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PubMed2003

Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maize.

A reliable, sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to determine four trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X and 3-acetyldeoxynivalenol) in maize. Sample preparation was performed by extracting the analytes with a mixture of acetonitrile and water, followed by a solid-phase extraction with Carbograph-4 cartridges as the purification step. For the LC/MS/MS analysis two interfacing systems, Turbo IonSpray (TISP) and atmospheric pressure chemical ionization (APCI), were compared in both negative and positive ion modes. LC and MS parameters were optimized to achieve better results and sensitivity. The effect of mobile phase modifiers such as ammonium acetate and formic acid on the ionization yield was also evaluated. The best results were obtained using the electrospray ionization (ESI) interface in negative ion mode and the multiple reaction monitoring mode (MRM) for the quantitation. The detection limits ranged between 10 ng/g for fusarenon X and 1.5 ng/g for deoxynivalenol. A linear working range was achieved with a standard deviation between 3 and 10% and recovery rates from the maize samples above 81%. The procedure was applied to the analysis of a set of maize samples collected from farms located in different areas of northern and central Italy. The investigated samples turned out to be contaminated primarily with deoxynivalenol and, to a minor extent, with its derivatives.

Chromatography, High Pressure Liquid↗
PubMed1996

Preconcentration and determination of Sn- and Pb-organic species in environmental samples by SPME and GC-AED.

A new sample preparation and preconcentration technique - solid phase microextraction (SPME) - is reported for the application of several tinorganic compounds and tetrabutyllead in aqueous samples. The solvent-free procedure is rapid in comparison with liquid-liquid extraction or SFE but also sensitive. Analytical variables of the extraction such as adsorption and desorption time, stirring rate and temperature has been investigated. The determination has been performed by GC coupled with atomic emission detection (AED). After optimization of the conditions of SPME a calibration was realized on the basis of a multicomponent standard solution, prepared by ethylation of organotin salts directly in the sample using sodium tetraethylborate (NaBEt(4)) without prior separation of the analytes from the matrix. The method permits preconcentration. Values of about 10 can be reached. A detection limit of 0.09 pg Sn and 0.08 pg Pb can be achieved under optimized conditions. The proposed procedure has been successfully applied to the analysis of organotin compounds in various slurry samples.

Journal Article↗
PubMed2001

Screening, confirmation and quantification of diuretics in urine for doping control analysis by high-performance liquid chromatography-atmospheric pressure ionisation tandem mass spectrometry.

A sensitive, selective, robust and fast method to identify 32 diuretics and masking agents in urine is described. The analytical procedure is reduced to a single XAD extraction step for sample preparation, followed by reversed-phase liquid chromatography in combination with atmospheric pressure ionisation/tandem mass spectrometry. This technique is, after minor modifications, suitable for screening analyses and confirmation of identity as well as quantitation of diuretics. Considerations relating to the stability and metabolism of the compounds are given if relevant for routine screening analyses.

Atmospheric Pressure↗
PubMed1999

Guidelines for in-house validation of analytical methods for pesticide residues in food and animal feeds.

Criteria are presented by which analytical methods may be judged to have been validated for the determination of pesticide residues. All stages of analysis are addressed, from initial preparation of samples to the production of results, but with a focus on simplicity and cost-effectiveness of the requirements. Criteria are provided for both quantitative and qualitative (screening) methods and they may be applied to single- or multi-residue methods.

Animal Feed↗
PubMed2003

The analysis of carbohydrate-deficient transferrin, marker of chronic alcoholism, using capillary electrophoresis.

Carbohydrate-deficient transferrin (CDT) is currently considered to be the best available marker for the diagnosis of chronic alcoholism. A large variety of methods have been developed, demonstrating the need for standardisation. Commercially available anion-exchange chromatographic-based assays are easy to use and require no specialised, expensive instruments. However, these methods cannot identify genetic transferrin variants or the carbohydrate-deficient glycoprotein syndrome. In 1989, a capillary isoelectric focusing method was developed for quantitative measurement of CDT. Despite the optimal resolution, this method is not easily applied in a clinical routine environment due to the complexity of analysis. Capillary electrophoresis in a polymer network using coated capillaries allowed full resolution of the sialoforms of human transferrin. The drawbacks due to an expensive and time-consuming sample preparation were eliminated when a method in neat serum was developed. Capillary zone electrophoresis allowed full resolution of the transferrin isoforms with a high analytical performance in a short analysis time thanks to a strong electroosmotic flow. Genetic transferrin variants were easily detected, avoiding false-positive results. Also, using capillary zone electrophoresis, it was shown that CDT is a suitable marker of chronic alcohol abuse detection in transferrin CD (common/cathodal) variants.

