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1.
J Agric Food Chem ; 67(33): 9362-9370, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31368700

RESUMO

This manuscript presents an effective and robust method for simultaneous stereoselective determination of two pyrethroid insecticides, tetramethrin and α-cypermethrin in different food products by high-performance liquid chromatography. Enantioseparation was carried out using reversed-phase chromatography, and the influences of four polysaccharide-based chiral columns, mobile phase composition, and column temperature on retention were fully investigated. Satisfactory separation was obtained on Chiralpak IG column using acetonitrile-water (75:25, v/v) under isocratic conditions. To extract and purify the target analytes from food matrices, matrix solid-phase dispersion was employed with C18 as dispersant and primary secondary amine as well as graphitized carbon black as cleanup sorbents. Response surface method based on Box-Behnken design was implemented to assist optimization of the extraction variables. Then, method validation was done in real samples including specificity, linearity, sensitivity, trueness, precision, as well as stability, and its analytical performance fulfills the criteria recommended by the European Union SANTE/11945/2015, demonstrating its applicability in studying the stereochemistry of chiral tetramethrin and α-cypermethrin in food products.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Grão Comestível/química , Frutas/química , Inseticidas/química , Piretrinas/química , Verduras/química , Cromatografia de Fase Reversa/métodos , Contaminação de Alimentos/análise , Inseticidas/isolamento & purificação , Piretrinas/isolamento & purificação , Extração em Fase Sólida , Estereoisomerismo
2.
J Chromatogr A ; 1602: 397-408, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31285058

RESUMO

In reversed-phase liquid chromatography, the performance for basic compounds is affected by the interaction of the protonated (cationic) species with the anionic free silanols on the alkyl-bonded stationary phases. Using aqueous-organic mobile phases in the absence of additives, the retention may be too high, and the peaks be broad and asymmetric. The performance is improved by addition to the mobile phase of ionic liquids, from which 1-hexyl-3-methylimidazolium chloride ([C6MIm][Cl]) has especially good characteristics. A recent report has also revealed that the use of the phosphate system as buffer, at varying concentration and pH, may have a significant role in the chromatographic performance of basic compounds, with effects on both retention and peak shape. In this work, this study has been extended to other three buffer systems (acetate, citrate, and formate), at increasing concentrations and pH 3 and 7, in the presence and absence of [C6MIm][Cl]. The results have been compared with those obtained with the phosphate system. The retention increases by addition of larger concentration of all buffers, in both absence and presence of [C6MIm][Cl]. Without additive, peak performance is also enhanced significantly. This effect is minimal in the presence of [C6MIm][Cl], which yields highly symmetrical peaks at all buffer concentrations, due to an effective blocking of the silanol activity.


Assuntos
Boratos/química , Cromatografia de Fase Reversa/métodos , Imidazóis/química , Acetonitrilos/química , Antagonistas Adrenérgicos beta/análise , Tampões (Química) , Cromatografia Líquida de Alta Pressão/métodos , Concentração de Íons de Hidrogênio , Líquidos Iônicos/química , Solventes , Água/química
3.
Food Chem ; 298: 125010, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31284091

RESUMO

Vitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. An HPLC-DAD method has been developed and validated, per AOAC SMPR 2016.017 (Standard Method Performance Requirements), for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. The method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005%w/w-85%w/w), high precision (reproducibility relative standard deviations ranged from 1.43% to 4.67%), and high accuracy (>96% spike recovery rate for 11 out of 12 accuracy testing data points). The method detection and quantification limits are less than 0.16 and 0.52 µg/mL, respectively. To our best knowledge, it is simpler, less time-consuming, and more economical than other published methods for its intended uses.


Assuntos
Cromatografia de Fase Reversa/métodos , Suplementos Nutricionais/análise , Vitamina B 12/análise , Cobamidas/análise , Laboratórios , Limite de Detecção , Vitamina B 12/análogos & derivados , Complexo Vitamínico B/análise , Complexo Vitamínico B/química
4.
Anal Chim Acta ; 1078: 200-211, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358220

