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PubMed1997

Analytical considerations for trace determinations of drugs in breast milk.

Recent scientific and regulatory interest in lacteal excretion of drugs has prompted this review of bioanalytical sample preparation techniques for milk. The composition and properties of milk are reviewed, with emphasis on how the sample preparation is affected. The most important principals of mammary gland pharmacology, including protein binding, ion trapping and liquid solubility, are described. Because adequate milk volume is difficult to obtain from some smaller rodent species, special arrangements for sample collection, control preparation and assay standardization often need to be made. Several commonly-used sample preparation approaches for drugs in milk, including direct injection, dialysis and ultrafiltration, protein precipitation, liquid-liquid extraction, solid-phase extraction and immunoaffinity extraction. Have been reported with varying degrees of success. The advantages and disadvantages of each of these approaches is discussed.

Animals↗
PubMed2000

High-performance liquid chromatography-tandem electrospray mass spectrometry for the determination of lidocaine and its metabolites in human plasma and urine.

A sensitive, selective and accurate high-performance liquid chromatographic-tandem mass spectrometric assay was developed and validated for the determination of lidocaine and its metabolites 2,6-dimethylaniline (2,6-xylidine), monoethylglycinexylidide and glycinexylidide in human plasma and urine. A simple sample preparation technique was used for plasma samples. The plasma samples were ultrafiltered after acidification with phosphoric acid and the ultrafiltrate was directly injected into the LC system. For urine samples, solid-phase extraction discs (C(18)) were used as sample preparation. The limit of quantification (LOQ) was improved by at least 10 times compared to the methods described in the literature. The LOQ was in the range 1.6-5 nmol/l for the studied compounds in plasma samples.

Calibration↗
PubMed2002

Detection of single nucleotide polymorphisms using electrospray ionization mass spectrometry: validation of a one-well assay and quantitative pooling studies.

Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of-principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high-throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated.

Alleles↗
PubMed1993

Determination of a new antibacterial agent (Ro 23-9424) by multidimensional high-performance liquid chromatography with ultraviolet detection and direct plasma injection.

Analysis of a new antibacterial agent, Ro 23-9424 (I), in plasma has been complicated by the fact that its metabolite, fleroxacin (II), is formed not only in vivo, but also nonenzymatically by the hydrolysis of the ester bond of I. In order to minimize sample preparation time and possible hydrolysis during sample preparation, a high-performance liquid chromatographic procedure was developed which features direct injection of plasma and multidimensional chromatography. The first dimension size-exclusion separation allows plasma proteins to elute with the column void volume. The second dimension reversed-phase column provides a high-resolution separation dependent upon the hydrophobicity of the sample species. With a 5-microliters injection, the limit of quantitation of the method is 0.35 microgram/ml for I and 0.27 microgram/ml for II. The method was used to determine steady state plasma vs. time profiles for I and II from 750 mg i.v. doses of I administered twice daily.

Anti-Infective Agents↗
PubMed2006

In situ derivatization hollow fiber mediated liquid phase microextraction of alkylphosphonic acids from water.

Alkylphosphonic acids (APAs), particularly the methyl-, ethyl-, isopropyl- and n-propyl-phosphonic acids are important markers of extremely toxic nerve agents. Hence, their detection and identification is of vital importance to verification of chemical weapons convention (CWC). Verification analysis of CWC requires development of fast, reliable, simple and reproducible sample preparation methods of water and soil samples. Present investigation is focused on the optimization of alkylation of APAs in water with subsequent extraction of alkylated acids by hollow fiber liquid phase microextraction (HF-LPME). This simple and sensitive sample preparation of APAs from water offered better recoveries in comparison to conventionally used extraction technique. Under optimized conditions, the APAs were detected at the concentration of 0.5-0.75 microg/mL with S/N ratio > or = 5, whereas the LODs for alkyl APAs (monobasic APAs) were achieved up to 0.1 microg/mL. The developed method was finally tested with water samples supplied in 19th official proficiency test conducted by the OPCW.

