930 resultados para matrix-assisted laser desorption-mass spectrometry (MALDI-MS)
Resumo:
Multi-element analysis of honey samples was carried out with the aim of developing a reliable method of tracing the origin of honey. Forty-two chemical elements were determined (Al, Cu, Pb, Zn, Mn, Cd, Tl, Co, Ni, Rb, Ba, Be, Bi, U, V, Fe, Pt, Pd, Te, Hf, Mo, Sn, Sb, P, La, Mg, I, Sm, Tb, Dy, Sd, Th, Pr, Nd, Tm, Yb, Lu, Gd, Ho, Er, Ce, Cr) by inductively coupled plasma mass spectrometry (ICP-MS). Then, three machine learning tools for classification and two for attribute selection were applied in order to prove that it is possible to use data mining tools to find the region where honey originated. Our results clearly demonstrate the potential of Support Vector Machine (SVM), Multilayer Perceptron (MLP) and Random Forest (RF) chemometric tools for honey origin identification. Moreover, the selection tools allowed a reduction from 42 trace element concentrations to only 5. (C) 2012 Elsevier Ltd. All rights reserved.
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Arrabidaea chica (crajiru) is an important Amazonian plant. Its extracts are used as red pigments, antimicrobial agents and astringents. Three different varieties of this species are cultivated in the Amazon region. In this work, direct infusions of A. chica extracts from these three varieties were analyzed via electrospray ionization mass spectrometry (ESI(+)-MS) fingerprinting. Derived data from the spectra were classified by using a multivariate method (PLS-DA, partial least squares-discriminant analysis). The direct method that is herein presented relies on extraction of dry, powdered leaves with acidified methanol/water solution with no further sample preparation. The resulting supernatants were analyzed by direct infusion ESI(+)-MS, which provides characteristic fingerprints of the sample composition. 3-Deoxyanthocyanidins are important substances in A. chica, their ions were used as markers in the PLS-DA data treatment. PLS-DA was able to differentiate the three varieties. ESI(+)-MS fingerprinting works as a simple and fast method to differentiate varieties of A. chica.
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STUDY BY MASS SPECTROMETRY OF SOLUTIONS OF [HYDROXY(TOSYLOXY)IODO]BENZENE: PROPOSED DISPROPORTIONATION MECHANISMS. Solutions of [hydroxy(tosyloxy)iodo]benzene (HTIB or Koser's reagent) in acetonitrile were analyzed using high resolution electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) under different conditions. Several species were characterized in these analyses. Based on these data, mechanisms were proposed for the disproportionation of the iodine(III) compounds in iodine(V) and iodine(I) species.
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Travelling wave ion mobility mass spectrometry (TWIM-MS) with post-TWIM and pre-TWIM collision-induced dissociation (CID) experiments were used to form, separate and characterize protomers sampled directly from solutions or generated in the gas phase via CID. When in solution equilibria, these species were transferred to the gas phase via electrospray ionization, and then separated by TWIM-MS. CID performed after TWIM separation (post-TWIM) allowed the characterization of both protomers via structurally diagnostic fragments. Protonated aniline (1) sampled from solution was found to be constituted of a ca. 5:1 mixture of two gaseous protomers, that is, the N-protonated (1a) and ring protonated (1b) molecules, respectively. When dissociated, 1a nearly exclusively loses NH3, whereas 1b displays a much diverse set of fragments. When formed via CID, varying populations of 1a and 1b were detected. Two co-existing protomers of two isomeric porphyrins were also separated and characterized via post-TWIM CID. A deprotonated porphyrin sampled from a basic methanolic solution was found to be constituted predominantly of the protomer arising from deprotonation at the carboxyl group, which dissociates promptly by CO2 loss, but a CID-resistant protomer arising from deprotonation at a porphyrinic ring NH was also detected and characterized. The doubly deprotonated porphyrin was found to be constituted predominantly of a single protomer arising from deprotonation of two carboxyl groups. Copyright (C) 2012 John Wiley & Sons, Ltd.
