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1.
Anal Bioanal Chem ; 411(27): 7221-7231, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31583449

RESUMO

DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS has emerged as an excellent method from the viewpoint of sensitivity, reproducibility, and cost. However, LC-MS/MS methods have limitations due to a lack of the stability and the standardization required for a laboratory assay. The present study aimed to establish a robust assay that guarantees highly accurate measurements of global DNA methylation levels. There are at least three facets of the developed method. The first is discovery of the solvent conditions to minimize sodium adducts. The second is improvement of separation of nucleosides by LC using the columns that had not been used in previous similar studies. The third is success in reduction of the uncertainty of the measurement results, which was achieved by the calibration using the ratio of mdC but not the absolute amount in the presence of internal standards. These facets represent the advantage over methods reported previously. Our developed method enables quantification of DNA methylation with a short time length (8 min) for one analysis as well as with the high reproducibility of measurements that is represented by the inter-day CV% being less than 5%. In addition, data obtained from measuring global DNA methylation levels in cultured cell lines, with or without pharmacological demethylation, support its use for biomedical research. This assay is expected to allow us to conduct initial screening of epigenetic alterations or aberration in a variety of cells.


Assuntos
Metilação de DNA , DNA/química , Espectrometria de Massas em Tandem/métodos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Citidina/análogos & derivados , Citidina/análise , Citidina/genética , DNA/genética , Humanos , Espectrometria de Massas em Tandem/economia , Fatores de Tempo
2.
Anal Bioanal Chem ; 411(25): 6697-6709, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401670

RESUMO

The adulteration of meat products by the undeclared addition of commercially available blood plasma powder is quite conceivable due to low costs, high protein contents (about 70%), and advantageous functional properties. This applies particularly to pork, which has the highest meat production rate in the European Union. Evidence of this type of food fraud has been rather difficult to identify due to the lack of appropriate analytical methods, especially when adding plasma to meat of the same animal species. Consequently, a rapid UHPLC-MS/MS method for the detection of porcine blood plasma in emulsion-type pork sausages was developed. After protein extraction and tryptic digestion in a quick and simple one-pot process, species-specific marker peptides for porcine blood cell proteins (four markers) and plasma proteins (12 markers) were measured by UHPLC-MS/MS. Emulsion-type pork sausages were produced from a variety of raw materials that differed in the age or sex of the slaughtered pigs. Sausages were spiked with 0.5, 1, 1.5, 2, 3, or 5% meat substitution by one of two plasma powders, or produced as corresponding blank samples, and subjected to different thermal treatments as full or semi-preserves. Four plasma peptides were identified for the overall sample that allowed detection down to 0.7% meat substitution from the sum of their peak areas, with 5% error probability for both false positives and negatives.


Assuntos
Proteínas Sanguíneas/análise , Contaminação de Alimentos/análise , Produtos da Carne/análise , Carne Vermelha/análise , Animais , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Emulsões/química , Feminino , Análise de Alimentos/economia , Análise de Alimentos/métodos , Masculino , Peptídeos/análise , Plasma/química , Suínos , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo
4.
Anal Bioanal Chem ; 411(15): 3241-3255, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31020368

RESUMO

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.


Assuntos
Manose/química , Oligossacarídeos/química , Espectrometria de Massas em Tandem/métodos , Configuração de Carboidratos , Sequência de Carboidratos , Isomerismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/economia
5.
Anal Bioanal Chem ; 411(8): 1503-1508, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30710208

RESUMO

Isocitrate dehydrogenase (IDH) I and II mutations in gliomas cause an abnormal accumulation of 2-hydroxyglutarate (2-HG) in these tumor cells. These mutations have potential prognostic value in that knowledge of the mutation status can lead to improved surgical resection. Information on mutation status obtained by immunohistochemistry or genomic analysis is not available during surgery. We report a rapid extraction nanoelectrospray ionization (nESI) method of determining 2-HG. This should allow the determination of IDH mutation status to be performed intraoperatively, within minutes, using a miniature mass spectrometer. This study demonstrates that the combination of tandem mass spectrometry with low-resolution mass spectrometry allows this analysis to be performed with confidence. Graphical Abstract.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isocitrato Desidrogenase/genética , Mutação , Espectrometria de Massas em Tandem/instrumentação , Desenho de Equipamento , Humanos , Papel , Espectrometria de Massas por Ionização por Electrospray/economia , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo
6.
J Agric Food Chem ; 67(9): 2691-2699, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30753780

