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PubMed2000

Laboratory database to manage electrophoresis and chromatography separations and the associated samples.

A database was developed to store, organize, and retrieve the data associated with electrophoresis and chromatography separations. It allows laboratories to store extensive data on separation techniques (analytical and preparative). The data for gel electrophoresis includes gel composition, staining methods, electric fields, analysis, and samples loaded. The database stores data on chromatography conditions, the samples used, and the fractions collected. The data structure of this database was designed to maintain the link between samples (including fractions) from chromatography separations and their analysis by gel electrophoresis. The database will allow laboratories to organize and maintain a large amount of separation and sample data in a uniform data environment. It will facilitate the retrieval of the separation history of important samples and the separation conditions used.

Chromatography↗
PubMed2000

Micellar electrokinetic chromatography for the analysis of cefpirome in microdialysis and plasma samples obtained in vivo from human volunteers.

Pharmacokinetics of drugs in the human interstitial space fluid can be monitored by means of microdialysis. However, the small-volume microdialysis samples containing low drug concentrations require a sensitive analytical method. In the present study, micellar electrokinetic chromatography (MEKC) is described for the quantification of cefpirome in human microdialysis and plasma samples. Sample preparation of human plasma samples by ultracentrifugation was suitable for comparison of plasma and microdialysate concentrations. Limits of quantification were 2 microg/mL and 0.3 microg/mL for plasma and microdialysate samples, respectively. The limit of detection (LOD) was estimated at 0.2 microg/mL for the plasma and microdialysate samples. In conclusion, MEKC is a reliable and reproducible technique for measuring cefpirome concentrations in microdialysates as well as centrifuged plasma samples.

Adsorption↗
PubMed2000

Analysis of catechins and caffeine in tea extracts by micellar electrokinetic chromatography.

Cancer chemotherapy is a new and important medical science and much interest has been focused on catechins, not only for their antioxidant activity, but also because of their known antimutagenic and antitumorigenic properties. Green tea and black tea, which are among the most popular beverages consumed worldwide, contain many different catechins. We developed an analytical method capable of separating six different catechins and caffeine in tea by micellar electrokinetic chromatography in only 20 min without extensive sample preparation. Furthermore, we compared the amount of catechins and caffeine in several teas and different preparation modes.

Caffeine↗
PubMed1999

The oral nitrate and nitrite intake in The Netherlands: evaluation of the results obtained by HPIC analysis of duplicate 24-hour diet samples collected in 1994.

In spring and autumn of 1994 duplicates of 24-h diets were collected from 123 respondents. One of the goals of this study was to determine the amount of nitrite and nitrate in the duplicates of 24-h diets to establish the oral daily intake of these analytes. For this purpose an HPIC/UV method for the determination of nitrate and nitrite in duplicate diets was developed and validated. The sample preparation procedure was derived from the in-house method used for the determination of nitrate and nitrite in human blood plasma. The sample is diluted with water, deproteinized with Carrez reagent, followed by chromatographic clean-up on an SPE C18-column. Both the nitrate and the nitrite results are quantitative. The recovery for nitrite was on average 104% (n = 21, spiking levels: 0.84-95 mg/kg) and for nitrate on average 103% (N = 21, spiking levels: 1.8-404 mg/kg). Samples of duplicates of 24-h diets were analysed according to the method developed. The median intake of nitrite calculated from the samples collected in spring 1994 was 0.6 mg/person day (range < 0.1-6.1 mg/person/day). For the samples collected in autumn 1994 these figures were < 0.2 mg/person/day (range < 0.1-16 mg/person/day). The mean intake of nitrate was 73 mg/person/day (range 7-322 mg/person/day) in spring 1994 and 87 mg/person/day (range 1-310 mg/person/day) in autumn 1994. The overall mean intake of nitrate in 1994 was 80 mg/person/day. The daily intake for nitrate was higher than that found in the duplicate diet study carried out in 1984/1985, when an average daily intake of 52 mg/person was measured. The intake of nitrite was also higher than found in the duplicate diets collected in 1984/1985. The findings of the study are discussed in the context of the ADI for nitrate and nitrite as well as the outcome of other recent European intake studies.

