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PubMed2004

Increasing the efficiency of pharmacokinetic sample procurement, preparation and analysis by liquid chromatography/tandem mass spectrometry.

The movement towards a 96-well format has greatly increased productivity and throughput in bioanalytical laboratories. Improvements in automated sample preparation and analytical methods have further contributed to increased productivity. We have focused on sample collection and transfer to the bioanalyst and have found improvements to the current available methods. The problem of manual transfers and plasma clotting issues can be overcome with the use of microtainers. Specifically, for illustrative purposes, three proprietary Theravance compounds were tested for stability, non-specific binding, and electrospray ion suppression in microtainers. There were no issues with stability, non-specific binding or ion suppression for the above compounds even after leaving plasma samples in the microtainers over long periods of time. The microtainers are robot-compatible and the resulting plasma can be transferred without clotting issues. To date, all in-house compounds successfully analyzed and tested using the microtainers have mass ranges between 200 and 1800 Da, pK(a) ranges between 3.8 and 10.3, and logD ranges between -1.7 and 4.2. Once samples are transferred into 96-well plates, flexibility in preparation and analysis is available. Together with automated sample preparation and the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) as an analytical tool, the use of microtainers as sample collection tubes and for sample storage saved considerable time, cost and effort in both of our pharmacokinetic (PK) and bioanalytical groups. This in turn has led to an increased efficiency and overall throughput in support of our drug discovery effort.

Animals↗
PubMed1993

Effects of preparation methods of biomedical samples on PIXE measurement sensitivity.

Instrumental sample preparation techniques for PIXE measurements of human blood serum and bovine muscle were investigated. The techniques studied were direct measurement of a drop, drying on a foil and on an Al cup, lyophilization on a foil, pellet pressing of dried material, microtome sectioning and biopsy preparation. The same serum and muscle were used in each procedure allowing the comparison of detection limits between different methods. The most beneficial technique for both samples was lyophilization and pellet pressing.

Animals↗
PubMed1983

[Nephritogenic glycoprotein isolated from human placenta (methods of isolation from placenta].

In our experiments, we succeeded in obtaining from human placenta a substance comparable to human renal nephritogenic glycoprotein prepared from GBM (glomerular basement membrane). The procedure was quite similar to that for preparation from the kidney. At first, placental terminal villi were isolated by the successive use of three metal sieves. Then trophoblast basement membranes (TrBM) were isolated from placental terminal villi by ultrasonic disruption. Finally TrBM were made soluble by trypsin digestion, after which they were further digested by pronase. Ouchterlony gel diffusion demonstrated the existence of a common precipitin line between antiserum to human renal nephritogenic glycoprotein prepared from GBM and antiserum to the sample prepared from human placental TrBM, and human renal nephritogenic glycoprotein prepared from GBM and the sample prepared from human placental TrBM. The monosaccharide composition of the sample prepared from human placental TrBM was rich in glucose (glucose, galactose and mannoside in the ratio of 1.00 : 0.74 : 1.00), and the amino acid composition of the sample contained no collagenous components. As a result of fractionation of the sample prepared from human placental TrBM by Bio Gel P200 column chromatography, the activity of renal nephritogenic glycoprotein was found within the void fraction.

Basement Membrane↗
PubMed2000

Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput.

Recent years have seen increasing usage of large particle size stationary phases and ultra-high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra-high flow rate liquid chromatography using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow-bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour.

Calibration↗
PubMed1998

Capillary electrophoresis-mass spectrometry coupling versus micro-high-performance liquid chromatography-mass spectrometry coupling: a case study.

Capillary zone electrophoresis (CZE) and micro-high-performance liquid chromatography (mu-HPLC) coupled to electrospray ionisation (ESI) mass spectrometry were compared with respect to their applicability to problems arising in pharmaceutical drug research and development. Both techniques, which are similar with regard to their operational parameters, were coupled to an API III plus triple quadrupole mass spectrometer using laboratory-built interfaces. The results achieved with the two combinations were compared for sensitivity and general applicability to the quantitative analysis of pharmaceuticals in biological fluids. Midazolam, the 8-chloro-6-(2-fluoro-phenyl)-1-methyl-4H-imidazo-[1,5-a][1,4]-benzodiaze pine, and three of its metabolites were used as test compounds, either as standard solution or after sample clean-up from human plasma. Following different sample preparation routes, liquid-liquid extraction or solid-phase extraction, differences in detection limits as well as robustness in CZE or mu-HPLC coupled with ion spray mass spectrometry (IS-MS) were investigated. Detection limits of about 500 pg/ml for the drug and 2 ng/ml for the metabolites were achieved, using 1 ml of human plasma, only when liquid-liquid extraction was used for sample preparation. Sample preparation using the simpler and faster solid-phase extraction route resulted in deterioration of the separation or clogging of the columns. In all cases, when standard solutions or sample extracts were used, CZE-ESI-MS provided both different selectivity and greater sensitivity.

