Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 145
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1114-1115: 31-44, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30927740

RESUMO

We describe a simplified approach for the purification and characterization of urinary albumin, a key biomarker currently used for understanding the onset and prognosis of microalbuminuria. Urinary albumin was purified from human urine collected from diabetic kidney disease patients by using 2-stage tangential flow filtration process and set of column chromatography steps. The relative molecular mass of urinary albumin is 66,871 Da (SYNAPT G2 High Definition Mass Spectrometry System). Isolated urinary albumin was analyzed by SDS-PAGE, Western blotting, immunoelectrophoresis, Ouchterlony double-immunodiffusion, single radial immunodiffusion, size-exclusion HPLC and peptide mass fingerprint analysis. The size-exclusion HPLC elution profile of the purified urinary albumin was similar to that of a reference form of native albumin. Peptide mass fingerprint analysis of the purified urinary albumin yielded peptides that partially matched with known sequence of ALBU_HUMAN (P02768). This is the first report of purification and validation of immunochemically reactive form of urinary albumin from a large volume of urine of diabetic kidney disease patients. In this purification approach, the cost of the purified albumin is significantly lower.


Assuntos
Albuminúria/urina , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Albumina Sérica Humana , Nefropatias Diabéticas/urina , Humanos , Imunoeletroforese , Reprodutibilidade dos Testes , Albumina Sérica Humana/economia , Albumina Sérica Humana/isolamento & purificação , Albumina Sérica Humana/urina
2.
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
3.
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
4.
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
5.
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
6.
Bioanalysis ; 10(14): 1129-1141, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29989424

RESUMO

Today, many LC-high-resolution MS instruments have become affordable, easy-to-use, sensitive and quantitative. Meanwhile, there is an increased need for more comprehensive approaches. However, omics analyses are still restricted to specialists whereas, in hospitals, routine analyses are targeted and quantitative and represent the main and heavy tasks. But the availability of fully automated LC-MS instruments that can handle independently from sample extraction to result reporting, as well as the increasing biomedical interest for global approaches, clinical analytical workflow should be reorganized. Bioanalysts are now in the position to develop/implement clinical metabolomics or proteomics as routine analyses. In this article, this coming evolution and the reasons to implement global/omics determinations as routine analysis, is described.


Assuntos
Automação , Técnicas de Laboratório Clínico , Metadona/sangue , Testosterona/sangue , Automação/economia , Cromatografia Líquida/economia , Técnicas de Laboratório Clínico/economia , Humanos , Espectrometria de Massas/economia , Metabolômica , Proteômica
7.
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
8.
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
9.
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
10.
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
11.
Anal Chem ; 90(3): 1870-1880, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29276835

RESUMO

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-µLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by µLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the µLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.


Assuntos
Cromatografia Líquida/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Espectrometria de Massas/instrumentação , Peptídeos/análise , Proteínas/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/análise , Biomarcadores/análise , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoglobulina G/análise , Limite de Detecção , Espectrometria de Massas/economia , Espectrometria de Massas/métodos , Camundongos , Ratos , Suínos
12.
Anal Chim Acta ; 999: 161-168, 2018 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-29254568

RESUMO

The value of exploring selectivity and solvent strength ternary gradients in enhanced fluidity liquid chromatography (EFLC) is demonstrated for the separation of inulin-type fructans from chicory. Commercial binary pump systems for supercritical fluid chromatography only allow for the implementation of ternary solvent strength gradients which can be restrictive for the separation of polar polymeric analytes. In this work, a custom system was designed to extend the capability of EFLC to allow tuning of selectivity or solvent strength in ternary gradients. Gradient profiles were evaluated using the Berridge function (RF1), normalized resolution product (NRP), and gradient peak capacity (Pc). Selectivity gradients provided the separation of more analytes over time. The RF1 function showed favor to selectivity gradients with comparable Pc to that of solvent strength gradients. NRP did not strongly correlate with Pc or RF1 score. EFLC with the hydrophilic interaction chromatography, HILIC, separation mode was successfully employed to separate up to 47 fructan analytes in less than 25 min using a selectivity gradient.