RESUMO

The effect of the ionization in the RP-HPLC retention of 66 acid-base compounds, most of them drugs of pharmaceutical interest, is studied. The retention time of the compounds can be related to the pH measured in the mobile phase (pwsH) through the sigmoidal equations derived from distribution of the neutral and ionic forms of the drug into the stationary and mobile phases. Fitting of the obtained retention vs. pH profiles provides the retention times of the ionic and neutral forms and the pKa values of the drugs in the mobile phase (pwsKa). The obtained pwsKa values are linearly correlated to the pKa values in water (pwwKa) with two different correlations, one for neutral acids and another for neutral bases that reflect the different influence of the dielectric constant of the medium in ionization of acids and bases. The retention of the neutral species is well correlated to the octanol-water partition coefficient of the drugs as measure of the lipophilicity of the drug, which affects chromatographic retention. Also, the retention time of the ionized forms is related to the retention time of the neutral forms by two different linear correlations, one for anions and the other for cations. These last correlations point out the different retention behaviour of anions and cations: anions are less retained than cations of the same lipophilicity, as measured by the octanol-water partition coefficient of the neutral form. The different retention behaviour of anionic, cationic and neutral forms is confirmed by the hold-up times obtained from different approaches: pycnometry and retention times of anionic (KBr and KI) and neutral (DMSO) markers. Hold-up times obtained by pycnometric measurements agree with those obtained by retention of neutral markers (0.83-0.85 min), whereas hold-up time for anions is mobile phase pH dependent. At acidic pH it is similar to the hold-up time for neutral markers (0.83 min), but then it decreases with the increase of mobile phase pH to 0.65 min at pH 11. The decrease can be explained by the ionization of the silanols of the column and exclusion of anions by charge repulsion. Although not directly measured, the obtained retention data and correlations indicate hold-up time for cations are similar or slightly lower than hold-up time for neutral compounds (0.77-0.83 min). The model proposed and the correlations obtained can be very useful for its implementation in retention prediction algorithms for optimization of separation purposes.


Assuntos
Aminas/química , Ácidos Carboxílicos/química , Fenóis/química , Piridinas/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 39-48, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31150952

RESUMO

Resveratrol and piperine are proven for their therapeutic benefits to treat various diseases. Due to their synergistic actions and combined drug delivery application, a rapid and specific RP-HPLC method was developed and validated as per ICH guidelines, by using an isosbestic point. The chromatographic separation was performed with Luna 5 µ 100 ŠC-18(2) HPLC column by using acetonitrile (ACN): phosphate buffer (0.01% orthophosphoric acid) (55:45) as mobile phase, at 1 mL/min of flow rate and 330 nm. The developed method was found to be linear over the concentration range of 0.25-8 µg/mL with correlation coefficient value >0.999. The developed method was accurate (percent recovery 98.06-101.74%), precise (percent relative standard deviation <2.0%), and robust. The limit of detection and limit of quantification for resveratrol were found to be 0.02 and 0.08 µg/mL, respectively and 0.04 and 0.11 µg/mL, for piperine, respectively. The developed method was also validated in human plasma as per ICH guidelines. Moreover, stress degradation studies of both phytoconstituents were studied and the relevancy of the developed method was analyzed on cubosome nanoformulation. A good separation of drug peaks was observed in the presence of the degradation products. This method could thus be used for regular in vitro and in vivo estimation of piperine and resveratrol.


Assuntos
Alcaloides/sangue , Benzodioxóis/sangue , Cromatografia Líquida de Alta Pressão/métodos , Piperidinas/sangue , Alcamidas Poli-Insaturadas/sangue , Resveratrol/sangue , Alcaloides/química , Benzodioxóis/química , Cromatografia de Fase Reversa/métodos , Estabilidade de Medicamentos , Humanos , Limite de Detecção , Modelos Lineares , Piperidinas/química , Alcamidas Poli-Insaturadas/química , Reprodutibilidade dos Testes , Resveratrol/química
6.
J Chromatogr A ; 1602: 253-265, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31178160