Chromatography, Liquid↗
PubMedDate not supplied

Simple and robust two-layer matrix/sample preparation method for MALDI MS/MS analysis of peptides.

Recent advances in MALDI MS/MS instrumentation allow a high degree of automation in the efficient detection of peptide fragment ions that can be used for protein identification. However, the performance of the technique is dependent on the MALDI sample preparation. We present a simple and robust two-layer sample preparation method tailored for sensitive and reproducible generation of MALDI MS/MS data. This method produces a strong and uniform crystal layer which allows acquisition of high quality MS/MS spectra over the entire sample surface area. Furthermore, due to its crystal strength, the matrix/sample layer can be washed extensively on target, enabling direct analysis of samples containing impurities, such as salts and surfactants. This method is demonstrated to be very useful in routine analysis of in-gel tryptic digests of silver-stained protein gel spots, without the need of desalting steps or hunting for "hot" spots. As an example, seven threonine-phosphorylated proteins involved in signal transduction in response to growth factor stimulation within the lipid raft fractions of the IMR5 neuroblastoma cells have been identified using differential gel display, in-gel digestion and MALDI MS/MS with the new two-layer sample preparation method. Some of these proteins have the functions of maintaining raft structure or cell signaling.

Cell Line, Tumor↗
PubMed2006

A novel technique for the rapid identification of alpha emitters released during a radiological incident.

Before the May 2003 TOPOFF II exercise in Seattle, the U.S. EPA's Laboratory in Las Vegas prepared five spiked samples to be analyzed by the Washington State Department of Health's (WSDOH) Radiation Laboratory. Two of these were simulated deposition samples prepared on packaging tape. Laboratories throughout the world are investigating rapid methods for analyzing plume-borne radioactive materials. While measuring gamma emitters in environmental samples is fairly straightforward, the potential presence of alpha emitters adds complexity. The short range of alpha particles and the high degree of energy interference between nuclides usually require chemical separations and very thin mounts for alpha spectrometry. Work published on Frisch-Grid alpha counting of transuranics in soil and the commercial development of radon-rejection for air filters indicate that alpha spectrometry can be used directly on some media with success. This suggests that plume-borne material sampled from air or freshly deposited surface layers may be counted directly by alpha spectrometry, making it possible to identify both alpha and gamma emitters and determine their relative concentrations. The long-range objective is to propose a sampling method that can be used for rapid qualitative and semi-quantitative analyses using conventional radioanalytical instruments, with minimal preparation. This paper describes experimentation with this approach during a real-time exercise. All samples were prepared by spiking with (137)Cs, (241)Am, (238)Pu, and (239)Pu and presented to the WSDOH's Radiation Laboratory for analysis during TOPOFF II. The laboratory quickly identified and determined the ratios of all four contaminants on the tape samples using sequential alpha spectrometry and gamma-ray spectroscopy without chemical separations.

Air Pollutants, Radioactive↗
PubMed1998

Identification of gel-separated proteins by liquid chromatography-electrospray tandem mass spectrometry: comparison of methods and their limitations.

We have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The efficiency of peptide recovery from trypsin-digested gel bands or electroblotted membrane slices was examined using 35S-labeled yeast proteins, and was found to be in excess of 80%. A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC-ESI-MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit. We were able to identify both standards at the 500 ng level in samples prepared from gel slices, using either a regular spray or a flow-split microspray HPLC-MS interface system. In samples prepared from membrane pieces, carbonic anhydrase was also identified at the 500 ng level, while bovine serum albumin could only be identified in samples of more than 1000 ng. In general, protein identification was slightly better in samples prepared from gels rather than membranes. A dilution series of lesser amounts of the same standard proteins was also analyzed using a gradient capillary LC system and we were able to successfully identify 50 ng of carbonic anhydrase and 100 ng of BSA.

