALDI target as small droplets. In this work, we describe a new approach in which an elastomeric device is reversibly sealed on the MALDI target to form a multi-well plate with the MALDI target as the base of the plate. The new format allows a larger volume (5-200 microL) of samples to be deposited on each spot and a series of sample handling processes, including desalting and concentrating, to be performed directly on the MALDI target. Several advantages have been observed: (i) multiple sample transferring steps are avoided; (ii) recovery of low-concentration peptides during sample preparation is improved using a novel desalting method that utilizes the hydrophobic surface of the elastomeric device; and (iii) sequence coverage of the peptide mass fingerprinting map is improved using a novel method in which proteins are immobilized on the hydrophobic surface of the elastomeric device for in-well trypsin digestion, followed by desalting and concentrating the digestion products in the same well.

Animals↗
PubMed1995

Automated sample preparation for cholesterol determination in foods.

An automated sample preparation system has been developed for the determination of cholesterol in a wide range of matrices. Isolation of cholesterol is performed with a robotic arm coupled with a series of modular stations. Samples are introduced into the system which adds the appropriate reagents, carries out the saponification, pH adjustment, solid-phase extraction and drying steps. This system was evaluated using 15 different food matrices. The average recovery for NIST standards exceeded 97%. A solution of n-hexane-2-propanol was substituted for the traditional methanol-chloroform extraction. Manual pH adjustment was replaced with a buffer. Manual and automated methods were compared and no difference was observed at the 95% confidence level.

Automation↗
PubMed2000

Degradation products generated by sonication of benzyl alcohol, a sample preparation solvent for the determination of residual solvents in pharmaceutical bulks, on capillary gas chromatography.

Benzyl alcohol used as the sample preparation solvent in the determination of residual solvents in pharmaceutical bulks yielded benzene, toluene, and benzaldehyde on capillary gas chromatography (GC) by sonication. The factors responsible for compounds generated are discussed. The quality of benzyl alcohol and the type of sonicator were not involved in the generation of benzene, toluene, and benzaldehyde, whereas matrix contributions were observed. The degradation profiles of benzyl alcohol and its analogous compounds obtained by pyrolysis-GC/mass spectrometric analysis were similar to those obtained by sonication, suggesting that benzyl alcohol is degraded by the high local heat generated by sonication. Consequently, no matter how long it may take to dissolve bulk substances in benzyl alcohol completely, we do not recommend the use of a sonicator in sample preparation for the determination of residual solvents in pharmaceutical bulks.

Benzyl Alcohols↗
PubMed2006

Determination of the DNA methylation level of the marbled crayfish: an increase in sample throughput by an optimised sample preparation.

Using a previously described capillary electrophoretic method with laser-induced fluorescence detection the genomic methylation level can be determined exactly. We present a sample preparation that eliminates the surplus of fluorescence marker used for coupling resulting in an increase of sample throughput from 75 to 250 analyses per week. The sensitivity of the method was also increased, which allows the determination of methylation levels under 1%. With these changes in sample preparation a methylation level of 1.64+/-0.03% in hepatopancreas DNA of the recently discovered marbled crayfish could be determined.

Animals↗
PubMed2006

Reliable on-line sample preparation of basic compounds from plasma using a reversed phase restricted access media in column-switching LC.

We investigated on-line sample preparation of basic compounds from plasma using a methylcellulose-immobilized reversed-phase restricted-access media in column-switching liquid chromatography (LC). Dilution of the plasma sample with phosphate buffered saline prevented or delayed the formation of fibrin clots at 4 degrees C and resulted in reproducible on-line sample preparation over a 30-h period. The use of an ion-pair reagent in the extraction LC enhanced recoveries of hydrophilic basic compounds. The ability of the methods to quantify compounds in plasma were validated and the method was successfully applied to the pharmacokinetic study of a hydrophilic basic compound injected into the bloodstream of rats.

Acetonitriles↗
PubMed1990

A sensitive high performance liquid chromatography microassay for bemoradan, a novel cardiotonic agent, in plasma/serum using a semi-automatic liquid/solid extraction sample preparation system--AASP.

A high performance liquid chromatographic method for the measurement of bemoradan levels in plasma/serum is described. This method uses Varian's AASP (Varian Associates, Sunnyvale, CA, USA), a semi-automatic liquid/solid extraction sample preparation system. It requires only small volumes of plasma/serum samples (0.2-1 mL) and needs no organic solvent for sample preparation. The mean recovery of bemoradan at plasma or serum concentrations of 0.5-100 ng/mL is 82%. The assay has a detection limit of 0.5 ng/mL (when 1 mL of plasma/serum is used) and is linear in the concentration range 0.5-500 ng/mL.

Animals↗
PubMed2006

Combining desorption electrospray ionization mass spectrometry and nuclear magnetic resonance for differential metabolomics without sample preparation.

Desorption electrospray ionization mass spectrometry (DESI-MS) and nuclear magnetic resonance (NMR) spectroscopy are used to provide data on urine examined without sample preparation to allow differentiation between diseased (lung cancer) and healthy mice. Principal component analysis (PCA) is used to shortlist compounds with potential for biomarker screening which are responsible for significant differences between control urine samples and samples from diseased animals. Similar PCA score plots have been achieved by DESI-MS and NMR, using a subset of common detected metabolites. The common compounds detected by DESI and NMR have the same changes in sign of their concentrations thereby indicating the usefulness of corroborative analytical methods. The effects of different solvents and surfaces on the DESI mass spectra are also evaluated and optimized. Over 80 different metabolites were successfully identified by DESI-MS and tandem mass spectrometry experiments, with no prior sample preparation.

Animals↗
PubMed2000

Comparison of sample preparation and signal evaluation methods for EPR analysis of tooth enamel.

In dose reconstruction by EPR dosimetry with teeth various methods are applied to prepare tooth enamel samples and to evaluate the dosimetric signal. A comparison of seven frequently used methods in EPR dosimetry with tooth enamel was performed. The participating Institutes have applied their own procedure to prepare tooth enamel samples and to evaluate the dosimetric signal. The precision of the EPR measurement and the dependence of the estimated dosimetric signal with irradiation up to 1000 mGy were compared. The obtained results are consistent among the different methods. The reproducibility of the dosimetric signal and its estimated relation with the absorbed dose was found to be very close for the applied methods with one possible exception.

Dental Enamel↗