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PubMed1987

Off-line automated sample preparation--experience in a clinical drug investigation unit.

The authors describe an evaluation of the Du Pont Prep. off-line automatic centrifugal sample processing system, used in the authors' Drug Investigation Unit over the past two years. In comparison to liquid-liquid extraction, the authors found that assay precision has generally been improved, absolute recovery was quantitative, reliability has been good and the use of the system has resulted in notable labour saving. However, the cost per test based on a seven-year amortization of capital cost shows that the system is more expensive. Assays for urinary opiates, serum tricyclic antidepressants, serum isoxicam, serum valproate, serum metoclopramide and serum pindolol are presented and the merits of the method are compared with those for manual liquid-liquid extraction procedures.

Journal Article↗
PubMed2000

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. I. Injection procedure and column stability.

We report a method for reliable routine polymer sample introduction with minimal bias, a separation method of the first six linear and cyclic oligomers by liquid chromatography, quantification using group equivalents and long term method performance. Injecting a polymer sample in a mobile phase containing an aqueous non-solvent often results in blocked systems as the polymer precipitates in the connecting capillaries. In this first part we focus on a new injection technique, in which the dissolved polyamide is placed between two zones of formic acid, preventing the polymer to precipitate before it reaches the column. Development of this sandwich injection method makes direct injection of the polymer into an aqueous acetonitrile gradient feasible. The oligomeric polyamide recovery of this technique, extraction, dissolution/precipitation and direct injection on a hexafluoro-isopropanol (HFIP) gradient were compared. With the sandwich injection method the polymer remains on the column, slowly changing the stationary phase. The influence of this on resolution and retention was studied. Column stability allows sixty injections before cleaning or replacing the column is necessary.

Acetonitriles↗
PubMed1997

Comparison and evaluation of the specificity and binding capacity of commercial and in house affinity columns used in sample preparation for analysis of growth-promoting drugs.

Immunoaffinity chromatography (IAC) and affinity chromatography (AC) are widely used for extraction of drugs from biological samples. Fifteen column types were purchased from five different manufacturers and their ability to bind specific drugs including beta-agonists and anabolic steroids over a range of analyte concentrations in fortified bovine urine samples was assessed. The performance data obtained from these columns were compared with columns produced in this laboratory (in house columns). The in house columns gave the highest recoveries, ranging from 92 to 100% at the 1 ng spiking concentration, for five of the seven analytes assessed. Forty percent (11 of 27) of all the commercial column assessments recorded recoveries of less than 50% even when the lowest spiking concentration was applied (1 ng). For one manufacturer, only one of seven different columns purchased delivered extraction efficiencies greater than 50%. The extraction efficiencies of the clenbuterol columns were the highest with all commercially prepared columns showing at least 50% binding of radiolabelled tracer. Recoveries of alpha-nortestosterone were the lowest. The variability of these products with respect to quality control requires constant monitoring.

Adrenergic beta-Agonists↗
PubMed2006

Evaluation of two sample preparation methods for prostate proteome analysis.

For laboratory techniques that require well-preserved proteins, such as 2-DE, fresh tissue must be harvested and processed as fast as possible to avoid proteolytic degradation. We describe a modified method for harvesting tissue from radical prostatectomy specimens for proteome analysis and compare it with the standard technique. Cells were scraped from cut surfaces of 11 prostate specimens. A fraction of the material was smeared on a glass slide and Giemsa stained for morphological control. The sample was collected in a medium with protease inhibitors, and the protein material was prepared for 2-DE. Filtering and Percoll centrifugation were omitted. Sample locations were noted on a specimen map. From the same area, a tissue block was harvested for comparison. The block was processed with the conventional technique including mechanical disintegration, filtering and Percoll centrifugation. Quality measures of 2-DE were similar with both methods. With the scrape sampling technique, control smears showed abundant epithelial cells and a cleaner background and processing was faster than with tissue block sampling. For proteomic analysis, the scrape sample technique has several advantages over the tissue block method.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2004

Urine sample preparation of tricyclic antidepressants by means of a supported liquid membrane technique for high-performance liquid chromatographic analysis.

