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PubMed2007

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing.

Mass spectrometry has become an invaluable tool for the identification of prohibited peptide hormones and proteins in doping control analysis. Regulatory authorities have established criteria for identifying banned drugs in doping control specimens, but these criteria do not address the specific issues for high molecular weight protein drugs such as molecular weight determination of multiply charged molecules, analysis of chemically or enzymatically derived degradation products, identification of amino acid sequence tags, etc. Technical considerations such as sample preparation methods (e.g. immunoaffinity purification), resulting analytes (e.g. intact compounds vs. chemically or enzymatically derived peptides), ionization modes, analyzer resolution, and the information provided by respective techniques are discussed in light of sports drug testing requirements using typical application examples.

Doping in Sports↗

PubMed1976

[Radiochemical investigations on the decomposition of (mono) methyl-mercury by means of acid with regard to the determination of total mercury in fish (author's transl)].

Considerable amonts of mercury in fish muscle tissue are organically bound i.e. appear as (mono)methylmercury-compounds. In order to make mercury of organic origin available for the determination of total mercury by the "cold vapour atomic absorption method", a splitting of the carbon-mercury bond by means of suitable chemical treatment must be maintained beforehand. The main subject of this article are investigations with special regard to the behaviour of (mono)methylmercurychloride during different wet digestion methods. The procedures under study involve wet digestion under reflux with HNO3, with mixtures of HNO3 and HC10(4) and HNO3 and H2SO4, as well as wet digestion with HNO3 in a closed system (pressure decomposition). The course of the decomposition of (mono)methylmercury dependent on time, temperature and concentration of reagents are discussed in detail. All experiments were controlled by measurement of the radioactivity of Hg-203 which had been added in the chemical form of CH3-Hg-Cl. From the analytical results obtained two methods of sample preparation have been derived that permit a reliable determination of total mercury in fish.

Fish Products↗

PubMed2006

Twenty years research in cholinesterase biosensors: from basic research to practical applications.

Over the last decades, cholinesterase (ChE) biosensors have emerged as an ultra sensitive and rapid technique for toxicity analysis in environmental monitoring, food and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. Over the years, engineering of more sensitive ChE enzymes, development of more reliable immobilization protocols and progress in the area of microelectronics could allow ChE biosensors to be competitive for field analysis and extend their applications to multianalyte screening, development of small, portable instrumentations for rapid toxicity testing, and detectors in chromatographic systems. In this paper, we will review the research efforts over the last 20 years in fabricating AChE biosensors and the recent trends and challenges encounter once the sensor is used outside research laboratory for in situ real sample applications. The review will discuss the generations of cholinesterase sensors with their advantages and limitations, the existing electrode configurations and fabrication techniques and their applications for toxicity monitoring. We will focus on low-cost electrochemical sensors and the approaches used for enzyme immobilization. Recent works for achieving high sensitivity and selectivity are also discussed.

Biosensing Techniques↗

PubMed2001

Occurrence of heterocyclic aromatic amines in the Swiss diet: analytical method, exposure estimation and risk assessment.

A total of 86 meat samples, prepared in restaurants or homes, ready to eat (including poultry and fish) and 16 commercial samples such as bouillon (cubes) were analysed for heterocyclic aromatic amines (HAA). The analytical method consisted of an acidic extraction, clean-up on a cation exchange cartridge followed by an analogous HPLC step to recover the following HAA: IQ, MeIQ, MeIQx, 4,8-DiMeIQx, PhIP and 7,8-DiMeIQx. The HAA containing HPLC-fractions were collected, the HAA identified and quantified using two RP-HPLC-systems of different retention properties (UV-detection). The limit of quantitation was in the range of 0.2-0.4 ng/g and the relative repeatability 6-15%. The recovery of PhIP was lower than for the other HAA analysed (less than 80%) and a correction factor was applied. No significant differences of the HAA-concentration were found in samples from homes and restaurants, half of the total samples contained HAA at the following frequencies: PhIP and MeIQx 33% (each), 4,8-DiMeIQx 11% and MeIQ 4%; 7,8-DiMeIQx and IQ were not detected. The frequencies in commercial products were for MeIQx 31%, 7,8-DiMeIQx 19%, IQ 13% and PhIP 6%; MeIQ and 4,8-DiMeIQx were not found. Based on these data, the average exposure of Swiss adults to HAA was estimated to be 5 ng/kg body mass per day, commercial products contributing less than 10%. The theoretical excess cancer risk due to this intake was estimated on the base of the carcinogenic potency of the HAA in long-term animal experiments by linear extrapolation. The resulting risk in the order of 10(-4) at the maximum is discussed in terms of Swiss epidemiological data.