Animals↗
PubMedDate not supplied

Determination of pesticides in composite dietary samples by gas chromatography/mass spectrometry in the selected ion monitoring mode by using a temperature-programmable large volume injector with preseparation column.

Use of a temperature-programmable preseparation column in the gas chromatographic (GC) injection port permits determination of a wide range of semi-volatile pesticides including organochlorines, organophosphates, triazines, and anilines in fatty composite dietary samples while reducing sample preparation time and solvent consumption. Dietary samples are mixed with diatomaceous earth and are Soxhlet-extracted with an azeotropic solution of hexane and acetone. Sample preparation uses liquid-liquid partitioning over diatomaceous earth followed by normal phase chromatography over partially deactivated alumina. The final cleanup step occurs in a preseparation column in the GC injector, which is able to perform splitless transfer of the analytes to the analytical column and purge 99% of the high molecular weight residue. Detection is performed by GC/mass spectrometry (MS) in the selected ion monitoring mode. Method detection limits were at or below 2 ng/g for 24 of 35 pesticides studied, with recovery between 70 and 125% for 27 pesticides in samples fortified at 10 ng/g. Recovery was not dependent on fat content when measured in laboratory fortified samples containing 1, 5, and 10% fat by weight. Precision over multiple injections was acceptable, with a relative standard deviation of 2.6-15% for 25 analytes.

Aluminum Oxide↗
PubMed2006

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry.

BACKGROUND: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. METHODS: Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. RESULTS: A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 microg/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. CONCLUSIONS: The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods.

Autoanalysis↗
PubMed2002

Quantitative noise analysis for gene expression microarray experiments.

A major challenge in DNA microarray analysis is to effectively dissociate actual gene expression values from experimental noise. We report here a detailed noise analysis for oligonuleotide-based microarray experiments involving reverse transcription, generation of labeled cRNA (target) through in vitro transcription, and hybridization of the target to the probe immobilized on the substrate. By designing sets of replicate experiments that bifurcate at different steps of the assay, we are able to separate the noise caused by sample preparation and the hybridization processes. We quantitatively characterize the strength of these different sources of noise and their respective dependence on the gene expression level. We find that the sample preparation noise is small, implying that the amplification process during the sample preparation is relatively accurate. The hybridization noise is found to have very strong dependence on the expression level, with different characteristics for the low and high expression values. The hybridization noise characteristics at the high expression regime are mostly Poisson-like, whereas its characteristics for the small expression levels are more complex, probably due to cross-hybridization. A method to evaluate the significance of gene expression fold changes based on noise characteristics is proposed.

Gene Expression↗
PubMed2001

Separation and identification methods for metalloproteinase inhibitors.

Metalloproteinase inhibitors are being explored for the treatment of a wide variety of human diseases including cancers, arthritis, cardiovascular disorders, human immunodeficiency virus infection, and central nervous system illnesses. This review provides an overview of various analytical sample preparation, separation, detection, and identification techniques employed for the quantitative and qualitative determination of these inhibitor compounds. Special emphasis is placed on biological sample preparation by automated solid-phase extraction, liquid-liquid extraction, and protein precipitation by centrifugation or filtration. Other sample preparation methodologies are also evaluated. Applications of high-performance liquid chromatography. gas chromatography, and capillary electrophoresis to the quantitative determination of metalloproteinase inhibitors are described. Examples of qualitative analysis of metalloproteinase inhibitors by hyphenated liquid chromatography with mass spectrometry and nuclear magnetic resonance are also presented. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.

Chromatography, Gas↗
PubMed2003

Size exclusion chromatography of cellulose in LiCl/N,N-dimethylacetamide.