Assuntos
Cromatografia Líquida/métodos , Inulina/isolamento & purificação , Solventes/química , Algoritmos , Cromatografia Líquida/economia , Cromatografia Líquida/instrumentação , Interações Hidrofóbicas e Hidrofílicas , Inulina/análise , Fatores de Tempo
13.
Artigo em Inglês | MEDLINE | ID: mdl-28898719

RESUMO

α-Amanitin is the main lethal component of amanita mushrooms, and data on its toxicokinetics are few. The aim of this study was to develop a sensitive and cost-effective method to identify α-amanitin and investigate its toxicokinetic parameters using liquid chromatography-triple quadrupole tandem mass spectrometry. The colchicine was used as the internal standard (IS). The compounds were extracted from plasma samples by protein precipitation with acetonitrile (containing 1% formic acid). The analysis was performed through multiple reactions monitoring. The molecular ions and fragment ions of α-amanitin could be used as characteristic ions to perform qualitative analysis of α-amanitin. The assay was successfully validated by selectivity, linearity, matrix effect, precision and accuracy, recovery and stability according to the U.S. Food and Drug Administration Guidance, and applied to study the toxicokinetic profile of α-amanitin in rats after a single intraperitoneal administration.


Assuntos
Alfa-Amanitina/sangue , Alfa-Amanitina/toxicidade , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Alfa-Amanitina/química , Alfa-Amanitina/farmacocinética , Animais , Cromatografia Líquida/economia , Estabilidade de Medicamentos , Limite de Detecção , Modelos Lineares , Ratos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/economia , Toxicocinética
14.
Biotechnol J ; 12(12)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28901658

RESUMO

Monoclonal antibodies currently dominate the biopharmaceutical market with growing sales having reached 80 billion USD in 2016. As most top-selling mAbs are approaching the end of their patent life, biopharmaceutical companies compete fiercely in the biosimilars market. These two factors present a strong motivation for alternative process strategies and process optimization. In this work a novel purification strategy for monoclonal antibodies comprising phenylboronic acid multimodal chromatography for capture followed by polishing by ion-exchange monolithic chromatography and packed bed hydrophobic interaction chromatography is presented and compared to the traditional protein-A-based process. Although the capital investment is similar for both processes, the operation cost is 20% lower for the novel strategy. This study shows that the new process is worthwhile investing in and could present a viable alternative to the platform process used by most industrial players.


Assuntos
Anticorpos Monoclonais , Reatores Biológicos , Cromatografia Líquida , Animais , Anticorpos Monoclonais/economia , Anticorpos Monoclonais/isolamento & purificação , Reatores Biológicos/economia , Reatores Biológicos/normas , Ácidos Borônicos/química , Células CHO , Cromatografia Líquida/economia , Cromatografia Líquida/normas , Cricetinae , Cricetulus , Interações Hidrofóbicas e Hidrofílicas
15.
Talanta ; 175: 550-556, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28842032

RESUMO

Fluoroquinolones (FQs) are a group of antimicrobial agents that have been widely used for therapeutic purposes in clinical medicine for human and veterinary diseases, as well as in aquaculture production. Their residues, however, may survive in foods of animal origin, thus causing health risks for human. In this study, a rapid and sensitive method based on in vivo solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to detect the residues of five FQs in cultured puffer fish (Takifugu obscurus). In vitro fiber evaluation experiment demonstrated that, compared with the thicker polydimethylsiloxane (PDMS) coating (165µm), the custom-made biocompatible C18-PAN fibers (45µm) exhibited much higher extraction efficiencies towards FQs (approximately 9-31 times). The custom-made C18-PAN coating also possessed excellent reproducibility in fish muscle with the intra-fiber relative standard deviations (RSDs) ranging from 11.2% to 14.3% (n = 6) and inter-fiber RSDs ranging from 13.1% to 16.1% (n = 6), which was suitable for in vivo sampling. The custom-made SPME fibers were subsequently applied to determine fluoroquinolones in dorsal-epaxial muscle of living puffer fish. The accuracies were verified through the comparison with traditional liquid extraction (LE) method, and the sensitivities were demonstrated to be satisfying with the limits of detection (LODs) ranging from 0.3ngg-1 to 1.5ngg-1. In general, this study presented a convenient and high-efficient method to determine fluoroquinolones in puffer fish by in vivo sampling.