RESUMO

For comparable surface coverage of alkyl-bonded chains (∼3 µmol/m2), the dewetting of 100% aqueous mobile phases from the mesopores of octyl(C8)-bonded silica particles is found 70 times faster than that from the same but octadecyl(C18)-bonded silica particles. This observation was made in this work for both fully porous (5 µm Symmetry) and superficially porous (2.7 µm CORTECS) particles. This experimental result is paradoxical because (1) the average pore size of C8-bonded materials is 10-15 Šlarger than that of C18-bonded materials for the same unbounded silica gel and (2) the contact angle of water measured on smooth and planar C8-bonded surface is about 6° smaller than that on the same but C18-bonded surface (104° versus 110°). The equilibrium Laplace pressure is then expected to be smaller and the kinetics of water dewetting to be slower for silica-C8 than for silica-C18 stationary phases used in RPLC. The solution to this riddle is investigated based on (1) the calculation of the dewetting time assuming that the pores are monosized and the process is driven by the Laplace pressure, (2) the measurement of the advancing and receding contact angles of three different C18- and C8-bonded silica gels (4 µm NovaPak, 5 µm Symmetry, and 2.7 µm CORTECS) from the water porograms measured in a range of water pressure from normal pressure to 500 bar, and (3) on the calculation of the pore connectivity for both C8 and C18-bonded silica. First, the experimental results show that the observed dewetting times are of the order of minutes or even hours instead of millisecond as predicted by the dewetting model. Secondly, the advancing and receding contact angles of water onto the C8-bonded silicas are found larger (by an average of +7° and +2°, respectively) than those measured for the same but C18-bonded silica (average of 112° and 92°). Finally, the calculated pore connectivity is decreasing by about 30% for 90 Šunbounded silica materials from C8 to C18-bonded RPLC phases. Overall, the observed and much faster dewetting of water from C8 column than that from C18 column is primarily explained by a higher internal pore connectivity due to the thinner thickness of the alkyl-bonded layer (7 Šversus 15 Å) and, to a lesser extent, by a higher extrusion Laplace pressure of water (≃+10 bar).


Assuntos
Cromatografia de Fase Reversa/métodos , Dióxido de Silício/química , Água/química , Cinética , Porosidade , Pressão
7.
Chem Pharm Bull (Tokyo) ; 67(5): 439-444, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061368

RESUMO

An ultra-performance liquid chromatography (UPLC) method was developed and validated for the quantification of linezolid, PNU-142300, and PNU-142586 in human plasma. After protein precipitation using acetonitrile, the protein-free supernatant was separated using reverse-phase chromatography using an ACQUITY UPLC HSS T3 column and monitored at 254 nm. p-Toluic acid was used as the internal standard. No interference peak was observed at the retention times of linezolid, PNU-142300, PNU-142586, and p-toluic acid from blank plasma. The calibration curve of linezolid was linear from 0.2 to 50.0 µg/mL (coefficient of determination (r2) > 0.9999) and those of PNU-142300 and PNU-142586 were linear from 0.2 to 20.0 µg/mL (r2 > 0.9996 and > 0.9998, respectively). The intra- and inter-assay accuracy (%) and precision (relative standard deviation (RSD) %) of the three components were confirmed to meet the criteria of the U.S. Food and Drug Administration guidelines. Tests confirmed the stability of linezolid, PNU-142300, and PNU-142586 in plasma during three freeze-thaw cycles and long-term storage of frozen plasma for up to 30 d; in extracts they were stable in the UPLC autosampler for over 48 h at 4°C. Furthermore, plasma concentrations of linezolid, PNU-142300 and PNU-142586 in patients treated with linezolid could be measured using the UPLC method developed in this study. This assay would be a powerful tool for therapeutic drug monitoring and clinical pharmacokinetic/pharmacodynamic (PK/PD) analyses in the optimization of linezolid treatment.


Assuntos
Antibacterianos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Linezolida/análogos & derivados , Linezolida/sangue , Acetonitrilos/química , Precipitação Química , Cromatografia de Fase Reversa/métodos , Humanos , Limite de Detecção , Reprodutibilidade dos Testes
8.
Artigo em Inglês | MEDLINE | ID: mdl-31082718