Adolescent↗
PubMed2006

Specific detection of anabasine, nicotine, and nicotine metabolites in urine by liquid chromatography-tandem mass spectrometry.

The sensitive and specific detection of nicotine, its metabolites, and the tobacco alkaloid, anabasine, is useful in evaluating the success of smoking cessation treatments and detecting tobacco use, passive exposure, and nontobacco nicotine exposure in potential transplant recipients, insurance clients, and elective surgical patients. Rapid sample preparation and extended high-performance liquid chromatographic separation of tobacco alkaloids and metabolites was interfaced with tandem mass spectrometry. By using deuterated internal standards and appropriate confirmatory ion mass transitions, direct injection of centrifugally clarified urine was possible. The method had excellent precision, limit of quantitation, and linearity. The rigorous separation method revealed an interferent of nicotine that had coeluted with anabasine in more rapid chromatography and that may result in tobacco use misclassification. The method provides more specific detection of tobacco exposure and illustrates the potential of centrifugal clarification for sample preparation in the detection of multiple analytes in urine.

Anabasine↗
PubMed2003

High-throughput analysis of protein/peptide complexes by immunoprecipitation and automated LC-MS/MS.

BACKGROUND: The identification of interacting proteins within protein complexes is key to understanding the transduction and regulation of cell signaling pathways, and is also a useful tool for identifying novel disease markers. Immunoprecipitation of protein complexes followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to identify targets that bind to a protein of interest. Here, we report a high-throughput nanoflow LC-MS/MS method for analysis of protein/peptide complexes. APPROACH: To overcome the large dwell volume from connecting lines and the injector when using an autosampler in microcapillary LC-MS/MS, we employed a valve-controlled variable flow method with a peptide trap. This method enables fast trapping of peptides from samples injected by an autosampler within minutes followed by a split-controlled nanoflow of acetonitrile gradient through a microcapillary C18 column to separate and elute peptides into the ion-trap MS/MS. Over 40 protein/peptide samples at femtomole levels could be analyzed continuously using the same in-house packed microcapillary C18 column. The p38 mitogen-activated protein kinase (p38 MAP kinase) is a signaling protein that is involved in transduction of extracellular signals including oxidative stress, growth factors, and cytokines, with diverse cellular consequences such as cell proliferation, differentiation, or apoptosis. Immunocomplex of monoclonal anti-p38 beta antibodies from 2 mg total lysates from the OCI LY-1 lymphoma cell line was resolved in 1D-PAGE gel followed by silver staining of the gel. Protein bands were excised and digested with trypsin. Peptides were extracted and analyzed using the automated LC-MS/MS method. RESULTS AND CONCLUSIONS: From 37 excised protein bands in the 1D-PAGE gel, we identified more than 50 proteins, including cytoskeletal proteins, ribosomal proteins, transcription factors, and KIAA potential signaling proteins. These proteins are the potential targets that may interact directly or indirectly with the p38 MAP kinase proteins. Our studies demonstrate the utility of automated nanoflow LC-MS/MS for sensitive and high-throughput analysis of protein/peptide complexes. Utilization of methods based on this principle would greatly facilitate peptide interaction mapping and other proteomic studies requiring high-throughput pre-analytical sample preparation.

Actins↗
PubMed2005

[The effect of preparation method on the quality of shen-mai injection].