Assuntos
Anti-Infecciosos/análise , Fluoroquinolonas/análise , Músculos/química , Microextração em Fase Sólida/métodos , Takifugu , Espectrometria de Massas em Tandem/métodos , Animais , Aquicultura , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Limite de Detecção , Reprodutibilidade dos Testes , Microextração em Fase Sólida/economia , Takifugu/metabolismo , Espectrometria de Massas em Tandem/economia
16.
Anal Bioanal Chem ; 409(26): 6141-6148, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28842738

RESUMO

A first of its kind, simple, rapid, and sensitive liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for quantification of perindopril and perindoprilat in both human plasma and breast milk. The analytes and internal standards (phenazone and acetyl salicylic acid) were extracted from biological matrices by protein precipitation. A Phenomenex® C-18 column was used to provide an appropriate chromatographic separation of the analytes, followed by detection with tandem mass spectrometry. Gradient chromatographic and mass spectrometric detection conditions with mobile phases (A: 5% methanol + 0.1% formic acid in water v/v, and B: 95% methanol + 0.1% formic acid in water v/v) were developed to achieve a LOQ of 0.5 ng/mL in both human plasma and milk. The method was suitable of evaluating clinical samples. The mass transition was followed as m/z 369.10/172.00 for perindopril, m/z 339.00/168.10 for perindoprilat, m/z 188.90/55.95 for phenazone, and m/z 179.04/137.02 for acetyl salicylic acid. The developed method was optimized and validated with a linear range of 0.1-200 ng/mL (r 2 = better than 0.99 for both perindopril and perindoprilat). The precision and accuracy values were within 15% CV. The overall recovery of the analytes was 80-110%. The method has good specificity and repeatability. Stability studies were conducted in both human plasma and bovine milk for up to 3 months, at the storage conditions of 25, 4, and -80 °C.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/análise , Inibidores da Enzima Conversora de Angiotensina/sangue , Indóis/análise , Indóis/sangue , Leite Humano/química , Perindopril/análise , Perindopril/sangue , Aleitamento Materno , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Feminino , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos
17.
Artigo em Inglês | MEDLINE | ID: mdl-28368805

RESUMO

IEF LC-MS/MS is an analytical method that incorporates a two-step sample separation prior to MS identification of proteins. When analyzing complex samples this preparatory separation allows for higher analytical depth and improved quantification accuracy of proteins. However, cost and analysis time are greatly increased as each analyzed IEF fraction is separately profiled using LC-MS/MS. We propose an approach that selects a subset of IEF fractions for LC-MS/MS analysis that is highly informative in the context of a group of proteins of interest. Specifically, our method allows a significant reduction in cost and instrument time as compared to the standard protocol of running all fractions, with little compromise to coverage. We develop algorithmics to optimize the selection of the IEF fractions on which to run LC-MS/MS. We translate the fraction optimization task to Minimum Set Cover, a well-studied NP-hard problem. We develop heuristic solutions and compare them in terms of effectiveness and running times. We provide examples to demonstrate advantages and limitations of each algorithmic approach. Finally, we test our methodology by applying it to experimental data obtained from IEF LC-MS/MS analysis of yeast and human samples. We demonstrate the benefit of this approach for analyzing complex samples with a focus on different protein sets of interest.


Assuntos
Cromatografia Líquida , Focalização Isoelétrica , Proteômica , Espectrometria de Massas em Tandem , Linhagem Celular Tumoral , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Análise por Conglomerados , Custos e Análise de Custo , Humanos , Focalização Isoelétrica/economia , Focalização Isoelétrica/métodos , Proteômica/economia , Proteômica/métodos , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/métodos
18.
J Chromatogr A ; 1499: 48-56, 2017 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-28420531

RESUMO

Therapeutic drug monitoring (TDM) is one of the most important services of clinical laboratories. Two main techniques are commonly used: the immunoassay and chromatography method. We have developed a cost-effective system of two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) for high-throughput determination of vancomycin in human plasma that combines the automation and low start-up costs of the immunoassay with the high selectivity and sensitivity of the liquid chromatography coupled with mass spectrometric detection without incurring their disadvantages, achieving high cost-effectiveness. This 2D-LC system offers a large volume injection to provide sufficient sensitivity and uses simulated gradient peak compression technology to control peak broadening and to improve peak shape. A middle column was added to reduce the analysis cycle time and make it suitable for high-throughput routine clinical assays. The analysis cycle time was 4min and the peak width was 0.8min. Compared with other chromatographic methods that have been developed, the analysis cycle time and peak width for vancomycin was reduced significantly. The lower limit of quantification was 0.20µg/mL for vancomycin, which is the same as certain LC-MS/MS methods that have been recently developed and validated. The method is rapid, automated, and low-cost and has high selectivity and sensitivity for the quantification of vancomycin in human plasma, thus making it well-suited for use in hospital clinical laboratories.