Amines↗

PubMed2004

PLE in the analysis of plant compounds. Part II: One-cycle PLE in determining total amount of analyte in plant material.

Pressurised liquid extraction (PLE) is recognised as one of the most effective sample preparation methods. Despite the enhanced extraction power of PLE, the full recovery of an analyte from plant material may require multiple extractions of the same sample. The presented investigations show the possibility of estimating the true concentration value of an analyte in plant material employing one-cycle PLE in which plant samples of different weight are used. The performed experiments show a linear dependence between the reciprocal value of the analyte amount (E*), extracted in single-step PLE from a plant matrix, and the ratio of plant material mass to extrahent volume (m(p)/V(s)). Hence, time-consuming multi-step PLE can be replaced by a few single-step PLEs performed at different (m(p)/V(s)) ratios. The concentrations of rutin in Sambucus nigra L. and caffeine in tea and coffee estimated by means of the tested procedure are almost the same as their concentrations estimated by multiple PLE.

Chromatography, High Pressure Liquid↗

PubMed2002

Determination of total cholesterol content in food by flow injection analysis with immobilized cholesterol oxidase enzyme reactor.

A new analytical method has been developed--including a new sample preparation procedure--for the automatic determination of total (free and bound) cholesterol in food by flow injection analysis (FIA) with immobilized (cholesterol oxidase) enzyme reactor (IMMER). A suitable sample preparation procedure has been applied to eliminate the problems derived from sensitivity of FIA equipment to common organic solvents used for dissolving of cholesterol: after direct saponification the non-saponificable fraction was dissolved in water phase detergent (sodium cholate) solution. The analytical method is based on the oxidation by cholesterol oxidase followed by the photometric determination of hydrogen peroxide (at 500 nm) using the indicator reaction with peroxidase. The new FIA method was tested for commercial food samples such as whole egg powder and dried pasta. The results were compared with data obtained by GC-determination. It was found that this new procedure is suitable for rapid automated measurement of total cholesterol content in foodstuff and consequently, the FIA technique with immobilized enzyme reactor could be an alternative to the widely used gas-chromatographic (GC) method.

Cholesterol↗

PubMed2003

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

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

Atrazine↗

PubMed2000

Comparison of zinc reduction with platinum reduction for analysis of deuterium-enriched water samples for the doubly labeled water technique.

OBJECTIVE: Isotope ratio mass spectrometry of hydrogen and oxygen is frequently used to determine total energy expenditure (TEE) using doubly labeled water. Conventionally, hydrogen isotope ratio is determined in hydrogen gas generated from water samples using zinc reduction. We compare this with a new automated platinum method to determine the ratios of hydrogen isotopes in deuterium-enriched water samples. RESEARCH METHODS AND PROCEDURES: The platinum method of sample preparation was compared with the zinc method in three ways: analytical variation in deuterium enrichment (within sample; n = 51), analytical variation in TEE estimates (within sample set; n = 10), and level of agreement of TEE estimates between both methods (n = 14). RESULTS: For the zinc method, the standard deviation for multiple sets of triplicate 2H2O sample analysis was +/-4.36 per thousand and +/-2.07 per thousand for platinum. The correlation between TEE estimates when sample sets were analyzed in duplicate was r = 0.89 for zinc and r = 0.83 for platinum. The intercept and slope of the regression line were significantly different from the line of identity for duplicate TEE estimates by zinc but were not different from the line of identity for platinum. After correction for the intra-assay variation of each method, the correlation between zinc and platinum for TEE was 0.77, and the intercept, but not the slope, of the regression was significantly different from the line of identity. The mean difference between the zinc method and the platinum method was 56 kcal/day, and the 95% confidence interval was -438 to 550 kcal/day. DISCUSSION: These data suggest that the platinum method is at least as reliable as the zinc method as a sample preparation technique for isotope ratio mass spectrometry of deuterium-enriched water samples. The platinum method is also less costly and less labor-intensive than the zinc method.

Autoanalysis↗

PubMed1994

Determination of oxytetracycline and its decomposition products in Tetran vulnerary powder.

A simple and fast HPLC method was developed for the determination of oxytetracycline and its decomposition products in pharmaceutical preparations. For this purpose an octadecyl modified silica stationary phase and a terner eluent containing aceto-nitrile-methanol-aqueous oxalic acid/EDTA solution was employed. Using this method, a stability study of oxytetracycline-containing powder was carried out. In this paper the analytical method and its performance data, the sample preparation and the obtained stability data are presented. The literature published on this topic is also shortly overviewed and discussed.