RESUMO

A fast, less expensive, analytical approach with high metrologic reliability was developed to assist an official program for 21 marine biotoxins, monitoring in bivalve mollusks. The simultaneous analysis of lipophilic and hydrophilic marine biotoxins was achieved using a sample preparation protocol based on solid-liquid extraction and low-temperature cleanup, followed by liquid chromatography coupled to tandem mass spectrometry. Samples were extracted with acidified methanol/water (90:10), followed by low-temperature cleanup. Chromatographic separation was obtained using a cyano-bonded silica phase. The mobile phase was composed of water and acetonitrile, with both 0.1% formic acid and 2.5 mmol L-1 ammonium formate. Electrospray ionization was used in both negative and positive modes. The single-laboratory validation approach enabled method performance assessment, and the necessary data to design a model for result expression were yielded. With this purpose, a systematic study of errors and uncertainties was performed. This new analytical approach aimed to minimize the use of highly expensive analytical standards, promoting economic viability to be applied by high-throughput routine laboratories. After its implementation on the Brazilian official monitoring program, positive results near the regulatory limits were obtained, demonstrating the fit for purpose of the method as a surveillance tool.


Assuntos
Bivalves/química , Cromatografia Líquida/economia , Toxinas Marinhas/análise , Espectrometria de Massas em Tandem/economia , Animais , Brasil , Cromatografia Líquida/métodos , Análise Custo-Benefício , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem/métodos
7.
J Pharm Biomed Anal ; 163: 105-112, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30292136

RESUMO

A new ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry was developed to evaluate the quality of Tanreqing injection. Seven flavonoids (Rutin, Baicalin, Scutellarin, Chrysin-7-O-Beta-d-glucoronide, Oroxylin A-7-O-ß-d-glucoronide, Wogonin, Luteolin-7-O-glucoside), two phenolic acids (Chlorogenic acid, Caffeic acid) and two cholesterines (Ursodeoxycholic acid, Chenodeoxycholic acid) in Tanreqing injection could be measured simultaneously. For the determination of the eleven compounds, the conditions were set as follows: The mobile phase was a gradient of 0.1% aqueous formic acid solution (A) and acetonitrile (B); the flow rate was 0.2 mL min-1, the column was Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm); and the multiple-reaction monitoring (MRM) with a negative electro spray ionization interface (ESI-) was selected. Within the test ranges, all the standard regression curves showed excellent linear regression (r > 0.99). In terms of (relative standard deviation) RSDs, the precision, repeatability and stability of the eleven compounds were all lower than 3%. The recovery rates of Tanreqing injection and the RSD were 97.8-103.7% and 0.4%-2.0%, respectively. The RSD value was in accordance with the requirements of less than 3.0%. This method has been successfully used in the analysis of Tanreqing injection. In summary, a fast, accurate and reliable UPLC-ESI--MS/MS method was successfully developed for the simultaneous detection of the eleven major active ingredients with different chemical structures in Tanreqing injection, and can be used for the quality control of Tanreqing injection as well.


Assuntos
Fracionamento Químico/métodos , Ácido Desoxicólico/análise , Medicamentos de Ervas Chinesas/análise , Flavonoides/análise , Hidroxibenzoatos/análise , Fracionamento Químico/instrumentação , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Composição de Medicamentos/normas , Contaminação de Medicamentos/prevenção & controle , Medicamentos de Ervas Chinesas/química , Controle de Qualidade , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo
8.
J Pharm Biomed Anal ; 164: 223-230, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30391811