RESUMO

A simple, rapid, accurate, reproducible and sensitive reverse phase HPLC method for the estimation of fusidic acid (FA) by means of Analytical Quality by Design (AQbD) was the aim of the present study. Initially, the vital pre-requisites for AQbD like analytical method target profile and critical analytical attributes (CAAs) like theoretical plates, tailing factor and percent assay were defined. An octadecyl silyl silica C18 column with a packing size of 5 µm was employed and the detection was performed at 235 nm using UV-detector. The separation was performed with isocratic elution employing mixture of methanol: acetonitrile (5: 95, v/v) and an aqueous phase with pH of 2.8 containing 0.1% orthophosphoric acid in the ratio of 60: 40 (v/v). Ishikawa fish-bone diagram provided the basis of the variation in CAAs with various inputs. Taguchi Design was selected as the initial screening design to select critical method parameters (CMPs) affecting method development and Central Composite Design (CCD) was further applied for systematic optimization of chromatographic method by evaluating CAAs. Crucial parameters viz. limit of detection, limit of quantification, specificity, linearity and sensitivity were employed to validate the method in accordance with the ICH guidelines. Stress degradation studies were performed and the developed method was able to successfully differentiate the degraded products from the parent drug, that too in a topical gel. In conclusion, the findings of the present study validated the utility of AQbD in the systematic design of a liquid chromatographic method with fine sensitivity for FA estimation in medicinal products.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Cromatografia de Fase Reversa/métodos , Cromatografia de Fase Reversa/normas , Ácido Fusídico , Ácido Fusídico/análise , Ácido Fusídico/química , Ácido Fusídico/farmacocinética , Cinética , Limite de Detecção , Modelos Lineares , Modelos Químicos , Reprodutibilidade dos Testes , Projetos de Pesquisa
9.
J Chromatogr Sci ; 57(6): 575-581, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31095673

RESUMO

The human body's vitamin D levels are determined by measuring the level of 25-hydroxy-vitamin D3 (25-OH-vitamin D3) in human serum. In this research, a fast, simple, efficient and highly sensitive low-density solvent based on dispersive liquid-liquid microextraction (DLLME) was employed for the successful determination of 25-OH-vitamin D3 from complex human serum matrices. Reversed phase high-performance liquid chromatography was used as a powerful technique. The important parameters in the low-density solvent-DLLME method were optimized using response surface methodology based on central composite design. The calibration curves displayed a high level of linearity (R2 > 0.997) for 25-OH-vitamin D3 in the range of 2-500 ng mL-1. The limit of detection and limit of quantitation were 0.6 ng mL-1 and 1.9 ng mL-1, respectively. The relative standard deviation for the seven analyses was 7.1%. The relative recoveries of vitamin D3 in spiked human serum samples were between 85% and 97%. The amount of 25-OH-vitamin D3 in samples was determined using the proposed method and acceptable results were reported.


Assuntos
25-Hidroxivitamina D 2/sangue , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Microextração em Fase Líquida/métodos , 25-Hidroxivitamina D 2/isolamento & purificação , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Projetos de Pesquisa
10.
Cell Mol Biol Lett ; 24: 30, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31131010

RESUMO

Background: Gemcitabine (2',2'-difluoro-2'-deoxycytidine;dFdC) is a first-line chemotherapy drug for pancreatic cancer. Recently, a synergistic anti-tumor treatment of dFdC and hyperthermia has achieved good clinical results, but there are few reports on the molecular mechanism influenced by hyperthermia. This study is an initial exploration of the effects of hyperthermia on changes in the concentration of dFdC and its metabolites in pancreatic cancer cells. The aim is to provide a theoretical basis for clinical detection and pharmacokinetic research. Methods: PANC-1 cells at logarithmic growth phase were used as the experimental object. The MTT assay was performed to determine the half maximal inhibitory concentration (IC50) of dFdC. After PANC-1 cells were cultured in DMEM medium containing IC50dFdC and treated with hyperthermia at 41 °C or 43 °C, changes in the concentration of dFdC, 2',2'-difluorodeoxyuridine (dFdU) and difluorodeoxycytidine triphosphate (dFdCTP) in the cells were tested using an optimized reverse phase high-performance liquid chromatography (RP-HPLC) protocol. Results: We found that 41 °C and 43 °Chyperthermia gave rise to a decrease in dFdC and dFdU content. At 41 °C, the levels respectively fell to 9.28 and 30.93% of the baseline, and at 43 °C, to 24.76 and 57.80%, respectively. The dFdCTP content increased by 21.82% at 41 °C and 42.42% at 43 °C. Conclusion: The two heat treatments could alter the mechanism of dFdC metabolism in PANC-1 cells. The effect of 43 °C hyperthermia is more significant. Our observations may be instrumental to explaining the higher anti-tumor efficacy of this combination therapy.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Desoxicitidina/análogos & derivados , Hipertermia Induzida , Metaboloma , Neoplasias Pancreáticas/metabolismo , Calibragem , Linhagem Celular Tumoral , Desoxicitidina/metabolismo , Humanos , Limite de Detecção , Modelos Lineares , Padrões de Referência
11.
Eur J Mass Spectrom (Chichester) ; 25(3): 339-353, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31096786