Chromatography, High Pressure Liquid↗

PubMed2004

Solid-phase microextraction (SPME) of permethrin residues from cucumber using a silica-bonded phase-coated stainless steel fibre.

Solid-phase microextraction (SPME) is a rapid, economic and solvent-free sample preparation method for the isolation of an analyte from its matrix. The technique uses a few centimetres of some adsorptive materials such as activated charcoal, polydimethylsiloxane or octadecyl silane coated onto fused silica optical fibres or, more recently, stainless steel fibres mounted into a microsyringe. The proposed method allows the extraction of permethrin from cucumber matrix into the coating, avoiding sample handling and saving evaporation of solvents and concentration steps. Adsorbed permethrin was desorbed in the split/splitless injection port of the gas chromatograph. The time required for each run was about 1.5 h, which gave a preconcentration of several orders of magnitude. The method could separate and quantify cis- and trans-isomers of permethrin. The calibration curve showed linearity in the range 1-9 micrograms ml-1, with detection limits of 0.03 and 0.05 microgram ml-1 for the cis- and trans-isomers of permethrin, respectively. The method had a recovery rate of about 70% and a relative standard deviation of less than 13%. Results suggest that this procedure provides a rapid and sensitive alternative method to those currently available.

Adsorption↗

PubMed2003

[Determination of cotinine in human urine by gas chromatograph-mass spectrometry].

A gas chromatograph-mass spectrometric assay has been developed for the quantitation of cotinine, the major metabolite of nicotine, in human urine. Extraction or condensing procedure was not required and the method reduced time involved in sample preparation. The analytes were separated on the fused-silica capillary column. The operating conditions were: injector, 250 degrees C; detector, 280 degrees C; column, 50-250 degrees C. The total gas flow-rate of helium (carrier) was 50 ml.min-1 and the pressure of column inlet was 100-200 kPa. The retention time was 18.1 min and the limit of quantitation was 5 ng.ml-1. This method provides an easy and simple assay for the detection of cotinine in clinical settings.

Adolescent↗

PubMed2002

[A simple method for determination of flurbiprofen in human plasma by gas chromatography-mass spectrometry].

A method for gas chromatograph-mass spectrometric assay has been developed for the quantitation of flurbiprofen in human plasma. Extraction or condensing procedure was not required and the method reduced time involved in sample preparation. The analytes were separated on the fused-silica capillary column. The operating conditions were: injector, 250 degrees C; detector, 300 degrees C; and column, 50-280 degrees C. The total gas flow-rate of helium (carrier) was 50 ml.min-1 and the pressure of column inlet was 100-200 kPa. The retention time was 18.1 min and the limit of quantitation was 0.5 microgram.ml-1. This method provides an easy and simple method for the detection of flurbiprofen.

Flurbiprofen↗

PubMed2004

Miniaturised sample preparation of fatty foodstuffs for the determination of polychlorinated biphenyls.

A miniaturised analytical method allowing the exhaustive extraction of environmentally relevant polychlorinated biphenyls (PCBs) from fatty foodstuffs and the purification of the extracts in a single step has been developed. After dispersion of the freeze dried sample on silica modified with 44% (w/w) of sulphuric acid, the mixture was packed in a glass column on top of a multilayer silica column used for removal of the lipids and biogenic co-extracted material. Using this arrangement, a complete sample preparation can be accomplished by two successive 10 min static extractions with hexane followed by a brief dynamic step to ensure purging of the sample and sorbents. The analytical method showed a satisfactory performance, with recoveries of the endogenous PCBs studied in the 81-134% range of those found using a more conventional off-line procedure, even though as small an amount of sample as 0.1 g was used. Detection limits by gas chromatography with micro-electron capture detection (GC-ECD) were in all cases lower than 0.3 ng/g sample (freeze dried basis) and the repeatability of the complete analytical procedure better than 14% (except for PCB 167). When combined with GC and ion trap detection in the tandem mass spectrometry mode, the miniaturised method has been proved to be a valuable alternative to the more expensive high resolution mass spectrometry for fast screening of PCBs 77, 126, and 169, even if these congeners were not isolated from the bulk of PCBs.

Chromatography, Gas↗

PubMed2002

A strategy for a post-method-validation use of incurred biological samples for establishing the acceptability of a liquid chromatography/tandem mass-spectrometric method for quantitation of drugs in biological samples.