Size exclusion of cellulose in LiCl/N,N-dimethylacetamide has been used for the past 15 years, yet much of the current research is still devoted to fundamental studies, as many issues regarding column calibration, separation mechanisms and solution behavior have not been resolved yet. The paper reviews practical aspects of sample preparation and it is demonstrated that sample heating and several techniques to aid solvent exchange call for reevaluation. It is further shown that the use of internal standard may introduce minor improvements in repeatability. The commonly used column calibration procedures, chromatographic conditions and applications are also reviewed. Further research is needed to understand the mechanisms of separation, to optimize column calibration and to facilitate and optimize sample preparation.

Acetamides↗
PubMed2006

Direct sampling of chemical weapons in water by photoionization mass spectrometry.

The vulnerability of water supplies to toxic contamination calls for fast and effective means for screening water samples for multiple threats. We describe the use of photoionization (PI) mass spectrometry (MS) for high-speed, high-throughput screening and molecular identification of chemical weapons (CW) threats and other hazardous compounds. The screening technology can detect a wide range of compounds at subacute concentrations with no sample preparation and a sampling cycle time of approximately 45 s. The technology was tested with CW agents VX, GA, GB, GD, GF, HD, HN1, and HN3, in addition to riot agents and precursors. All are sensitively detected and give simple PI mass spectra dominated by the parent ion. The target application of the PI MS method is as a routine, real-time early warning system for CW agents and other hazardous compounds in air and in water. In this work, we also present comprehensive measurements for water analysis and report on the system detection limits, linearity, quantitation accuracy, and false positive (FP) and false negative rates for concentrations at subacute levels. The latter data are presented in the form of receiver operating characteristic curves of the form of detection probability P(D) versus FP probability P(FP). These measurements were made using the CW surrogate compounds, DMMP, DEMP, DEEP, and DIMP. Method detection limits (3sigma) obtained using a capillary injection method yielded 1, 6, 3, and 2 ng/mL, respectively. These results were obtained using 1-microL injections of water samples without any preparation, corresponding to mass detection limits of 1, 6, 3, and 2 pg, respectively. The linear range was about 3-4 decades and the dynamic range about 4-5 decades. The relative standard deviations were generally <10% at CW subacute concentrations levels.

Chemical Warfare Agents↗
PubMed1992

Solid-phase extraction and HPLC determination of 4-vinyl guaiacol and its precursor, ferulic acid, in orange juice.

This study is undertaken to develop a simplified, rapid method to determine both immediate and potential off odors due to 4-vinyl guaiacol and its odorless precursor, ferulic acid, from a single sample preparation and chromatographic analysis. Orange juice sample preparation consists of a simple, C18 solid phase extraction. Utilizing a 5-microns, 25-cm, C18 column, both compounds can be separated within 40 min using a one-step, linear gradient beginning with an aqueous 12% tetrahydrofuran (THF)-5% acetonitrile mixture and ending with 35% aqueous THF. Hesperidin and nariutin have been identified as the compounds that interfered with the ultraviolet (UV) determination of sinapic and caffeic acids. Fluorescence detection with wavelength programming offers optimal sensitivity and selectivity. Recoveries of 4-vinyl guaiacol and ferulic acid range from 90 to 103%. Detection limits are 1 ppm and 5 ppm for ferulic acid and 4-vinyl guaiacol, respectively. Other hydroxycinnamic acids such as coumaric, sinapic, and caffeic acids may also be determined from the same chromatogram.

Beverages↗
PubMed2004

Human sperm responses to calcitonin, angiotensin II and fertilization-promoting peptide in prepared semen samples from normal donors and infertility patients.