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A rapid, sensitive and specific method for quantifying hydroxocobalamin in human plasma using paracetamol as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethanol 100%; -20°C). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on Prevail C8 3 μm, analytical column (2.1×100 mm i.d.). The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 5-400 ng.mL-1 (r>0.9983). The limit of quantification was 5 ng.mL-1. The method was also validated without the use of the internal standard. The precision in the intra-batch validation with IS was 9.6%, 8.9%, 1.0% and 2.8% whereas without IS was 9.2%, 8.2%, 1.8% and 1.5% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in intra-batch validation with IS was 108.9%, 99.9%, 98.9% and 99.0% whereas without IS was 101.1%, 99.3%, 97.5% and 92.5% for 5, 15, 80 and 320 ng/mL, respectively. The precision in the inter-batch validation with IS was 9.4%, 6.9%, 4.6% and 5.5% whereas without IS was 10.9%, 6.4%, 5.0% and 6.2% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in inter-batch validation with IS was 101.9%, 104.1%, 103.2% and 99.7% whereas without IS was 94.4%, 101.2%, 101.6% and 96.0% for 5, 15, 80 and 320 ng/mL, respectively. This HPLC-MS-MS procedure was used to assess the pharmacokinetics of Hydroxo cobalamin following intramuscular injection 5000 μg in healthy volunteers of both sexes (10 males and 10 females). The volunteers had the following clinical characteristics (according to gender and expressed as mean ± SD [range]): males: age: 32.40 ± 8.00 y [23.00-46.00], height: 1.73 ± 0.07 m [1.62-1.85], body weight: 72.48 ± 10.22 Kg [60.20- 88.00]; females: age: 28.60 ± 9.54 y [18.00-44.00], height: 1.60 ± 0.05 m [1.54-1.70], body weight: 58.64 ± 6.09 Kg [51.70- 66.70]. The following pharmacokinetic parameters were obtained from the hydroxocobalamin plasma concentration vs. time curves: AUClast, T1/2, Tmax, Vd, Cl, Cmax and Clast. The pharmacokinetic parameters were 120 (± 25) ng/mL for Cmax, 2044 (± 641) ng.h/mL for AUClast, 8 (± 3.2) ng.mL-1 for Clast, 38 (± 15.8) hr for T1/2 and 2.5 (range 1-6) hr for Tmax. Female volunteers presented significant (p=0.0136) lower AUC (1706 ± 704) ng.h/mL) and larger (p=0.0205) clearance (2.91 ± 1.41 L/hr), as compared to male 2383 ± 343 ng.h/mL and 1.76 ± 0.23 L/hr, respectively. These pharmacokinetic differences could explain the higher prevalence of vitamin B12 deficiency in female patients. The method described validated well without the use of the internal standard and this approach should be investigated in other HPLC-MS-MS methods.
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[EN]Until recently, sample preparation was carried out using traditional techniques, such as liquid–liquid extraction (LLE), that use large volumes of organic solvents. Solid-phase extraction (SPE) uses much less solvent than LLE, although the volume can still be significant. These preparation methods are expensive, time-consuming and environmentally unfriendly. Recently, a great effort has been made to develop new analytical methodologies able to perform direct analyses using miniaturised equipment, thereby achieving high enrichment factors, minimising solvent consumption and reducing waste. These microextraction techniques improve the performance during sample preparation, particularly in complex water environmental samples, such as wastewaters, surface and ground waters, tap waters, sea and river waters. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and time-of-flight mass spectrometric (TOF/MS) techniques can be used when analysing a broad range of organic micropollutants. Before separating and detecting these compounds in environmental samples, the target analytes must be extracted and pre-concentrated to make them detectable. In this work, we review the most recent applications of microextraction preparation techniques in different water environmental matrices to determine organic micropollutants: solid-phase microextraction SPME, in-tube solid-phase microextraction (IT-SPME), stir bar sorptive extraction (SBSE) and liquid-phase microextraction (LPME). Several groups of compounds are considered organic micropollutants because these are being released continuously into the environment. Many of these compounds are considered emerging contaminants. These analytes are generally compounds that are not covered by the existing regulations and are now detected more frequently in different environmental compartments. Pharmaceuticals, surfactants, personal care products and other chemicals are considered micropollutants. These compounds must be monitored because, although they are detected in low concentrations, they might be harmful toward ecosystems.