RESUMO

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/análise , Avaliação Pré-Clínica de Medicamentos/métodos , Enzimas Imobilizadas/química , Peptidil Dipeptidase A/química , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Benzilisoquinolinas/análise , Benzilisoquinolinas/química , Benzilisoquinolinas/farmacologia , Berberina/análogos & derivados , Berberina/análise , Berberina/química , Berberina/farmacologia , Cromatografia de Afinidade/economia , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Análise Custo-Benefício , Avaliação Pré-Clínica de Medicamentos/economia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Enzimas Imobilizadas/isolamento & purificação , Células HEK293 , Humanos , Oligopeptídeos/química , Peptidil Dipeptidase A/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
9.
J Pharm Biomed Anal ; 165: 198-206, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30553110

RESUMO

Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography-mass spectrometry (LC-MS) bioanalytical assays. Recently, the application of SIL drugs and LC-MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes. It also eliminates potential metabolite interference with AMS-based assay. However, one major challenge in the application of this approach is the potential interference between the unlabeled drug, the microdose SIL drug, and the SIL-IS during LC-MS analysis. Here we report a convenient and cost-effective strategy to overcome the interference by monitoring the isotopic ion (instead of the commonly used monoisotopic ion) of the interfered compound in MS analysis. For the BMS-986205 absolute BA case study presented, significant interference was observed from the microdose IV drug [13C7,15N]-BMS-986205 to its SIL-IS, [13C7,15N, D3]-BMS-986205, since the difference of nominal molecular mass between the two compounds is only 3 mu, and there is a Cl atom in the molecules. By applying this strategy (monitoring the 37Cl ion for the analysis of the IS), a 90-fold reduction of interference was achieved, which allowed the use of a synthetically accessible SIL compound and enabled the fast progress of the absolute BA study. This strategy minimizes the number of stable isotope labels used for avoiding interference, which greatly reduces the difficulty in synthesizing the SIL compounds and generates significant time and cost savings. In addition, this strategy can also be used to reduce the MS response of the analyte, therefore, avoiding the detector saturation issue of LC-MS/MS assay for high concentration BMS-986205. A LC-MS/MS assay utilizing this strategy was successfully developed for the simultaneous analysis of BMS-986205 and [13C7, 15N]-BMS-986205 in dog plasma using [13C7,15N, D3]-BMS-986205 as the IS. The assay was successfully applied to a microdose absolute BA study of BMS-986205 in dogs. The assay was also validated in human plasma and used to support a human absolute BA study. The same strategy can also be applied to other compounds, including those not containing Cl or other elements with abundant isotopes, or other applications (e.g. selection of internal standard), and the applications were presented.


Assuntos
Acetamidas/análise , Cromatografia Líquida/métodos , Inibidores Enzimáticos/análise , Quinolinas/análise , Espectrometria de Massas em Tandem/métodos , Acetamidas/administração & dosagem , Acetamidas/farmacocinética , Animais , Disponibilidade Biológica , Cromatografia Líquida/economia , Análise Custo-Benefício , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Marcação por Isótopo , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Espectrometria de Massas em Tandem/economia
10.
J Sep Sci ; 42(2): 491-500, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30462887

RESUMO

Rapid, economic, and highly effective determination of multiple mycotoxins in complex matrices has given huge challenges for the analytical method. In this study, an economic analytical strategy based on sensitive and rapid ultrafast liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometry technique was developed for the determination of seven mycotoxins of different chemical classes (aflatoxin B1 , B2 , G1 , and G2 , ochratoxin A, T-2 toxin, and HT-2 toxin) in Polygonum multiflorum. Target mycotoxins were completely extracted using a modified quick, easy, cheap effective, rugged, and safe method without additional clean-up steps. The types of extraction solvents and adsorbents for the extraction procedure were optimized to achieve high recoveries and reduce coextractives in the final extracts. Due to significant matrix effects for all analytes (≤68.9% and ≥110.0%), matrix-matched calibration curves were introduced for reliable quantification, exploring excellent linearity for the seven mycotoxins with coefficients of determination >0.9992. The method allowed high sensitivity with limit of detection in the range of 0.031-2.5 µg/kg and limit of quantitation in the range of 0.078-6.25 µg/kg, as well as satisfactory precision with relative standard deviations lower than 8%. Recovery rates were between 74.3 and 119.8% with relative standard deviations below 7.43%. The proposed method was successfully applied for 24 batches of P. multiflorum samples, and six samples were found to be positive with aflatoxin B1 , B2 , G1 , or ochratoxin A. The method with significant advantages, including minimum analytical time, low time and solvent consumption, and high sensitivity, would be a preferred candidate for economic analysis of multiclass mycotoxins in complex matrices.


Assuntos
Micotoxinas/análise , Polygonum/química , Cromatografia Líquida/economia , Espectrometria de Massas em Tandem/economia
11.
Pain Physician ; 21(6): E583-E592, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30508989

RESUMO

BACKGROUND: The technical advantages of direct-to-definitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) urine testing for monitoring patient compliance in pain management are well known. However, the design and implementation of LC-MS/MS methods are more controversial, including factors such as determining appropriate cutoffs, specimen processing (e.g., specimen hydrolysis), reporting of qualitative and/or quantitative results, and test menu. OBJECTIVES: The objective of the research was to compare the clinical performance of our previous urine pain toxicology panel, a combination of immunoassay (IA) screens and LC-MS/MS, to our current pain toxicology panel, which features direct-to-definitive LC-MS/MS for 34 drugs and metabolites. STUDY DESIGN: Six months of results from our previous pain toxicology panel were compared to 5.5 months of results from our current pain toxicology panel, enabling us to make conclusions regarding clinical performance. SETTING: The research took place at Brigham and Women's Hospital in Boston, MA. METHODS: The percentage of false positive IA results was evaluated for our previous pain toxicology panel. The positivity rates for each drug and/or metabolite were calculated for both the previous and current panels, including rates of detection of both prescribed and illicit drugs. The turnaround time (TAT), direct and send-out costs associated with each approach, as well as projected cost savings were also determined. RESULTS: False positive rates with IA ranged from 0% to 29%; the highest false positive rate was seen for 6-acetylmorphine (6-AM). The elimination of IA, addition of metabolites, and/or lowering of cutoffs increased the detection rate of 6-AM, benzoylecgonine (cocaine metabolite), fentanyl, morphine, and oxycodone. The ability to differentiate compliance from simulated compliance improved after eliminating specimen hydrolysis. The TAT improved significantly and projected yearly cost savings with the current panel was $95,003 (USD). In our opinion, qualitative results appeared sufficient to assess compliance in the majority of cases. LIMITATIONS: Our study was performed in a single academic center in a specific geographic region; therefore, our results may not be generalizable to other types of centers or regions. CONCLUSION: Direct-to-definitive LC-MS/MS testing has several clinical benefits, including reduction of false positive results, improved assessment of patient compliance, decreased TAT, and increased detection of drug use and abuse. Cost savings were also realized using this approach. KEY WORDS: Direct-to-definitive, LC-MS/MS, immunoassay, sensitivity, cost, pain management, turnaround time, patient compliance.


Assuntos
Cromatografia Líquida/métodos , Imunoensaio/métodos , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos , Urinálise/métodos , Cromatografia Líquida/economia , Reações Falso-Positivas , Feminino , Humanos , Imunoensaio/economia , Manejo da Dor , Detecção do Abuso de Substâncias/economia , Transtornos Relacionados ao Uso de Substâncias/diagnóstico , Transtornos Relacionados ao Uso de Substâncias/urina , Espectrometria de Massas em Tandem/economia , Urinálise/economia
12.
Anal Bioanal Chem ; 410(27): 7239-7247, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30151683

RESUMO

An automated on-line solid-phase extraction (SPE) following liquid chromatography tandem mass spectrometry was established for the fast determination of plant growth regulator residues in soybean sprout and mung bean sprout. The crude extracted specimens were directly purified on a poly (2-(dimethylamino) ethyl methacrylate-co-ethylene dimethacrylate) monolithic column which was well-defined as the on-line SPE adsorbent. Under the optimized conditions, the developed method gave the linear range of 0.3-50 ng/mL for gibberellin and 2,4-dichlorophenoxyacetic acid, 0.2-50 ng/mL for 4-chlorophenoxyacetic acid, and 0.5-50 ng/mL for 1-naphthaleneacetic acid (r ≥ 0.998). The detection limits (S/N = 3) ranged from 1.0 to 2.5 µg/kg and the recoveries for spiked soybean sprout samples were in the range of 75.0-93.3%. Besides, the total time for one analysis was 16 min. The reusability of the monolith was up to 600 extractions. The proposed process facilitated fully automated SPE and accurate determination in one step with rapidity, simplicity, and reliability. Graphical abstract ᅟ.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Reguladores de Crescimento de Planta/análise , Extração em Fase Sólida/métodos , Soja/química , Espectrometria de Massas em Tandem/métodos , Vigna/química , Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Ácido 2,4-Diclorofenoxiacético/análise , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/instrumentação , Desenho de Equipamento , Giberelinas/análise , Limite de Detecção , Ácidos Naftalenoacéticos/análise , Plântula/química , Extração em Fase Sólida/economia , Extração em Fase Sólida/instrumentação , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/instrumentação , Fatores de Tempo
13.
Rapid Commun Mass Spectrom ; 32(17): 1514-1520, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29873842

RESUMO

RATIONALE: Non-Saccharomyces yeasts are widespread microorganisms that nowadays have gained importance for their ability to produce volatile compounds which in alcoholic beverages improve aromatic complexity and therefore the overall quality. Their rapid identification and differentiation in fermentation processes is vital for timely decision making. METHODS: A total of 19 strains of Pichia kluyveri isolated from mezcal, tejuino and cacao fermentations were analyzed with rep-PCR fingerprinting using the primer (GTG)5 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Microflex LT mass spectrometer using Biotyper 3.1 software (Bruker Daltonics). RESULTS: The comparative analysis between MS spectra and rep-PCR patterns obtained from these strains showed a high similarity between both methods. However, minimal differences between the obtained rep-PCR and MALDI-TOF MS clusters could be observed, especially by the presence and/or absence of one or more discriminating peaks even when they have similarities in their main spectra projection, observing that isolates from the same fermentative process were grouped into the same sub-cluster based on their MALDI-TOF MS profiles. CONCLUSIONS: The data shown suggests that MALDI-TOF MS is a promising alternative technique for rapid, reliable and cost-effective differentiation of native yeast strains isolated from different traditional fermented foods and beverages.


Assuntos
/microbiologia , Técnicas de Tipagem Micológica/métodos , Pichia/química , Pichia/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Cacau/microbiologia , Fermentação , Técnicas de Tipagem Micológica/economia , Pichia/genética , Pichia/metabolismo , Reação em Cadeia da Polimerase/economia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/economia
14.
Anal Chem ; 90(14): 8553-8560, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29924586

RESUMO

High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.


Assuntos
Neoplasias Colorretais/patologia , Proteínas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Neoplasias Colorretais/química , Transporte de Elétrons , Elétrons , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Processos Fotoquímicos , Processamento de Proteína Pós-Traducional , Proteômica/economia , Espectrometria de Massas em Tandem/economia
15.
Anal Chem ; 90(14): 8487-8494, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29920210

RESUMO

There has been an increasing interest during recent years in the role of the gut microbiome on health and disease. Therefore, metabolites in human feces related to microbial activity are attractive surrogate marker to track changes of microbiota induced by diet or disease. Such markers include 5α/ß-stanols as microbiome-derived metabolites of sterols. Currently, reliable, robust, and fast methods to quantify fecal sterols and their related metabolites are missing. We developed a liquid chromatography-high-resolution mass spectrometry (LC-MS/HRMS) method for the quantification of sterols and their 5α/ß-stanols in human fecal samples. Fecal sterols were extracted and derivatized to N, N-dimethylglycine esters. The method includes cholesterol, coprostanol, cholestanol and sitosterol, 5α/ß-sitostanol, campesterol and 5α/ß-campestanol. Application of a biphenyl column permits separation of isomeric 5α- and 5ß-stanols. Sterols are detected in parallel reaction monitoring (PRM) mode and stanols in full scan mode. HRMS allows differentiation of isobaric ß-stanols and the [M + 2] isotope peak of the coeluting sterol. Performance characteristics meet the criteria recommended by Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. Analysis of fecal samples from healthy volunteers revealed high interindividual variability of sterol and stanol fractions. Interestingly, cholesterol and sitosterol showed similar fractions of mainly 5ß-stanols. In contrast, campesterol is substantially converted to 5α-campestanol and might be a poorer substrate for bacterial metabolism. Robust and fast quantification of fecal sterols and their related stanols by LC-MS/HRMS offers great potential to find novel microbiome-related biomarker in large-scale studies.