Assuntos
Análise Química do Sangue/economia , Análise Química do Sangue/métodos , Cromatografia Líquida , Monitoramento de Medicamentos/economia , Monitoramento de Medicamentos/métodos , Vancomicina/sangue , Análise Química do Sangue/normas , Cromatografia Líquida/economia , Cromatografia Líquida/normas , Humanos , Imunoensaio/economia , Reprodutibilidade dos Testes
19.
Drug Test Anal ; 9(4): 588-595, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27368111

RESUMO

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the qualitative and quantitative analysis of nine barbiturates (barbital, phenobarbital, pentobarbital, amobarbital, secobarbital, thiopental, butalbital, butabarbital, and hexobarbital) in human whole blood. Barbiturates were extracted from 100 µL of human whole blood samples using a simple liquid-liquid extraction (LLE) procedure, and detected by LC-MS/MS. An UPLC C18 (2.1 mm × 100 mm, 1.7 µm) column was used at 40 °C for the separation and acetonitrile/water system was used as the mobile phase with gradient elution. This method showed excellent accuracy (86-111%) and precision (relative standard deviation <15%). The limits of detection (LODs) were 0.2 ng/mL for barbital and secobarbital and 0.5 ng/mL for the other barbiturates. The linearity ranged from 2 ng/mL to 2000 ng/mL, with r2 > 0.99 over the range. This method achieved the separation and detection of pentobarbital and amobarbital at the same time in a convenient way. Moreover, it was both simple and sensitive for the determination of nine most commonly used barbiturate drugs, which was meaningful in the field of clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Barbitúricos/sangue , Cromatografia Líquida/métodos , Hipnóticos e Sedativos/sangue , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/economia , Toxicologia Forense/economia , Toxicologia Forense/métodos , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Detecção do Abuso de Substâncias/economia , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/economia , Fatores de Tempo
20.
Ann Clin Biochem ; 54(1): 170-173, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27166307

RESUMO

Background The Endocrine Society Clinical Practice Guideline on Phaeochomocytoma and Paraganglioma recommends phlebotomy for plasma-free metanephrines with patients fasted and supine using appropriately defined reference intervals. Studies have shown higher diagnostic sensitivities using these criteria. Further, with seated-sampling protocols, for result interpretation, reference intervals that do not compromise diagnostic sensitivity should be employed. Objective To determine the impact on diagnostic performance and financial cost of using supine reference intervals for result interpretation with our current plasma-free metanephrines fasted/seated-sampling protocol. Methods We conducted a retrospective cohort study of patients who underwent screening for PPGL using plasma-free metanephrines from 2009 to 2014 at Galway University Hospitals. Plasma-free metanephrines were measured using liquid chromatography-tandem mass spectrometry. Supine thresholds for plasma normetanephrine and metanephrine set at 610 pmol/L and 310 pmol/L, respectively, were used. Results A total of 183 patients were evaluated. Mean age of participants was 53.4 (±16.3) years. Five of 183 (2.7%) patients had histologically confirmed PPGL (males, n=4). Using seated reference intervals for plasma-free metanephrines, diagnostic sensitivity and specificity were 100% and 98.9%, respectively, with two false-positive cases. Application of reference intervals established in subjects supine and fasted to this cohort gave diagnostic sensitivity of 100% with specificity of 74.7%. Financial analysis of each pretesting strategy demonstrated cost-equivalence (€147.27/patient). Conclusion Our cost analysis, together with the evidence that fasted/supine-sampling for plasma-free metanephrines, offers more reliable exclusion of PPGL mandates changing our current practice. This study highlights the important advantages of standardized diagnostic protocols for plasma-free metanephrines to ensure the highest diagnostic accuracy for investigation of PPGL.


Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Biomarcadores Tumorais/sangue , Metanefrina/sangue , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico , Postura , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Cromatografia Líquida/economia , Jejum , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Paraganglioma/sangue , Paraganglioma/patologia , Posicionamento do Paciente/métodos , Feocromocitoma/sangue , Feocromocitoma/patologia , Guias de Prática Clínica como Assunto , Valores de Referência , Estudos Retrospectivos , Sensibilidade e Especificidade , Decúbito Dorsal , Espectrometria de Massas em Tandem/economia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA