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1.
Molecules ; 24(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261913

RESUMO

A novel strategy was developed to identify hepatotoxic compounds in traditional Chinese medicines (TCMs). It is based on the exposure of HL-7702 cells to a TCM extract, followed by the identification and further determination of potential hepatotoxic compounds accumulated in the cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a case study, potential hepatotoxic components in Chelidonium majus L. were screened out. Five alkaloids (sanguinarine, coptisine, chelerythrine, protopine, and chelidonine) were identified by LC-MS/MS within 10 min, and their intracellular concentrations were first simultaneously measured by LC-MS/MS with a run time of 4 min. A cell viability assay was performed to assess the cytotoxicity of each alkaloid. With their higher intracellular concentrations, sanguinarine, coptisine, and chelerythrine were identified as the main hepatotoxic constituents in Ch. majus. The study provides a powerful tool for the fast prediction of cytotoxic components in complex natural mixtures on a high-throughput basis.


Assuntos
Alcaloides/análise , Alcaloides/toxicidade , Chelidonium/química , Fígado/citologia , Benzofenantridinas/análise , Benzofenantridinas/toxicidade , Berberina/análogos & derivados , Berberina/análise , Berberina/toxicidade , Alcaloides de Berberina/análise , Alcaloides de Berberina/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Avaliação Pré-Clínica de Medicamentos , Humanos , Isoquinolinas/análise , Isoquinolinas/toxicidade , Fígado/química , Fígado/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Espectrometria de Massas em Tandem , Testes de Toxicidade
2.
Biomed Chromatogr ; 33(9): e4565, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31032988

RESUMO

In previous structure-activity relationship studies to identify new and selective 5-HT7 receptor (5-HT7 R) ligands, we identified the chiral compound, 5-chloro-2-{2-[3,4-dihydroisoquinoline-2(1H)-yl]ethyl}-2-methyl-2,3-dihydro-1H-inden-1-one (SYA 40247), with high-affinity binding to the 5-HT7 R. Thus, it was of interest to separate the enantiomers in order to evaluate their affinity at the 5-HT7 R. To achieve this separation, a normal-phase analytical method using HPLC-PDA and a 4.6 × 250 mm Chiralpak AD-H column was developed. Optimized isocratic conditions of 1.00 mL/min 95:5:0.1 v/v/v hexane-ethanol-diethylamine and a 254 nm analysis wavelength yielded a 6.07 min baseline separation. The method was scaled up to a 10 × 250 mm Chiralpak AD-H column, allowing 3 mg of racemate to be separated with a single injection, and 6 mg for an overlapping double injection in the same run. The separated enantiomers were reinjected into the analytical HPLC system, peak identities confirmed by retention time and PDA UV spectra, and the enantiomeric purities determined to be 100% for peak 1 and 100% for peak 2. A Jasco P-1020 polarimeter was used to determine the specific rotation [α] of the enantiomers of peaks 1 and 2, which were -86.2 and +93.3 (deg mL)/(g dm) respectively. No racemization was observed, and the enantiomeric purity remained at 100% for each peak.


Assuntos
Amilose/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Indenos/química , Indenos/isolamento & purificação , Isoquinolinas/química , Isoquinolinas/isolamento & purificação , Fenilcarbamatos/química , Amilose/química , Isoquinolinas/análise , Ligantes , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Estereoisomerismo
3.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1118-1119: 33-39, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31005772