Assuntos
Fezes/química , Microbioma Gastrointestinal , Esteróis/análise , Espectrometria de Massas em Tandem/métodos , Colesterol/análogos & derivados , Colesterol/análise , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Fezes/microbiologia , Humanos , Limite de Detecção , Fitosteróis/análise , Sitosteroides/análise , Espectrometria de Massas em Tandem/economia
16.
J AOAC Int ; 101(5): 1578-1583, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29852890

RESUMO

BACKGROUND: Biotin and folate are B-group vitamins that play a critical role in numerous metabolic reactions, and they are supplemented to infant and adult nutritional formulas as free biotin and folic acid. OBJECTIVE: We describe a rapid method for the analysis of biotin and folic acid that is applicable to liquid milk, milk powders, infant formula, and milk-based nutritional products. METHODS: Samples are autoclaved, centrifuged, filtered, and analyzed by HPLC-MS/MS, with quantitation accomplished by the internal standard technique. RESULTS: The method was shown to be accurate, with acceptable spike recovery (biotin: 96.5-108.2%; folic acid: 92.6-104.4%), and no bias (α = 0.05) against either a certified reference material (biotin: P = 0.70; folic acid: P = 0.23) or established analytical method (biotin: P = 0.10; folic acid: P = 0.48) was found. Acceptable precision was confirmed with repeatability relative standard deviation (RSDr) and Horwitz ratio (HorRat) values (biotin: RSDr = 0.5-5.6%, HorRatr = 0.1-0.6; folic acid: RSDr = 2.0-3.1%, HorRatr = 0.3-0.5). Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. CONCLUSIONS: This rapid method is intended for use in high-throughput laboratories as part of the routine product compliance release testing of biotin and folic acid in the manufacturing of infant formulas and adult nutritional products.


Assuntos
Biotina/análise , Cromatografia Líquida de Alta Pressão/métodos , Ácido Fólico/análise , Análise de Alimentos/métodos , Fórmulas Infantis/análise , Leite/química , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida de Alta Pressão/economia , Análise de Alimentos/economia , Humanos , Lactente , Limite de Detecção , Espectrometria de Massas em Tandem/economia , Fatores de Tempo
17.
Anal Bioanal Chem ; 410(20): 4967-4978, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29947895

RESUMO

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL-1 and 3.2 to 160 ng mL-1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and - 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.


Assuntos
Benzodiazepinas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Drogas Desenhadas/análise , Hipnóticos e Sedativos/sangue , Membranas Artificiais , Espectrometria de Massas em Tandem/métodos , Benzodiazepinas/análise , Benzodiazepinas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/economia , Drogas Desenhadas/isolamento & purificação , Desenho de Equipamento , Humanos , Hipnóticos e Sedativos/análise , Hipnóticos e Sedativos/isolamento & purificação , Limite de Detecção , Extração Líquido-Líquido/economia , Extração Líquido-Líquido/instrumentação , Espectrometria de Massas em Tandem/economia , Fatores de Tempo
18.
Anal Bioanal Chem ; 410(12): 2971-2979, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29532193

RESUMO

D-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some D-amino acids are metabolized by D-amino acid oxidase (DAO), while D-Asp and D-Glu are metabolized by D-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO+/+), mutant mice lacking DAO activity (ddY/DAO-/-), and the heterozygous mice (ddY/DAO+/-) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for L-amino acid levels among the three strains except for L-Trp which was markedly elevated in the DAO+/- and DAO-/- mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of L-amino acids were L-Glu, L-Asp, and L-Gln in all the three strains. The lowest L-amino acid level was L-Cys in ddY/DAO+/- and ddY/DAO-/- mice, while L-Trp showed the lowest level in ddY/DAO+/+mice. The highest concentration of D-amino acid was found to be D-Ser, which also had the highest % D value (~ 25%). D-Glu had the lowest % D value (~ 0.01%) in all the three strains. Significant differences of D-Leu, D-Ala, D-Ser, D-Arg, and D-Ile were observed in ddY/DAO+/- and ddY/DAO-/- mice compared to ddY/DAO+/+ mice. This work provides the most complete baseline analysis of L- and D-amino acids in the brains of ddY/DAO+/+, ddY/DAO+/-, and ddY/DAO-/- mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.