Validated liquid chromatography/tandem mass spectrometric (LC/MS/MS) methods are now widely used for quantitation of drugs in post-dose (incurred) biological samples for the assessment of pharmacokinetic parameters, bioavailability and bioequivalence. In accordance with the practice currently accepted within the pharmaceutical industry and the regulatory bodies, validation of a bioanalytical LC/MS/MS method is performed using standards and quality control (QC) samples prepared by spiking the drug (the analyte) into the appropriate blank biological matrix (e.g. human plasma). The method is then declared to be adequately validated for analyzing incurred biological samples. However, unlike QC samples, incurred samples may contain an epimer or another type of isomer of the drug, such as a Z or E isomer. Such a metabolite will obviously interfere with the selected reaction monitoring (SRM) transition used for the quantitation of the drug. The incurred sample may also contain a non-isomeric metabolite having a molecular mass different from that of the drug (such an acylglucuronide metabolite) that can still contribute to (and hence interfere with) the SRM transition used for the quantitation of the drug. The potential for the SRM interference increases with the use of LC/MS/MS bioanalytical methods with very short run times (e.g. 0.5 min). In addition, a metabolite can potentially undergo degradation or conversion to revert back to the drug during the multiple steps of sample preparation that precede the introduction of the processed sample into the LC/MS/MS system. In this paper, we recommend a set of procedures to undertake with incurred samples, as soon as such samples are available, in order to establish the validity of an LC/MS/MS method for analyzing real-life samples. First, it is recommended that the stability of incurred samples be investigated 'as is' and after sample preparation. Second, it is recommended that potential SRM interference be investigated by analyzing the incurred samples using the same LC/MS/MS method but with the additional incorporation of the SRM transitions attributable to putative metabolites (multi-SRM method). The metabolites monitored will depend on the expected metabolic products of the drug, which are predictable based on the functional groups present in the chemical structure of the drug. Third, it is recommended that potential SRM interference be further investigated by analyzing the incurred samples using the multi-SRM LC/MS/MS method following the modification of chromatographic conditions to enhance chromatographic separation of the drug from any putative metabolites. We will demonstrate the application of the proposed strategy by using a carboxylic acid containing drug candidate and its acylglucuronide as a putative metabolite. Plasma samples from the first-in-man (FIM) study of the drug candidate were used as the incurred samples.

Animals↗

PubMed2005

Extending the solvent-free MALDI sample preparation method.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an important technique to characterize many different materials, including synthetic polymers. MALDI mass spectral data can be used to determine the polymer average molecular weights, repeat units, and end groups. One of the key issues in traditional MALDI sample preparation is making good solutions of the analyte and the matrix. Solvent-free sample preparation methods have been developed to address these issues. Previous results of solvent-free or dry prepared samples show some advantages over traditional wet sample preparation methods. Although the results of the published solvent-free sample preparation methods produced excellent mass spectra, we found the method to be very time-consuming, with significant tool cleaning, which presents a significant possibility of cross contamination. To address these issues, we developed an extension of the solvent-free method that replaces the mortar and pestle grinding with ball milling the sample in a glass vial with two small steel balls. This new method generates mass spectra with equal quality of the previous methods, but has significant advantages in productivity, eliminates cross contamination, and is applicable to liquid and soft or waxy analytes.

Chemistry Techniques, Analytical↗

PubMed2000

Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard.

We describe the first results of a quantitative LC-tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was approximately 0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of approximately 75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.

Calibration↗

PubMed2003

Before the injection--modern methods of sample preparation for separation techniques.

The importance of sample preparation methods as the first stage in an analytical procedure is emphasised and examined. Examples are given of the extraction and concentration of analytes from solid, liquid and gas phase matrices, including solvent phase extractions, such as supercritical fluids and superheated water extraction, solid-phase extraction and solid-phase microextraction, headspace analysis and vapour trapping. The potential role of selective extraction methods, including molecular imprinted phases and affinity columns, are considered. For problem samples alternative approaches, such as derivatisation are discussed, and potential new approaches minimising sample preparation are noted.

Chromatography↗

PubMed1998

A novel ETA-AAS background reduction method for determination of lead and cadmium in urine.

Urinary lead and cadmium level were determined in double diluted urine samples using two modifiers: 1% HNO3 (own modification) and 1.6% NH4H2PO4 that is generally used for matrix modification. The background signal is reduced in the samples diluted with 1% HNO3 because of the modifying effect of nitric acid. Lower variation coefficients were obtained in a run of 10 determinations of each of the samples processed by our method which is an advantageous feature compared with the method using 1.6% NH4H2PO4 as a modifier. Recovery is between 95 and 105% for both methods. The analytical performance, flexible pre-instrumental preparation and minimized risk of sample contamination make the use of 1% HNO3 modifier a reliable method in the routine practice of determination of the urinary Pb and Cd level.

Cadmium↗