RESUMO

There is an urgent need to implement holistic and untargeted doping control protocols with improved discriminatory power, compared to conventional methods that only target doping agents. Metabolomics, which aims to characterize all metabolites present in biological matrices, could fulfill this need. In this context, the aim of this study was to evaluate the impact of environmental factors on the ability to obtain a metabolic signature of stanozolol administration in horse doping situation. Urine samples from 16 horses breeded in two different places were collected over a one-year period, before, during and seven months after the administration of stanozolol, a horse doping agent. Metabolomic analysis was performed using ultra-high pressure reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (MS). Results showed a major impact of the nutritional regimen, drug administration (for de-worming purpose) and breeding place on the metabolite profiles of horse urines, which hampered the detection of metabolic perturbations induced by stanozolol administration. After having used MS/MS experiments to characterize some MS features related to these environmental factors, we showed that highlighting and then removing the features impacted by these confounding factors before performing supervised multivariate statistical analyses could address this issue. In conclusion, adequate consideration should be given to environmental and physiological factors; otherwise, they can emerge as confounding factors and conceal doping administration.


Assuntos
Cromatografia de Fase Reversa/métodos , Doping nos Esportes/métodos , Cavalos/urina , Espectrometria de Massas/métodos , Metabolômica/métodos , Prednisolona/urina , Detecção do Abuso de Substâncias/métodos , Animais , Cromatografia de Fase Reversa/veterinária , Limite de Detecção , Espectrometria de Massas/veterinária , Detecção do Abuso de Substâncias/veterinária
12.
Anal Bioanal Chem ; 411(17): 3857-3870, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31073732

RESUMO

The present paper describes a novel two-dimensional liquid chromatography (2D-LC) system, which is comprised of a first-dimensional ion exchange chromatography (IEX1) column, trap column, and second-dimensional reversed-phase chromatography (RP2) column system. The biological sample is separated by the first-dimensional LC using an IEX column to remove interferences. The analytes are transferred to the trap column after heart-cutting. Then, the analytes are transferred to the second-dimensional LC using an RP2 column for further separation and ultraviolet detection. This 2D-LC system can offer a large injection volume to provide sufficient sensitivity and exhibits a strong capacity for removing interferences. Here, the determination of three monoterpene indole alkaloids (MIAs; gelsemine, koumine, and humantenmine) from Gelsemium in biological matrices (plasma, tissue, and urine) was used this 2D-LC system. After a rapid and easy sample preparation method based on protein precipitation, the sample was injected into the 2D-LC. The method was developed and validated in terms of the selectivity, LOD, LOQ, linearity, precision, accuracy, and stability. The sample preparation time for the three MIAs was 15 min. The LOD for these compounds was 10 ng/mL, which was lower than the developed HPLC methods. The results showed that this method had good quantitation performance and allowed the determination of gelsemine, koumine, and humantenmine in biological matrices. The method is rapid, exhibits high selectivity, has good sensitivity, and is low-cost, thus making it well-suited for application in the pharmaceutical and toxicological analysis of Gelsemium. Graphical abstract.


Assuntos
Alcaloides/análise , Cromatografia por Troca Iônica/instrumentação , Cromatografia de Fase Reversa/instrumentação , Alcaloides Indólicos/análise , Alcaloides/sangue , Alcaloides/normas , Alcaloides/urina , Cromatografia por Troca Iônica/métodos , Cromatografia de Fase Reversa/métodos , Alcaloides Indólicos/sangue , Alcaloides Indólicos/normas , Alcaloides Indólicos/urina , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta/métodos
13.
Artigo em Inglês | MEDLINE | ID: mdl-30953918

RESUMO

During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 µM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.


Assuntos
Cromatografia de Fase Reversa/métodos , Contaminação de Medicamentos , Sulfonas/análise , Fármacos Anti-HIV/química , Fármacos Anti-HIV/normas , Anticorpos Neutralizantes/química , Anticorpos Anti-HIV/química , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Tecnologia Farmacêutica
14.
Artigo em Inglês | MEDLINE | ID: mdl-30981182