Assuntos
Aminoácidos/análise , Química Encefálica , D-Aminoácido Oxidase/genética , Deleção de Genes , Animais , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Masculino , Camundongos , Estereoisomerismo , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos
19.
Anal Bioanal Chem ; 410(10): 2517-2531, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29492623

RESUMO

A validated liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of D- and L-amino acids in human serum. Under the optimum conditions, except for DL-proline, L-glutamine, and D-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for L-amino acids (including glycine) and D-amino acids were 5-56.25 µM and 0.625-500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from -12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of D- and L-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of L-arginine, L-isoleucine, L-aspartate, L-tryptophan, L-alanine, L-methionine, L-serine, glycine, L-valine, L-leucine, L-phenylalanine, L-threonine, D-isoleucine, D-alanine, D-glutamate, D-glutamine, D-methionine, and D-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P < 0.01). D-Glutamate and D-glutamine were identified as the most downregulated serum markers (fold change greater than 1.5), which deserves further attention in hepatocellular carcinoma research. Graphical abstract Simultaneous determination of D- and L-amino acids in human serum from hepatocellular carcinoma patients and healthy individuals. AA amino acid, HCC hepatocellular carcinoma, LC liquid chromatography, MS/MS tandem mass spectrometry, NC normal control, TIC total ion chromatogram.


Assuntos
Aminoácidos/sangue , Carcinoma Hepatocelular/sangue , Cromatografia Líquida/métodos , Neoplasias Hepáticas/sangue , Espectrometria de Massas em Tandem/métodos , Aminoácidos/análise , Cromatografia Líquida/economia , Humanos , Limite de Detecção , Estereoisomerismo , Espectrometria de Massas em Tandem/economia , Fatores de Tempo
20.
Talanta ; 180: 108-119, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29332787

RESUMO

The aim of the present work was to develop a rapid and interference-free method based on liquid chromatography-mass spectrometry (LC-MS) for the simultaneous determination of nine B-group vitamins in various energy drinks. A smart and green strategy that modeled the three-way data array of LC-MS with second-order calibration methods based on alternating trilinear decomposition (ATLD) and alternating penalty trilinear decomposition (APTLD) algorithms was developed. By virtue of "mathematical separation" and "second-order advantage", the proposed strategy successfully solved the co-eluted peaks and unknown interferents in LC-MS analysis with the elution time less than 4.5min and simple sample preparation. Satisfactory quantitative results were obtained by the ATLD-LC-MS and APTLD-LC-MS methods for the spiked recovery assays, with the average spiked recoveries ranging from 87.2-113.9% to 92.0-111.7%, respectively. These results acquired from the proposed methods were confirmed by the LC-MS/MS method, which shows a quite good consistency with each other. All these results demonstrated that the developed chemometrics-assisted LC-MS strategy had advantages of being rapid, green, accurate and low-cost, and it could be an attractive alternative for the determination of multiple vitamins in complex food matrices, which required no laborious sample preparation, tedious condition optimization or more sophisticated instrumentations.


Assuntos
Cromatografia Líquida/métodos , Bebidas Energéticas/análise , Análise de Alimentos/métodos , Espectrometria de Massas em Tandem/métodos , Complexo Vitamínico B/análise , Algoritmos , Calibragem , Cromatografia Líquida/economia , Análise de Alimentos/economia , Limite de Detecção , Modelos Lineares , Niacinamida/análise , Ácido Pantotênico/análise , Espectrometria de Massas por Ionização por Electrospray/economia , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/economia , Tiamina/análise , Vitamina B 12/análise , Vitamina B 6/análise
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