OBJECTIVE: To investigate the effect of different preparation method on the quality of Shen-mai injection. METHOD: The Shen-mai injection samples were prepared using three different methods. Fingerprints of Shen-mai extracts red ginseng, and its intermediates were obtained using an HPLC analytical procedure. The contents of ginsenoside Rg1, Rc and Rb1, and the gross saponins of Shen-mai extract were quantitatively mensured with HPLC procedures. RESULT: There was significant difference in fingerprints and chemical contents of the injections prepared by the three different methods. CONCLUSION: The quality of Shen-mai injection was greatly influenced by the preparation method. HPLC fingerprinting method can be applied for the determination of the Shen-mai preparations.

Chromatography, High Pressure Liquid↗
PubMed1991

Improved sample preparation for accurate determination of low concentrations of lead in whole blood by graphite furnace analysis.

The effect of low concentrations of lead on pre- and post-natal growth and development is a current concern. We describe a simple method of sample preparation for direct determination of lead in whole blood by Zeeman graphite-furnace atomic absorption spectrometry. This procedure improves analytical precision and accuracy of lead determinations at low concentrations as compared with published furnace data. At blood lead concentrations of 0.25, 1.98, and 3.76 mumol/L, within-run CVs were 3.2%, 1.8%, and 1.4% respectively; between-run CVs were 7.3%, 2.9%, and 2.2%. Accuracy, as demonstrated by analytical recovery, ranged from 99% to 102%. Our reproducibility/accuracy score in the 1989 Quebec interlaboratory comparison program was 96% compared with the target, second best of 66 participating laboratories.

Adult↗
PubMed2006

A sample preparation process for LC-MS/MS analysis of total protein drug concentrations in monkey plasma samples with antibody.

The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel "denaturing" step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC-MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC-MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9ng/mL and 53920.0ng/mL with a coefficient of determination (r(2)) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations.

Animals↗
PubMed1986

Measurement of WR-1065 in plasma: preliminary pharmacokinetics in the beagle.

Described is a high-performance liquid chromatographic (HPLC) method for the quantification of WR-1065 [2-(3-aminopropylamino)-ethanethiol] in plasma. After a brief sample preparation, the drug and internal standard were separated in less than 15 minutes using a reversed-phase column and ion-pairing reagent. An electrochemical detector provided a minimum quantifiable limit of 0.05 microgram/mL. The analytical method was applied in three experiments to measure drug concentrations in the plasma of beagle dogs following IV administration of WR-1065 (60 mg/kg). The concentration-time data, best described by a three-compartment open kinetic model, were used to estimate pharmacokinetic parameters. Mean values were: steady state volume of distribution--2.27 L X kg-1; clearance--0.064 L X min-1 X kg-1; and terminal elimination half-life--81.4 min. The high clearance is consistent with the formation of mixed disulfides in the circulation.

Animals↗
PubMed2005

On-probe sample preparation without washes for matrix-assisted laser desorption/ionization mass spectrometry using an anion exchange medium.

When the mass spectra of biological samples (proteins, peptides, and so on) are obtained routinely by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a serious problem is the reduction of the ionization efficiency by impurities, such as buffer salts and detergents. We focused our attention on devising a method to maintain the ionization efficiency of protein samples, even in the presence of sodium dodecyl sulfate (SDS), without any extra purification step. Although no protein ion peaks are observed in the presence of 2.5% SDS with the usual methods, the addition of a granular anion exchange silica gel to the matrix solution allowed the protein ion peaks to be obtained with an excellent signal-to-noise ratio. Together with other supporting experiments, we suggest that the positively charged surface (the basic environment derived from the anion exchange groups) and the roughness of the particles were important for good ionization in the presence of a high SDS concentration. For a very uneven surface, the SDS might be absorbed into the particle interiors during the process of cocrystallization with the matrix and analytes, which is known as the molecular sieve effect, and the SDS concentration in the surface crystalline film might be reduced. As a result, we developed an on-probe sample preparation method without washes for MALDI, using a strong anion exchange silica gel. This method is applicable even in the presence of 2.5% SDS, and is not only very simple but also inexpensive, because it can be used with the standard MALDI target plates.

Anions↗
PubMed2001

Application of on-site solid-phase microextraction in aquatic dissipation studies of profoxydim in rice.

The application of a manual operated solid-phase microextraction (SPME)-HPLC interface is discussed for the analysis of thermally labile analytes in aqueous matrices. The technique has been applied on-site at a flooded rice field to demonstrate its potential for real time extraction of the herbicide profoxydim. Thus, compounds which would otherwise easily degrade in the aqueous matrices within hours or days could be determined more accurately. The fibers were shipped back to the laboratory with express delivery where the target analyte was desorbed from the fiber and determined by HPLC-UV analysis. The SPME method was characterized by significant ruggedness where conventional techniques such as liquid-liquid extraction and solid-phase extraction require additional shipping and handling costs and time-consuming multiple sample preparation steps. In general, any delay in shipping the aqueous samples to the laboratory has the potential for sample degradation and a loss in accuracy when using non on-site extraction techniques. Fifty microm Carbowax-templated resin coatings were most suitable for coupling SPME to HPLC in order to achieve a high sensitivity for polar analytes. The SPME technique was characterized by a good sensitivity and a precision less than 10% RSD. The SPME-LC-UV method was linear over at least three orders of magnitude while achieving a limit of detection in the lower microg/l range. The on-site SPME method has shown significantly increased accuracy. Profoxydim was determined at concentrations of ca. 180 microg/l 3 h after an application on a flooded bare soil field.

Benzene Derivatives↗
PubMed1991

Immunoaffinity chromatography, its applicability and limitations in multi-residue analysis of anabolizing and doping agents.

The use of (multi-)immunoaffinity chromatography in residue analysis is discussed. After an introduction to the immunochemical background an overview of applications is given. A distinction is made between the following methods: (1) single-antibody, single-analyte procedures; (2) single-antibody, multi-analyte procedures; (3) multi-antibody, multi-analyte procedures. It is concluded that immunoaffinity chromatography is superior to most other techniques for sample preparation and extract clean-up. Its advantages in multi-residue procedures are most clear when compared with e.g. high-performance liquid chromatography. In combination with gas chromatography-low-resolution mass spectrometry, very effective multi-residue methods are possible. Most frequently they concern screening procedures which can fulfill the identification criteria for reference methods. It is concluded that the use of (multi-)immunoaffinity chromatography will proliferate further in the 1990s. However, its future viability is highly dependent on the interest of commercial firms and on the involvement of the EC Community Bureau of Reference in manufacturing and supplying the necessary materials.

Anabolic Agents↗
PubMed1999

Development and validation of a method for the analysis of a pharmaceutical preparation by near-infrared diffuse reflectance spectroscopy.

A near-infrared (NIR) spectroscopic method based on the use of a fiber optical probe for the analysis of a commercially available pharmaceutical preparation is proposed. The analyte is identified by comparison with a second-derivative spectral library, using the correlation coefficient as the discriminating parameter. Once a sample has been positively identified, the active principle is quantified with partial least-squares (PLS) calibration. The proposed method was validated for use as a control method; to this end, the selectivity of the identification process, and the repeatability, intermediate precision, accuracy, linearity, and robustness of the active principle quantitation, were assessed.

Sensitivity and Specificity↗
PubMed2005

Determination of atorvastatin and metabolites in human plasma with solid-phase extraction followed by LC-tandem MS.