Chromatography, High Pressure Liquid↗
PubMed2001

A comparative study of raw and prepared semen samples from two consecutive days.

OBJECTIVE: To study and compare raw and prepared semen samples from two consecutive days by conventional sperm parameters, various motility characteristics of spermatozoa determined by computer-aided sperm analysis and calcium ionophore-induced acrosome reaction. STUDY DESIGN: Semen samples of male partners in couples undergoing 81 cycles of double intrauterine insemination were studied. The first sample was produced after abstinence of 2-7 days and the second, 24 hours after the first. Both samples were processed by isolate sperm separation medium. RESULTS: Semen volume, sperm concentration and total motile spermatozoa were significantly reduced in day 2 raw and prepared samples, whereas normal morphology, motility characteristics and percentage of acrosome-reacted spermatozoa increased significantly in day 2 inseminated samples as compared to day 1. Oligospermic, asthenospermic and teratozoospermic samples showed a significant improvement in concentration, various motility characteristics and normal morphology of spermatozoa in day 2 samples as compared to day 1. CONCLUSION: Men with normal samples showed improvements in normal morphology and acrosome-reacted spermatozoa, whereas those with subnormal semen samples from day 1 showed a significant improvement in concentration, various spermatozoal velocities and normal morphology on day 2.

Acrosome Reaction↗
PubMed1989

Visual detection of microencapsulated insecticides with selective staining and scanning electron microscopy.

Selective staining with Sudan IV and methylene blue for light microscopy and scanning electron microscopy (SEM) were investigated to determine their potential for detecting and quantifying microencapsulated insecticides. Penncap-M (microencapsulated methyl parathion), Penncapthrin (microencapsulated permethrin), and Dyfonate (microencapsulated fonofos) were selectively stained with Sudan IV but not with methylene blue. Selective staining was not possible for Altosid SF-10 or SR-20 (microencapsulated methoprene) with either stain. Sudan IV enabled detection of some microencapsulated formulations in the digestive content of selected aquatic invertebrates and prepared contaminated pollen samples. Staining intensity with Sudan IV was greatest with acetone but capsular damage was high. A solvent ratio of 50:50 and 20:80 acetone/xylene minimized capsular collapse and maintained good staining intensity. The use of SEM for capsule identification and quantification depended upon the method of sample preparation: the slide smear method was superior to samples prepared by incision or microtomy. SEM was most suitable for investigation of formulations such as methoprene, for which selective staining was not possible. The chemical basis of staining with Sudan IV and potential application of both identification techniques are discussed.

Aedes↗
PubMed2005

A novel standard sample powder preparation method for quantitative analysis of polymorphs.

The purpose of this study was to improve the sensitivity and accuracy of the quantitative analysis of polymorphic mixtures. Calibration curve reliability has been improved through the advancement of standard mixture preparation techniques whereby samples at scales of several grams are homogeneously and accurately prepared without decrease in crystallinity. Crystalline powders are first micronized to anisotropic, nearly equal-sized (<10 microm) particles of almost random orientation in an air jet mill. These powders are then mixed in a vibration mill that includes rubber balls as mixing media. Using this technique, alpha and gamma forms of indomethacin (IMC) were prepared in various proportions from 0.5% to 50% alpha content and subjected to X-ray powder diffraction analysis. The validation data showed that these precisely prepared standard mixtures resulted in superior analytical sensitivity and accuracy compared to those prepared by agate mortar.

Calibration↗
PubMed1996

Preparation of samples for polymerase chain reaction in situ.

The purpose of this paper is to describe the key variables in sample and reagent preparation needed for successful polymerase chain reaction (PCR) in situ. Tissue or cell preparations should be fixed in a cross linking fixative, such as 10% buffered formalin, preferably from 15 to 48 hours. Tissues should be embedded in paraffin; cell preparations can be fixed when near confluence, then physically removed and processed. When possible three samples (4 microM tissue sections or 1-5000 cells) should be placed on silane coated glass slides. Digestion in pepsin (2 mg/ml) for 30 min is adequate for DNA detection by PCR in situ hybridization whereas optimal protease digestion time is variable and related to formalin fixation time for reverse transcriptase (RT) in situ PCR. RT in situ PCR requires an overnight digestion with DNase. The amplifying solution should contain 4.5 mM MgCl2, 0.05% bovine serum albumin, and, for RNA analysis, the reporter nucleotide. A false positive signal would be evident with incorporation of the reporter nucleotide for DNA targets due to DNA repair; this can be avoided with frozen, fixed tissues and the hot start maneuver. Otherwise, one needs to use a labeled probe and a hybridization step to detect amplified DNA targets in paraffin embedded tissues.

Brain↗
PubMed1991

[Some criteria for the planning and evaluation of the ruggedness test of HPLC methods].

Ruggedness is a measure of the reproducibility of the individual test results when the procedure is used repeatedly to determine the same homogeneous sample in a variety of specified experimental conditions. Ruggedness testing can be divided into two major groups: Type-A, when the influence of the changed environmental conditions (different equipment, analysts, etc.) on the analytical performance parameters and results are tested, and Type-B, when the effect of variation in the experimental conditions are checked. In the latter case, Type-B group can be further classified into two major subgroups: ruggedness testing of sample preparation and chromatographic separation. Checking sample preparation for ruggedness testing includes the investigation of various sample preparation procedures, such as: liquid-liquid extraction, solid phase extraction, direct sample introduction and derivative formation. The investigation of column-to-column variability, changes in the instrumental and experimental conditions belongs to the ruggedness testing of chromatographic separation. In this paper the basic principles of selection for experimental variables for various ruggedness tests are discussed introducing a new possibility to design multivariable-multicriteria system, and the evaluation of a single-variable-single-criterion system, as well. In general, from the work introduced in this paper, it can be concluded that when new analytical methods are developed, it is quite important to be aware the sensitivity of the method to variations in the specified conditions. For adequate performance of ruggedness testing: a) the factors (variables) which have significant influence on the chromatographic results can be firstly selected, b) maximum acceptable deviations from the prespecified experimental conditions can be decided, and c) the acceptable deviations from the values of the prespecified analytical performance parameters can be defined.

Chromatography, High Pressure Liquid↗
PubMed2006

High throughput screening of protein formulation stability: practical considerations.

The formulation of protein drugs is a difficult and time-consuming process, mainly due to the complexity of protein structure and the very specific physical and chemical properties involved. Understanding protein degradation pathways is essential for the success of a biopharmaceutical drug. The present review concerns the application of high throughput screening techniques in protein formulation development. A protein high throughput formulation (HTF) platform is based on the use of microplates. Basically, the HTF platform consists of two parts: (i) sample preparation and (ii) sample analysis. Sample preparation involves automated systems for dispensing the drug and the formulation ingredients in both liquid and powder form. The sample analysis involves specific methods developed for each protein to investigate physical and chemical properties of the formulations in microplates. Examples are presented of the use of protein intrinsic fluorescence for the analysis of protein aqueous properties (e.g., conformation and aggregation). Different techniques suitable for HTF analysis are discussed and some of the issues concerning implementation are presented with reference to the use of microplates.

Calorimetry↗
PubMed1995

Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.

Animals↗
PubMed2003

Unbiased whole-genome amplification directly from clinical samples.

Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods.

Blood Cells↗
PubMed1990

Pyrolytic methylation-gas chromatography of whole bacterial cells for rapid profiling of cellular Fatty acids.

A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) profiles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the filament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for time-consuming subculturing) as well as analysis of FAME profiles directly from complex environmental samples.

Journal Article↗
PubMed2003

Single-step perfusion chromatography with a throughput potential for enhanced peptide detection by matrix-assisted laser desorption/ ionization-mass spectrometry.

Mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis can be routinely performed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) which has become a standard tool. Since MALDI-MS detection relies heavily on the quality of the MALDI target, development of an efficient sample preparation technique for removal of sample contaminants is necessary. To date, among the several sample preparation techniques for MALDI targets available, multistep perfusion chromatography (MSPC) using Poros R2 and Oligo R3 has been most commonly used. However, MSPC requires at least four working steps and is not efficient for high-throughput analysis and recovery of low abundance proteins. During the course of proteomic analysis of a large set of rat liver tissues and the immortalized human sebaceous gland cells (SZ95 cells), we were interested in developing an alternative to MSPC. Here, we describe a single-step perfusion chromatography (SSPC) method for MALDI target preparation, which uses a tiny column packed with a mixture of Poros R2 and Oligo R3 resins. The SSPC method significantly improves not only detection of peptides but also efficiency of sample handling, thus enabling high-throughput sample preparation for analyzing large set of samples with high resolution and reproducibility.

Animals↗
PubMed2002

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis.

We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near-infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye-primer versus dye-terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross-linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near-IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye-terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye-primer within the gel lane.

Acrylic Resins↗
PubMed2006

Laser microdissection in translational and clinical research.

Laser microdissection (LMD) is now a well established method for isolating individual cells or subcellular structures from a heterogeneous cell population. In recent years, cell, DNA, RNA, and protein based techniques has been successfully coupled to LMD and important information has been gathered through the analysis of the genome, transcriptome, and more recently the proteome of individual microdissected cells. The aims of this review are to summarize and compare the principles of different laser microdissection instruments and techniques, to discuss sample preparation procedures for microdissection, and to provide wide variety of examples of translational/clinical research applications of LMD. Novel techniques specifically developed for the improved isolation of stained cells, living cells, or rare cells are also discussed.LMD has become an indispensable tool in the preparation of homogenous samples for sophisticated cell or molecular assays. Despite major technological advances, the labor requirements of LMD are still relatively high. However, understanding the advantages and disadvantages of LMD technology and associated sample preparation procedures may aid in the earlier introduction of this method into the routine clinical diagnostics.

Animals↗
PubMed2002

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry.

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.

Arsenates↗