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Nowadays obesity can be defined as a global epidemic. The precise identification of circulating biomarkers involved in this pathology could be essential to early diagnose potential co-morbidities and to better address the development of future therapeutic strategies. Published evidences show that circulating steroid hormones and endocannabinoids might have a role in the physiopathology of obesity; however, a precise and reliable quantification of these molecules is still lacking. In the first part of the present thesis, we developed a sensitive, specific and accurate quantification method for nine steroid hormones using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system. This method has been used first for a comparative study with immunoassays, currently used in the clinical practice to quantify these molecules and then to redefine circulating reference intervals in healthy subjects. Furthermore, we measured circulating steroid hormones in three groups of subjects: normo-weight, over-weight and obese, defining different steroid hormones profiles depending on the obesity state. The role of circulating endocannabinoids in humans is still unclear, however there are several evidences concerning their involvement in obesity. In the second part of the thesis, we determined changes of circulating endocannabinoids in obese patients after a weight loss induced by bariatric surgery, currently the most effective long-term treatment for obesity, using LC/MS-MS. We measured basal and dynamic endocannabinoids plasma levels in 12 patients with severe obesity before, one month after and six months after the Roux-en-Y gastric bypass intervention, currently one of the most performed types of bariatric surgery. All together the findings illustrated in this thesis project will help better define the role of steroid hormones and endocannabinoids in the framework of obesity in humans and the role that each type of molecule might have in its pathophysiology.
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A liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography-negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. All the corticosteroids can be detected at a concentration around 1 μg kg(-1); the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.
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A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.
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New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.
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A polyphasic taxonomic analysis was carried out on 11 uncommon Gram-stain-negative, non-motile, catalase- and oxidase-positive, but indole-negative, bacterial strains isolated from tortoises. Phenotypically and genetically they represented a homogeneous group of organisms most closely related to, but distinct from, Uruburuella suis. In a reconstructed 16S rRNA gene tree they clustered on a monophyletic branch next to U. suis with gene similarities between strains of 99.5-100%, and of up to 98.2% with U. suis . DNA-DNA hybridization indicated the organisms represented a novel species with only 40% DNA-DNA similarity with U. suis . Partial sequencing of rpoB resulted in two subclusters confirming the 16S rRNA gene phylogeny; both genes allowed clear separation and identification of the novel species. Furthermore, they could be unambiguously identified by matrix-assisted laser desorption ionization time-of-flight MS, where, again, they formed a highly homogeneous cluster separate from U. suis and other members of the family Neisseriaceae . The major fatty acids were C(16 : 0) and summed feature C(16 : 1)ω7c/iso-C(15 : 0) 2-OH. The DNA G+C content was 54.4 mol%. Based on phenotypic and genetic data we propose classifying these organisms as representatives of a novel species named Uruburuella testudinis sp. nov. The type strain is 07_OD624(T) ( = DSM 26510(T) = CCUG 63373(T)).
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OBJECTIVE To analytically validate a gas concentration of chromatography-mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at -80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at -80°C for 1 week had no effect on GC-MS results.
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In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric λ repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.
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Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is ΔU2SHNS,6SI2SHNS,6SI2SHNS,6SIHNAc,6SGHNS,3S,6S (±DDD4–7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.
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Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 106 DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 106 DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.