The aim of the present study was to develop a chromatographic method for the analysis of atorvastatin, o- and p-hydroxyatorvastatin (acid and lactone forms) in human plasma after administration of atorvastatin at the lowest registered dose (10 mg) in clinical studies. Sample preparation was performed by solid-phase extraction and was followed by separation of the analytes on an HPLC system with a linear gradient and a mobile phase consisting of acetonitrile, water and formic acid. Detection was achieved by tandem mass spectrometry operated in the electrospray positive ion mode. Validation of the method for the compounds for which reference compounds were available (acid forms of atorvastatin, o- and p-hydroxyatorvastatin) showed linearity within the concentration range (0.2-30 ng/ml for atorvastatin acid and p-hydroxyatorvastatin acid, and 0.5-30 ng/ml for o-hydroxyatorvastatin acid) (r2 > or = 0.99, n = 5 for all analytes). Accuracy and precision (evaluated at 0.5, 3 and 30 ng/ml for atorvastatin, p-hydroxyatorvastatin and 1, 3 and 30 ng/ml for o-hydroxyatorvastatin) were both satisfactory. The detection limit was 0.06 ng/ml for atorvastatin and p-hydroxyatorvastatin, and 0.15 ng/ml for o-hydroxyatorvastatin. The method has been successfully applied in a clinical study where atorvastatin, o- and p-hydroxyatorvastatin (both acid and lactone forms) could be detected in a 24-h sampling interval after administration of the lowest registered dose of atorvastatin (10 mg) for one week.

Atorvastatin↗
PubMed2003

ED-XRF as a tool for rapid minerals control in milk-based products.

An ED-XRF method for the rapid determination of a series of analytes (phosphorus, sulfur, chlorine, potassium, calcium, iron, zinc) in milk-based products has been developed and validated. The investigated samples were commercial products obtained from various parts of the world. Reference values measured by inductively-coupled plasma-optical emission spectroscopy and by potentiometry for chloride were used to calibrate the ED-XRF. Calibrations were established with 30 samples, and validation was made using a second set of 30 samples. An evaluation of this alternative method was done by comparison with data from the reference methods. Pellets of 4 g were prepared under 2 tons of pressure. For each sample, 3 pellets were prepared and analyzed. Limits of quantification and repeatabilities were evaluated for the described analytes.

Animals↗
PubMed2006

Determination of benzoxazinone derivatives in plants by combining pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray mass spectrometry.

A new analytical method based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction with LiChrolut RP C18 cartridges was evaluated for the sample preparation, extraction, and cleanup of eight naturally occurring benzoxazinone derivatives, 2-beta-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one, 2-beta-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, 2-hydroxy-1,4-benzoxazin-3-one, 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one, benzoxazolin-2-one, and 6-methoxybenzoxazolin-2-one in plant samples. Afterward, liquid chromatography-electrospray mass spectrometry, using the selected ion monitoring mode and internal standard (2-MeO-DIBOA, indoxyl-beta-D-glucoside, and quercetin-3-O-rutinoside) quantification method was performed. This paper demonstrates the effectiveness of the PLE method, in conjunction with sensitive and specific mass spectrometric detection, for the quantitative recovery of compounds of the benzoxazinone class from plants. The recoveries of the analytes ranged from 66 to 110% with coefficients of variation ranging from 1 to 14%. This method gave detection limits between 1 and 27 microg/g. The method was applied to foliage and roots of three different wheat cultivars, and the analytes were detected in the range of 11-3261 microg/g of dry weight.

Benzoxazines↗
PubMed2000

A RIA combined with SPE for the determination of a dual D2-receptor and beta2-adrenoceptor agonist, AR-C68397XX, in human plasma.

A radioimmunoassay has been developed for the determination of AR-C68397XX, a dual D2-receptor and beta2-adrenoceptor agonist, in human plasma. The method incorporates solid phase sample extraction and is suitable for the determination of the analyte at pg ml(-1) concentrations. The antiserum was raised in Suffolk cross sheep following primary and booster immunisations with an immunogen prepared by conjugating a carboxyphenylmethyl derivative of AR-C68397XX, to bovine serum albumin. The radioligand was prepared by the 125I-labelled iodination of a derivative of AR-C68397XX. The solid phase extraction procedure, using octadecyl sorbent, was introduced to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 20-500 pg ml(-1), using 0.5 ml of undiluted human plasma sample.

Adrenergic beta-2 Receptor Agonists↗