Supported liquid membrane (SLM) technique for sample work-up and enrichment was used for determination of tricyclic antidepressant drugs in urine by high-performance liquid chromatography (HPLC) with UV detection. The studied antidepressant drugs were amitriptyline, opipramol, noxiptyline and additionally diethazine was used as possible internal standard. Alkaline phosphoric buffer with urine sample, as the donor solution, was passed over the liquid membrane into which investigated substances were extracted. On the other side of the membrane, analyzed compounds were trapped due to creating non-extractable form in acidic acceptor solution. Enriched and cleaned up drugs were then injected into a HPLC system with ultraviolet detection to analyze of their concentration in acceptor solution. Optimum extraction efficiency was determined by changing acceptor and donor solutions pH, application of different flow rates of donor solution and by using different solvents in the membrane. Also, donor solution volume, extraction time and concentration of analytes were varied to check the linearity of extraction process. The highest extraction efficiency: 43% for opipramol, 56% for noxiptyline, 43% for amitriptyline and 42% for diethazine (R.S.D. values were <6% and n=3) was achieved when 0.05 M phosphate buffer pH 4.0 and 9.5 were used as donor and acceptor solutions, respectively, n-undecane with 5% tri-n-octylphosphine oxide (TOPO) was used as liquid membrane. Limit of quantification (LOQ) for tricyclic antidepressants after enrichment of 100ml of urine sample was about 1 ng/ml.

Amitriptyline↗
PubMed2000

Analysis of several phenolic compounds with potential antioxidant properties in grape extracts and wines by high-performance liquid chromatography-photodiode array detection without sample preparation.

A RP-HPLC method that allows the separation of several types of phenolic compounds present in grapes and wines by direct injection of samples, using a binary gradient with solvents free of salts and photodiode array detection is described. Results show that more than 15 different phenolic molecules with antioxidant properties (flavan-3-ols, anthocyanins, cinnamic acid derivatives, flavonol derivatives and trans-resveratrol) may be separated in a single run by direct injection of red wine. The method is also valuable for the analysis of these compounds in white wine and in skins, seeds and pulp extracts of red and white grapes.

Antioxidants↗
PubMed2002

Improved sample preparation for the quantitative analysis of paroxetine in human plasma by stable isotope dilution negative ion chemical ionisation gas chromatography-mass spectrometry.

An improved sample work-up and derivatisation procedure for the quantitative determination of paroxetine in human plasma by gas chromatography-negative ion chemical ionisation mass spectrometry is presented. Solvent extraction from plasma samples at alkaline pH was combined with derivatisation to the pentafluorobenzyl carbamate derivative in one step and subsequently analysed without any further purification. Thus, lengthy and time-consuming solvent evaporation steps are avoided to assure high-throughput analysis. Complete validation data are presented. The method is rugged, rapid and robust and has been applied to the batch analysis of paroxetine during pharmacokinetic profiling of the drug.

Calibration↗
PubMedDate not supplied

Simultaneous HPLC analysis of hydrophobic prodrugs and their hydrophilic metabolites in biological media without sample preparation. Application to the biotransformation studies of pronucleotides derived from 5-fluorouracil.

Improvements of an "on-line cleaning" HPLC method for analysis of biological samples are presented: (i) the use of cleaning precolumns filled with hydrophobic stationary phases instead of the hydrophilic ones previously used to eliminate the biological matrix: (ii) the combination in the mobile phase of anionic and cationic pairing reagents in order to retain on the precolumn all the metabolites, whatever their hydrophilicity and ionicity are. Such modifications allowed to study the biotransformation of prodrugs of 5-Fluorouracil, designed to act as antitumoral pronucleotides.

Antimetabolites, Antineoplastic↗
PubMedDate not supplied

Sample preparation improvement in polycyclic aromatic hydrocarbons determination in olive oils by gel permeation chromatography and liquid chromatography with fluorescence detection.

The determination of 15 polycyclic aromatic hydrocarbons (PAHs) in olive oil samples has been improved in order to obtain a fast methodology with a low limit of detection through the combination of liquid-liquid extraction with acetonitrile and preparative gel permeation chromatography (GPC) prior to the injection of purified extracts into a C18 column. Acetonitrile-water was used as the mobile phase with a gradient from 50 to 95%, w/w, acetonitrile in 30 min. The oven temperature was maintained at 15 degrees C, and fluorometric detection was made at a fixed excitation wavelength of 264 nm and variable, optimal emission wavelength for each analyte ranging from 352 nm for 11-H-benzo(b)fluorene to 500 nm for indeno(1,2,3-cd)pyrene. Recovery for all the compounds studied varied from 75 to 111%, and limit of detection values from 0.05 ng/g for benzo(k)fluoranthene to 0.48 ng/g for indeno(1,2,3-cd)pyrene, corresponding to 0.09 ng/g benzo(a)pyrene. Results were compared with those obtained by liquid-liquid extraction followed by a cleanup on silica and a direct GPC treatment of oil samples diluted in dichloromethane, 2 other methodologies that are appropriate for quantifying PAHs in olive oils. However, the proposed method improves the determination limits, reduces the time of analysis, and provides a highly stable baseline for sample chromatograms.

Acetonitriles↗
PubMed2005

Sample preparation and digestion for proteomic analyses using spin filters.

We describe the use of commercially available microcentrifugation devices (spin filters) for cleanup and digestion of protein samples for mass spectrometry analyses. The protein sample is added to the upper chamber of a spin filter with a > or = 3000 molecular weight cutoff membrane and then washed prior to resuspension in ammonium bicarbonate. The protein is then reduced, alkylated, and digested with trypsin in the upper chamber and the peptides are recovered by centrifugation through the membrane. The method provides digestion efficiencies comparable to standard in-solution digests, avoids lengthy dialysis steps, and allows rapid cleanup of samples containing salts, some detergents, and acidic or basic buffers.

Bicarbonates↗
PubMed2005

A rapid sample preparation method for mass spectrometric characterization of N-linked glycans.

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.

Carbohydrate Sequence↗
PubMed2004

Enzymatic probe sonication extraction of Se in animal-based food samples: a new perspective on sample preparation for total and Se speciation analysis.

This paper describes a fast, simple and novel extraction method for total selenium and selenium species determination in food samples. Parameters influencing extraction, such as sonication time, extracting media, temperature, sample mass, ultrasound amplitude and sample/enzyme mass ratio were investigated. The enzymatic hydrolysis proposed, enhanced by probe sonication, allowed the quantitative extraction of selenium in chicken muscle, liver, kidney and feed (97, 93, 95 and 102%, respectively) in 2 min, maintaining the original Se-species integrity. Total Se content of the samples was determined using inductively coupled plasma mass spectrometry. Se-species were identified and quantified using high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry. Chromatographic analyses were carried out under two chromatographic conditions and led to the identification of SeMet in all samples. The accuracy of the proposed method was assessed using certified reference materials as well as microwave digestion. Potential advantages of the proposed method over traditional hydrolysis are speed, simplicity and safety of the procedure.

Animal Feed↗
PubMed2006

Laser-assisted microdissection for real-time PCR sample preparation.

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.

Cell Line↗
PubMed2002

Sample preparation for the determination of 241Am in sediments utilizing gamma-spectroscopy.

This paper describes a procedure developed to separate americium-241 from the bulk of a sample by coprecipitation followed by high sensitivity gamma-counting of the concentrate in a well-type detector. It enables the measurement of 241Am at low concentrations, e.g. fallout levels in soils and sediments, or where large sample sizes are not available. The method is much faster and more reliable than those involving separation from other alpha-emitters, electroplating and alpha-spectrometry. A number of tracer experiments was performed in order to optimize the conditions for coprecipitation of 241Am from sediment leachates. The general outline of the determination of americium is also given.

Americium↗
PubMed2003

Analytical method for the determination of atrazine and its dealkylated chlorotriazine metabolites in water using SPE sample preparation and GC-MSD analysis.