RESUMO

Residual free-drug-related species that are present in antibody-drug conjugates (ADC) are a potential safety risk to patients and are therefore categorized as a critical quality attribute that must be strictly monitored and controlled. Among the many analytical methods developed for free-drug analysis, reversed-phase liquid chromatography (RP-LC) is the most common approach. Conventional RP-LC methods for free-drug analysis, however, involve labor-intensive sample preparation. Here we present a new RP-LC method to directly analyze free-drug-related species in an ADC sample without the need for sample preparation. In our work, free-drug-related species were very well separated from ADC peaks in the chromatography gradient. Typical performance issues observed in conventional RP-LC, such as column fouling, detection interference, and carryover, were not observed or were negligible with this new method. Three options were evaluated for free-drug quantitation: Strohl (2017) [1] use of an external free drug calibration curve for determination of absolute concentration; Perez et al. (2014) [2] calculation of relative percentage based on peak area ratio between free drug and ADC at a characteristic wavelength unique for drug payload; and (Beck et al., 2017) [3] calculation of relative percentage based on peak area ratio between free drug and corrected ADC peak area (at any wavelength). The method with calibration curve provides the highest sensitivity, the best accuracy and precision for determination of free drug present in the ADC. However, the second and third options were simpler because they eliminated the need for an external calibration curve, making them worth exploring. Due to its simplicity and compatibility with mass spectrometry, the new method is also a good choice for direct analysis of stability samples.


Assuntos
Imunoconjugados/análise , Imunoconjugados/química , Cromatografia de Fase Reversa/métodos , Limite de Detecção , Modelos Lineares , Espectrometria de Massas/métodos , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/química , Reprodutibilidade dos Testes
15.
Anal Bioanal Chem ; 411(15): 3321-3330, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989271

RESUMO

Hyaluronic acid and its acrylate derivatives are important intermediates for various pharmaceutical, biomedical, and cosmetic applications due to their biocompatibility and viscoelasticity properties. However, these polymers are inherently difficult to characterize due to their significant heterogeneity regarding molar mass and chemical composition (degree of substitution, DS). The present study describes the development of a comprehensive online two-dimensional liquid chromatography (2D-LC) approach to characterize hyaluronic acid and its acrylate derivatives (DS ranging from 0.4 to 3.1) in terms of molar mass and degree of substitution. In the first dimension of the 2D-LC method, separation according to chemical composition/DS was achieved by using a stepwise solvent gradient and a reversed phase C8 column. Fractions from the first dimension were automatically transferred to the second dimension comprising size exclusion chromatographic separation of the fractions according to molar mass. It was found that the hyaluronic acid derivatives were broadly distributed with regard to both chemical composition and molar mass. Fractions with different degrees of substitution were identified, and their molar mass distributions were determined. The study proved that comprehensive 2D-LC is a powerful approach to reveal the complex nature of hyaluronic acid and its derivatives. Graphical abstract.


Assuntos
Acrilatos/química , Cromatografia Líquida/instrumentação , Ácido Hialurônico/análogos & derivados , Cromatografia em Gel/instrumentação , Cromatografia em Gel/métodos , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/instrumentação , Cromatografia de Fase Reversa/métodos , Desenho de Equipamento , Ácido Hialurônico/química , Peso Molecular , Solventes
16.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1118-1119: 70-77, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31029036

RESUMO

Polar compounds are abundant in the water decoction of Traditional Chinese Medicines (TCMs) and possess important biological activities. However, purifying these compounds has faced great difficulties largely due to poor retention and insufficient selectivity. To solve this problem, an offline orthogonal 2-D RPLC coupled with hydrophilic interaction chromatography (HILIC) method was developed to achieve purification of polar compounds from Caulis Polygoni Multiflori. A polar-copolymerized XAqua C18 column and a zwitterionic Click XIon column exhibited satisfactory retention and separation for polar compounds. Therefore, they were adopted to construct the offline 2-D LC system. Furthermore, the method presented a high orthogonality, which was calculated to be 72%. The XAqua C18 column was used in the first dimension to fractionate the 5.1 g polar fraction with a recovery of 85% within 10 h. In the second dimension, three representative fractions were purified using the Click XIon column. Finally, three compounds with purity higher than 95% were identified for the first time from this plant. This offline 2-D RPLC/HILIC method was shown to be an effective approach to purifying polar compounds from Caulis Polygoni Multiflori.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Medicamentos de Ervas Chinesas/análise , Alcaloides/química , Alcaloides/isolamento & purificação , Medicamentos de Ervas Chinesas/química , Glicosídeos/química , Glicosídeos/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Polifenóis/química , Polifenóis/isolamento & purificação
17.
Anal Bioanal Chem ; 411(15): 3383-3394, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31020370