Alcoholism↗
PubMed2003

Determining whether transgenic and endogenous plant DNA and transgenic protein are detectable in muscle from swine fed Roundup Ready soybean meal.

Questions regarding the digestive fate of DNA and protein from transgenic feed have been raised in regard to human consumption and commercial trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops. Using highly sensitive, well-characterized analytical methods, pork loin samples were analyzed for the presence of fragments of transgenic and endogenous plant DNA and transgenic protein from animals fed meal prepared from conventional or glyphosate-tolerant Roundup Ready (RR) soybeans. Pigs were fed diets containing 24, 19, and 14% RR or conventional soybean meal during grower, early-finisher, and late-finisher phases of growth, respectively, and longissimus muscle samples were collected (12 per treatment) after slaughter. Total DNA was extracted from the samples and analyzed by PCR, followed by Southern blot hybridization for the presence of a 272-bp fragment of the cp4 epsps coding region (encoding the synthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase derived from Agrobacterium sp. strain CP4) and a 198-bp fragment of the endogenous soybean gene le1 (encoding soy lectin). Using 1 microgram of input DNA per reaction, none of the extracted samples was positive for cp4 epsps or le1 at the limit of detection (LOD) for these PCR/Southern blot assays. The LOD for these assays was shown to be approximately one diploid genome equivalent of RR soybean DNA, even in the presence of 10 micrograms of pork genomic DNA. A 185-bp fragment of the porcine preprolactin (prl) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification. Using a competitive immunoassay with an LOD of approximately 94 ng of CP4 EPSPS protein/g of pork muscle, neither the CP4 EPSPS protein nor the immunoreactive peptide fragments were detected in loin muscle homogenates from pigs fed RR soybean meal. Taken together, these results show that neither small fragments of transgenic DNA nor immunoreactive fragments of transgenic protein are detectable in loin muscle samples from pigs fed a diet containing RR soybean meal.

Animal Feed↗
PubMed2002

Metabolomics--the link between genotypes and phenotypes.

Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. In parallel to the terms 'transcriptome' and proteome', the set of metabolites synthesized by a biological system constitute its 'metabolome'. Yet, unlike other functional genomics approaches, the unbiased simultaneous identification and quantification of plant metabolomes has been largely neglected. Until recently, most analyses were restricted to profiling selected classes of compounds, or to fingerprinting metabolic changes without sufficient analytical resolution to determine metabolite levels and identities individually. As a prerequisite for metabolomic analysis, careful consideration of the methods employed for tissue extraction, sample preparation, data acquisition, and data mining must be taken. In this review, the differences among metabolite target analysis, metabolite profiling, and metabolic fingerprinting are clarified, and terms are defined. Current approaches are examined, and potential applications are summarized with a special emphasis on data mining and mathematical modelling of metabolism.

Arabidopsis↗
PubMed1996

A radioimmunoassay combined with solid-phase extraction for the determination of a novel anti-obesity agent, ARL 15849XX, in dog plasma.

A radioimmunoassay has been developed for the determination of ARL 15849XX, a cholecystokinin-8 (CCK-8) analogue, in dog plasma. The method incorporates solid-phase sample extraction and is suitable for the determination of the analyte at picogram per millilitre concentrations. The antiserum was raised in Suffolk-cross sheep following primary and booster immunisations with an immunogen prepared by conjugating ARL 16935XX, an analogue of ARL 15849KF, to bovine serum albumin. The radioligand was prepared by the no-carrier-added 125I iodination of a non-sulphated derivative, ARL 15745XX. The solid-phase extraction procedure, carried out using ion-exchange aminopropyl and octadecyl sorbents sequentially, was introduced to remove matrix interferences in the plasma and to enhance the method sensitivity. The calibration range is 20-1000 pg ml-1, using a 1 ml sample of undiluted dog plasma.

Amino Acid Sequence↗
PubMed2004

Quantitative determination of dityrosine in milk powders by liquid chromatography coupled to tandem mass spectrometry using isotope dilution.

An analytical method to quantify dityrosine (DiTyr) in milk powder samples is presented. The assay is based on isotope dilution liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESIMS/MS). The sample preparation entails acid hydrolysis of milk proteins followed by a solid phase-extraction (SPE) step. Neither artifactual formation nor degradation of DiTyr were observed during the proteolysis step. Mass spectral detection was performed in the positive ion mode by recording five transition reactions for DiTyr, in order to unambiguously confirm the presence of DiTyr by correct ion ratios. Under the analytical conditions used, the limit of detection (LOD) and limit of quantification (LOQ) for DiTyr were estimated at ca. 2 and 6 micromol DiTyr per mol of Tyr (using ca. 500 microg of milk proteins), with a mean recovery of ca. 90%. Quantification was conduced in eight different commercial milk powder samples, and the level of DiTyr ranged from below the LOQ up to 393.0 +/- 9.1 micromol DiTyr per mol of Tyr.

Animals↗
PubMed2006

Liquid chromatographic method for the simultaneous determination of cefalexin and trimethoprim in dog plasma and application to the pharmacokinetic studies of a coformulated preparation.

A liquid chromatographic method is described for the simultaneous determination of cefalexin and trimethoprim in dog plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recoveries for both analytes. Chromatographic separation of the analytes was achieved on a C(18) column using a mixture of 2 mol/l formate buffer (pH 3.5), methanol and acetonitrile (22:7:7, v/v/v) containing a 0.002 mol/l sodium dodecyl sulfate as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration ranges of 1.0-100.0 microg/ml for cefalexin and 0.5-50.0 microg/ml for trimethoprim. For each level of QC samples including the lower limit of quantification, both inter- and intra-day precisions (R.S.D.) were < or =14.0% for cefalexin and < or =11.4% for trimethoprim, and accuracy (RE) was -1.4% for cefalexin and -3.0% for trimethoprim. The present LC method was successfully applied to the pharmacokinetic studies of coformulated cefalexin dispersible tablets after oral administration to beagle dogs.

Animals↗
PubMed1999

Extraction and isolation of linear alkylbenzenesulfonate and its sulfophenylcarboxylic acid metabolites from fish samples.

Linear alkylbenzenesulfonate (LAS) is the most widely used synthetic surfactant. In fish, assessment of the environmental risk and investigation of the biotransformation behavior of LAS require compound-specific methods for extraction and isolation of LAS and its biotransformation products, sulfophenylcarboxylic acids (SPC). Matrix solid-phase dispersion (MSPD) extraction with subsequent ion-pair liquid-liquid (IP-LL) partitioning of the extract was a time-efficient sample preparation method for analysis of LAS. The recovery of parent LAS from spiked fish exceeded 70%, and the limit of quantitation ranged around 0.2 mg.kg-1 corresponding to 0.6 mumol.kg-1. In a simultaneous determination of LAS and SPC in fish, the analytes were MSPD extracted in different fractions. The target compounds were separated from the sample matrix by protein precipitation and subsequent isolation of (a) SPC by graphitized carbon black solid-phase extraction of the supernatant and (b) parent LAS by IP-LL partitioning of the pellet obtained after protein precipitation. The recoveries of the model compounds C12-2-LAS and C4-3-SPC were 84 +/- 6 and 65 +/- 11%, respectively. The use of C3-3-SPC as an internal standard corrected for the loss of the biotransformation product during sample workup. The suitability of both methods was demonstrated by analyzing fish containing LAS and SPC incurred during aqueous exposure.

Animals↗
PubMed2004

[Determination of the content of wear metal in lubrication oil by ICP-AES].

An analytical method for determining wear metal elements directly by ICP-AES was proposed in this paper. The optimum condition was selected. The Conostan S-21 and Conostan 75 (oil-base standard samples) prepared by multi-element oil-base reserved standard samples were applied to make calibration curve. Oil samples were diluted 5 times by kerosene. The contents of wear metals in lubrication oil were determined by ICP-AES. The relative standard deviation (RSD) of all elements was less than 1%. In addition, the authors represented the analytical efficacy of Leeman labs multi-track profile ICP, and its rapid and accurate characters of simultaneous multi-element measurement.

Calibration↗
PubMed2001

Low-level determination of polymorph composition in physical mixtures by near-infrared reflectance spectroscopy.

Near-infrared reflectance spectroscopy (NIRS) was employed to quantify sulfathiazole (STZ) forms I and III in binary physical mixtures in which one form was the dominant component. Physical mixtures of the polymorph pair were made by weight, ranging from 0 to 5% STZ form I mixed with STZ form III, and near-infrared spectra of the powder samples contained in glass vials were obtained over the wavelength region of 1100 to 2500 nm. A calibration plot was constructed by plotting STZ form I weight percent against a ratio of second-derivative values of log (1/R') (where R' is the relative reflectance) versus wavelength. The coefficients of determination, R(2), were > 0.9983 and standard errors were low for these calibration models. The instrument reproducibility, method error, and limits of detection (LOD) and quantification (LOQ) of the method were assessed. The LOD and LOQ were determined from the standard deviation of the response of the 0% analyte sample (0% STZ form I containing 100% STZ form III). The LOQ was subsequently validated with independently prepared samples. The results show that polymorphs can be quantified in binary physical mixtures in the 0.3% polymorph composition range. These studies indicate that NIRS is a precise and accurate quantitative tool for determination of polymorphs in the solid state, is comparable to other characterization techniques, and is more convenient to use than many other methods.

Calibration↗
PubMed2001

Liquid-phase microextraction of phenolic compounds combined with on-line preconcentration by field-amplified sample injection at low pH in micellar electrokinetic chromatography.

This paper describes a novel method that applies field-amplified sample injection (FASI) in micellar electrokinetic chromatography (MEKC) with a low pH background electrolyte (BGE). Six phenolic compounds prepared in water or NaOH solution were used as the test analytes. Sample was injected electrokinetically after the introduction of a plug of water. During the injection, the water plug was pumped out of the capillary inlet by the electroosmotic flow, and the phenolic anions migrated very quickly in the direction of the outlet. When the anions reached the boundary between the water plug and BGE, they were neutralized and ceased moving. Thereafter, MEKC was initiated for the separation. This on-line preconcentration method could be conveniently coupled with a liquid-liquid-liquid microextraction procedure, in which a hollow fiber was used as an extraction solvent support to extract the analytes from the water sample. The acceptor phase consisted of 8 mM NaOH. After extraction, the extract was analyzed directly by MEKC, as described.

Journal Article↗
PubMed2001

Separation methods for methotrexate, its structural analogues and metabolites.

Methotrexate (MTX) is the prototype folate antagonist cytotoxic drug, employed in the therapy of solid tumors and leukaemias, and recently also as an immunosuppressive agent in organ transplantation, in the treatment of some autoimmune diseases and in the therapy of severe asthma. MTX is one of the very few antineoplastic drugs the therapeutic concentration monitoring of which is currently employed in clinical practice and can be routinely measured in biological samples by a number of different analytical techniques, among which are immunoenzymatic and chromatographic methods. Each technique has of course its own advantages in terms of sensitivity, specificity, speed, cost and level of expertise required. Along with therapeutic drug concentration monitoring and clinical pharmacology, fundamental research into the mechanism of action of antifolate drugs is still a field which requires the measurement of MTX, of its new analogues and of their metabolites in biological samples. This review summarizes the instrumental conditions and the performance of several published chromatographic methods employed to measure MTX, its metabolites and some analogues in clinical and biological research. More than 70 papers describing chromatographic assays for MTX and its metabolites have been published in the literature between 1975 and 2000. A wide array of experimental conditions for sample preparation, analyte separation and detection have been employed. According to their chemical properties, MTX, its metabolites and analogue drugs present in several biological samples (plasma, serum, saliva, urine, cerebrospinal fluid, tissue specimens) can be extracted, separated and detected under a variety of chromatographic conditions, i.e. on different stationary phases, under a wide choice of mobile phase conditions (acidic or neutral, employing ion-pair or micellar chromatography), followed by several detection techniques (UV-Vis spectrophotometry, pre- or post-column oxidation and fluorimetry, electrochemistry, mass spectrometry). Optimized methods allow simultaneous measurement within a few minutes of the plasma levels of MTX and its main metabolites at concentrations in the low-nM range. One special field which needs sensitive, fast and inexpensive methods for the detection and measurement of MTX is the monitoring of contamination in workplace environments, such as pharmaceutical industries and oncological hospital pharmacies, and in sewage waters. The measurement of the intracellular gamma-oligo-glutamate metabolites of biological folates, of MTX and of some analogue drugs is of great importance in basic pharmacological research. The existence of empirical quantitative relationships between the retention of individual oligomers under different chromatographic conditions and the number of added glutamic acid units allows identification of the metabolites even when authentic standards are not available.

Folic Acid Antagonists↗
PubMed1979

Isotachophoresis in capillary tubes of CSF proteins -- especially gammaglobulins.

Isotachophoresis in polyacrylamide gel tubes (PAG-ITP) and in capillary tubes (Tachophor, LKB) have previously been found by the authors, to be very promising high-separation methods for CSF and serum proteins, especially regarding the diagnosis of MS. PAG-ITP methods for analytical and preparative use have been described by the authors elsewhere, while in this paper proper cationic systems for ITP in capillary tubes for studying gammaglobulins in microliter amounts of CSF and serum are described, i.e. the albumin injection-clog problem is avoided and the preparation time can be forced. By using microdialysis of the CSF samples for desalting, with a technique easy to perform and with high reproducibility, microliter amounts of native CSF can be performed in less than half an hour. The method seems to be even more applicable for clinical and scientific use if the capillary isotachophoretic apparatus is connected to a synchronized equipment (LKB Tachophrac) with a cellulosa acetate strip onto which the separated fractions are ejected for further analysis by immunological tests. The analytical systems used have been especially directed to gammaglobulins in CSF and serum regarding further studies on demyelinating and infectious disorders of the nervous system.

Cerebrospinal Fluid Proteins↗
PubMed2006

Rapid, micro-scale preparation and very fast gas chromatographic separation of cod liver oil fatty acid methyl esters.

The present research is focussed on the optimization of a fast and economically convenient method for the sample preparation and gas chromatographic separation of fatty acids contained in lipidic samples. This, as part of a wider project that has, as ultimate goal, the automation of this specific analytical procedure. The developed approach was applied to the analysis of cod liver oil, a highly complex lipid characterized by a wide nutritional interest. Derivatization was carried out by using low quantities (microL amounts) of a reagent and solvent, while the derived fatty acid methyl esters (FAMEs) were separated in 120 s on a 10 m x 0.1 mm i.d. polar micro-bore column. The total analysis time required for six samples was approximately 45 min (7.5 min/sample), considering a simultaneous process of methylation and GC separation of previously prepared samples. The results obtained were compared to those derived from conventional applications on the same sample. With regard to the validation of the rapid method, peak area/retention time repeatability, linear range, limit of detection (LOD) and quantitation (LOQ) were determined. Peak assignment was carried out by exploiting bidimensional group-type mapping information obtained in a comprehensive gas chromatographic application.

Animals↗
PubMed1998

Results of a European interlaboratory study for the determination of oxytetracycline in pig muscle by HPLC.

An interlaboratory study was organised in 1996 in order to determine laboratory testing performance for measuring oxytetracycline (OTC) and 4-epioxytetracycline (4-epiOTC) residues in pig muscle. The organisation was designed according to the 'International Harmonised Protocol for Proficiency Testing of (Chemical) Analytical Laboratories' produced by the Association of Official Analytical Chemists (AOAC International) and the International Union of Pure and Applied Chemistry (IUPAC). Fourteen National Reference Laboratories (NRLs) out of the 15 asked to participate agreed to analyse the samples. These laboratories were from 13 different European Union (EU) Member States and each participant was allowed to use the extraction method of their own choice but had to use liquid chromatography as the analytical technique. Most of the methods used were based on UV detection (simple wavelength or diode-array detection) but some involved fluorimetric detection. The production of incurred samples was prepared on-site. The OTC and 4-epiOTC concentrations present in the samples were determined by taking the mean of the results (excluding outliers) and the deviation of each result from the assigned value was measured. The performance of the laboratories was evaluated by calculating the 'z-scores'. The results were globally satisfactory and showed that all 14 laboratories were capable of determining OTC and 4-epiOTC in pig muscle with satisfactory accuracy. Only two laboratories obtained a questionable result in terms of repeatability.

Animals↗