A method is reported for the determination of atrazine and its dealkylated chlorotriazine metabolites in ground, surface, and deionized water. Water samples are adjusted to pH 3-4 prior to loading onto two SPE cartridges in series: C-18 and C-18/cation exchange mixed-mode polymeric phases. The analytes are eluted from each of the two cartridges separately, and the pooled and concentrated fraction is analyzed using gas chromatography-mass selective detection in the selected ion monitoring mode. The lower limit of method validation is 0.10 micrograms/L (ppb) for 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 2-amino-4-chloro-6-(isopropylamino)-s-triazine (G-30033, deethylatrazine), 2-amino-4-chloro-6-(ethylamino)-s-triazine (G-28279, deisopropylatrazine), and 2,4-diamino-6-chloro-s-triazine (G-28273, didealkyatrazine). The overall mean procedural recoveries (and standard deviations) are 96 (6.9), 96 (5.5), 95 (6.8), and 100% (10%) for atrazine, G-30033, G-28279, and G-28273, respectively (n = 49). The method validation study was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160. The reported procedure accounts for residues of G-28273 in water.

Atrazine↗
PubMed2005

Effects of sample preparation on chemistry, cytotoxicity, and inflammatory responses induced by air particulate matter.

Methanol is used for high-efficiency extraction of air particulate (PM) mass from the sampling substrate in the high-volume cascade impactor. Sonication is needed during extraction and when dissolving dried PM samples in liquids used in exposure studies. We investigated the effects of these procedures on the PM chemistry and PM-induced cytotoxic and inflammatory responses in mouse macrophages. Untreated and methanol-treated ambient air reference PM samples (SRM1649a, EHC-93) and diesel PM (SRM1650) were tested after different sonication durations (530 min). Furthermore, the time dependency of the responses to SRM1649a, EHC-93, and a fine PM sample from Helsinki was investigated. Methanol pretreatment increased on average by 24% and 21% the recovery of water-soluble metals from SRM1649a and EHC-93, but not SRM1650. It had no systematic effect on the recoveries of inorganic secondary ions (NO3-, SO4(2-), NH4+) or the sum of genotoxic PAH compounds from the three reference samples. Nitric oxide (NO) response to SRM1650 was strongly enhanced by methanol pretreatment, whereas the cytotoxic or inflammatory responses to the ambient air PM samples (EHC-93, SRM1649a) were only slightly modified. Sonication duration was a modifying factor only in connection to SRM1650. Maximal interleukin (IL)-1 production was observed earlier (8 h) than maximal tumor necrosis factor (TNF) alpha and IL-6 productions (24 h), which indicates the importance to know the optimal time points for measurement of the selected response parameters. In conclusion, methanol extraction and reasonable sonication duration are not likely to modify the cytotoxic and inflammatory potency of ambient air PM samples, but some responses to air PM, rich in organic compounds, can be modified.

Air Pollutants↗
PubMed1972

Stomaching: a new concept in bacteriological sample preparation.

An entirely new mixing device, particularly suitable for preparing bacterial suspensions from foods, fabrics, swabs, and other fairly soft materials, has been developed. With this technique the sample and diluent are put into an inexpensive, sterile plastic bag which is vigorously pounded on its outer surfaces by paddles when placed inside the machine. The resulting compression and shearing forces effectively remove even deep-seated bacteria. After samples are taken for analysis the bag and its remaining contents are thrown away. Labor involved in cleaning and sterilizing reusable homogenizer cups or probes is eliminated, and the device is immediately ready for reuse. Running costs are thus drastically reduced, compared with conventional homogenizers. Additional advantages of this device, which is simple and inexpensive to manufacture, are low noise level, negligible temperature rise, and the small storage space required for bags.

Journal Article↗
PubMed2000

Comparison of membrane filtration rates and hydrophobic grid membrane filter coliform and Escherichia coli counts in food suspensions using paddle-type and pulsifier sample preparation procedures.

Food suspensions prepared by Pulsifier contained less debris and filtered 1.3x to 12x faster through hydrophobic grid membrane filters (HGMFs) than those prepared by Stomacher 400. Coliform and Escherichia coli counts made by an HGMF method yielded 84 and 36 paired samples, respectively, positive by both suspending methods. Overall counts of pulsificates and stomachates did not differ significantly for either analysis, though coliform counts by Pulsifier were significantly higher in mushrooms and significantly lower in ground pork (P = 0.05). Regression equations for log10 counts of coliform and E. coli by Pulsifier and Stomacher were: Pulsifier = 0.12 + 0.97 x Stomacher, and Pulsifier = 0.01 + 1.01 x Stomacher, respectively.

Animals↗