RESUMO

This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Oligonucleotídeos Fosforotioatos/química , Butilaminas/química , Cromatografia por Troca Iônica/métodos , Cromatografia de Fase Reversa/métodos , Etilaminas/química , Indicadores e Reagentes , Oligonucleotídeos Fosforotioatos/isolamento & purificação , Estereoisomerismo , Enxofre/análise
18.
Anal Bioanal Chem ; 411(14): 3103-3113, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30972471

RESUMO

13C metabolite tracer and metabolic flux analyses require upfront experimental planning and validation tools. Here, we present a validation scheme including a comparison of different LC methods that allow for customization of analytical strategies for tracer studies with regard to the targeted metabolites. As the measurement of significant changes in labeling patterns depends on the spectral accuracy, we investigate this aspect comprehensively for high-resolution orbitrap mass spectrometry combined with reversed-phase chromatography, hydrophilic interaction liquid chromatography, or anion-exchange chromatography. Moreover, we propose a quality control protocol based on (1) a metabolite containing selenium to assess the instrument performance and on (2) in vivo synthesized isotopically enriched Pichia pastoris to validate the accuracy of carbon isotopologue distributions (CIDs), in this case considering each isotopologue of a targeted metabolite panel. Finally, validation involved a thorough assessment of procedural blanks and matrix interferences. We compared the analytical figures of merit regarding CID determination for over 40 metabolites between the three methods. Excellent precisions of less than 1% and trueness bias as small as 0.01-1% were found for the majority of compounds, whereas the CID determination of a small fraction was affected by contaminants. For most compounds, changes of labeling pattern as low as 1% could be measured. Graphical abstract.


Assuntos
Isótopos de Carbono/análise , Cromatografia por Troca Iônica/métodos , Cromatografia de Fase Reversa/métodos , Espectrometria de Massas/métodos , Estudos de Validação como Assunto , Resinas de Troca de Ânions/química , Isótopos de Carbono/normas , Interações Hidrofóbicas e Hidrofílicas , Pichia/química , Padrões de Referência , Selênio/química
19.
Anal Bioanal Chem ; 411(18): 4167-4173, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30989264

RESUMO

Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with negative mode electrospray ionization mass spectrometry (ESI-MS) due to their highly negative charged phosphodiester backbones. Herein, the signal suppression effect of triethylamine (TEA) adducts caused the ion-pair reagent TEA/hexafluoroisopropanol (HFIP) is greatly alleviated after improving the in-source energy in positive mode ESI-MS. This strategy is applied for different RNA sequencing through analyzing their formic acid hydrolysates via IP-RPLC MS. Comparing with negative ion mode, we demonstrate that IP-RPLC MS analysis in positive ion mode is more suitable for RNA sequencing with fewer contaminant interferences. Finally, simultaneous online separation and detection of oligonucleotides and protein digests are achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.


Assuntos
Cromatografia de Fase Reversa/métodos , Oligonucleotídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos
20.
Pak J Pharm Sci ; 32(1(Supplementary)): 333-338, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30829212

RESUMO

A simple, rapid and accurate reverse phase high performance liquid chromatographic (RP- HPLC) method was developed for the quantification of lornoxicam in oral disintegrating tablets (ODTs) and in rabbit's plasma. C18 Hypersil™ column was used as stationary phase to separate the drug. Mobile phase methanol: acetonitrile: water (60:30:10) was run isocratically at flow rate of 1 mL/min at room temperature. Mean retention time was 4.23 minutes and minimum amount of lornoxicam that can be measured was 7 ng/mL in rabbit's plasma. Good linearity was observed in concentration range of 10-100 ng/mL with regression coefficient R2 value of 0.9989 and slope value 23773. As per ICH norms, developed method was validated in terms of interday, intraday precision, accuracy, specificity, limit of detection (LOD), limit of quantification (LOQ) and drug plasma stability studies. All the data obtained revealed that this method can be used for in-vitro and in-vivo determination of lornoxicam in various pharmaceutical preparations.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Piroxicam/análogos & derivados , Administração Oral , Animais , Calibragem , Estabilidade de Medicamentos , Limite de Detecção , Piroxicam/administração & dosagem , Piroxicam/sangue , Coelhos , Sensibilidade e Especificidade , Comprimidos/administração & dosagem
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