BACKGROUND: Fertilization-promoting peptide (FPP), angiotensin II (AII) and calcitonin, present in seminal plasma, have significant effects on mouse sperm function in vitro. This study investigated responses of uncapacitated and capacitated human sperm to these peptides, initially using samples from donors with normal semen parameters and then samples from men attending infertility clinics. METHODS: Prepared suspensions were incubated in the presence/absence of a range of peptide concentrations and assessed using chlortetracycline (CTC) analysis and the hamster oocyte penetration test. RESULTS: In uncapacitated suspensions, maximal stimulatory responses (CTC) were obtained with calcitonin at 0.5-15 nmol/l and AII at 0.3-100 nmol/l; FPP is known to be most effective at 100 nmol/l. All peptides also significantly stimulated sperm penetrating ability. Combinations of peptides at low concentrations, having no detectable effect when used singly, elicited significant responses, suggesting that they work via the same signalling pathway. In suspensions incubated in the presence of fucose to accelerate capacitation and acrosome reactions, both FPP and calcitonin, but not AII, inhibited acrosome loss; however, AII did not interfere with responses to FPP and calcitonin. Unlike samples from 15 donors, some samples from >70 patients had high proportions of capacitated and/or acrosome-reacted cells when assessed immediately following preparation. Even so, the peptides usually elicited responses similar to those obtained with donor samples and combinations of peptides inhibited spontaneous acrosome loss for at least 3 h. CONCLUSIONS: The responses obtained in vitro suggest that these peptides could have significant effects on human sperm function in vivo and could also be used effectively in infertility clinics.

Angiotensin II↗
PubMed2006

Development of a novel 2D proteomics approach for the identification of proteins secreted by primary chondrocytes after stimulation by IL-1 and oncostatin M.

OBJECTIVES: To develop a proteomics approach to study changes in the secreted protein levels of primary human chondrocytes after stimulation by the pro-inflammatory cytokines interleukin-1 and oncostatin M. METHODS: Using both the primary human articular and bovine nasal chondrocyte-conditioned mediums, methods were investigated to enable the separation of proteins by two-dimensional (2D) gel electrophoresis. Differentially regulated proteins were identified using tandem electrospray mass spectrometery. RESULTS: We discovered that proteoglycans and glycosylaminoglycans (GAGs) secreted by chondrocytes significantly interfered with 2D gel focusing. Several different methods for GAG removal were attempted including enzymic digestion, cetyl pyridinium chloride precipitation and anion exchange in high salt. The anion exchange proved to be the most effective. Even from these initial gels, we were able to identify eight proteins produced by human chondrocytes: matrix metalloproteinase (MMP)-1, MMP-3, YKL40, cyclophilin A, beta2-microglobulin, transthyretin, S100A11, peroxidine 1 and cofilin. MMP-1, MMP-3, YKL40 and cyclophilin A were all identified as processed, smaller peptide fragments. CONCLUSIONS: We were able to develop a novel sample preparation protocol to allow the reproducible sample preparation of secreted proteins from human chondrocytes. From the initial data, we were able to show that at least some of the proteins produced were cleaved to smaller fragments as a result of proteolysis. Therefore, this technique provides valuable information about protein processing which gene-based arrays do not.

Aged↗
PubMed2002

Biocompatibility of dense hydroxyapatite prepared using an SPS process.

In the present study, the preparation of dense hydroxyapatite (HAp) materials at relative low temperatures using a spark plasma sintering was carried out. The bioactivity of HAp samples prepared by a spark plasma sintering method was investigated by in vitro tests and compared with HAp obtained by a conventional hot-pressing method. No growth of bone-like HAp crystals on surface of HAp sintered by a conventional hot-pressing method at 1200 degrees C was observed after immersion in a simulated body fluid (SBF) for 2 days. However, many large bone-like HAp crystals were observed on the surface of HAp samples prepared by a spark plasma sintering at 1200 degrees C after 2 days in the SBF immersion test. Especially, the negatively charged surface of the HAp samples prepared by spark plasma sintering was covered with larger HAp crystals compared with the positively charged surface. The electric poling of HAp was measured using a thermally simulated current technique. This rapid growth of bone-like HAp crystals of the HAp samples made by spark plasma sintering was believed to be related with the OH(-) and/or Ca(2(+) ) ion deficiency at the grain boundaries of the HAp matrix grains as well as a small electric poling effect resulting during the spark plasma sintering process.

Biocompatible Materials↗