RESUMO

In Positron Emission Tomography (PET) research, it is important to assess not only pharmacokinetics of a radiotracer in vivo, but also of the drugs used in blocking/displacement PET studies. Typically, pharmacokinetic/pharmacodynamic (PK/PD) analyses of drugs used in rodent PET studies are based on population average pharmacokinetic profiles of the drugs due to limited blood volume withdrawal while simultaneously maintaining physiological homeostasis. This likely results in bias of PET data quantification, including unknown bias of target occupancy (TO) measurements. This study aimed to develop a High Performance Liquid Chromatography (HPLC) method for PK/PD quantification of drugs used in preclinical rodent PET research, specifically the translocator 18 kDa protein (TSPO) selective drug, PK11195, that used sub-millilitre blood volumes. The lowest detection limit for the proposed HPLC method ranged between 7.5 and 10 ng/mL depending on the method used to calculate the limit of detection, and the measured average relative standard deviation for intermediate precision was equal to 17.2%. Most importantly, we were able to demonstrate a significant difference between calculated PK11195 concentrations at 0.5, 1, 2, 3, 5, 15 and 30 min post-administration and individually measured whole blood levels (significance level range from p < 0.05 to p < 0.001; one-way ANOVA, Dunnet's post hoc test, p < 0.05). The HPLC method developed here uses sub-millilitre sample volumes to reproducibly assess PK/PD of PK11195 in rodent blood. This study highlights the importance of individually measured PK/PD drug concentrations when quantifying the TO from blocking/displacement rodent PET experiments.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Isoquinolinas/análise , Isoquinolinas/farmacocinética , Administração Intravenosa , Animais , Isoquinolinas/administração & dosagem , Limite de Detecção , Modelos Lineares , Masculino , Tomografia por Emissão de Pósitrons , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Distribuição Tecidual
4.
Electrophoresis ; 40(4): 582-586, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30488648

RESUMO

Far infrared radiation was employed in the rapid removal of the solvents in the extracts of Plumula Nelumbinis and standard mixture solutions to prevent the interference of the solvent peaks toward their capillary electrophoretic measurements. The sample solutions in small vials were exposed to far infrared ray at 60°C for 3 min to remove solvent. The dried samples in the vials were each dissolved into running buffer with the aid of ultrasonication for capillary electrophoresis analysis. The far infrared-assisted solvent removal approach was sucessfully applied in the rapid determination of neferine, liensinine, isoliensinine, rutin and hyperoside in Plumula Nelumbinis. The five analytes could be well separated within 12 min in a 40 cm long fused silica capillary at a separation voltage of 12 kV in a 50 mM borate buffer (pH 9.2). The results indicated that the interferences of the solvent peaks in the capillary electropherograms of the herbal drugs were eliminated completely.


Assuntos
Eletroforese Capilar/métodos , Metanol/química , Nelumbonaceae/química , Extratos Vegetais , Solventes/química , Desenho de Equipamento , Flavonóis/análise , Flavonóis/química , Flavonóis/isolamento & purificação , Raios Infravermelhos , Isoquinolinas/análise , Isoquinolinas/química , Isoquinolinas/isolamento & purificação , Limite de Detecção , Modelos Lineares , Extratos Vegetais/análise , Extratos Vegetais/química , Reprodutibilidade dos Testes
5.
J Pharm Biomed Anal ; 151: 200-208, 2018 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-29353808

RESUMO

This paper describes the development of a simple reversed-phase HPLC method that can quantitate trace amounts of a polymeric degradants (BMT-041910) in asunaprevir drug substance and formulated drug product with quantitation limits of ∼0.05% w/w. The method has overcome several challenges of polymer quantitation such as band broadening, peak coeluting and low sensitivity. The hydrophobic function group (BOC) of BMT-041910 is removed to increase its aqueous solubility by a simple sample treatment procedure (des-BOC). The des-BOC polymer (BMT-052076) is excluded from stationary phase pores and eluted as a single peak before solvent front, and then its peak area response can be used to determine BMT-041910 amount. The HPLC conditions were optimized using a 250 × 4.6 mm Waters XSelect CSH column maintained at 30 °C with a mobile phase of water-acetonitrile-trifluoroacetic acid (20:80:0.1 v/v/v). The feasibility of other HPLC approaches including size exclusion chromatography and normal phase chromatography were also investigated and found to be less suitable for this particular application. Validation data for this method in terms of precision, linearity, accuracy and sensitivity are also presented.


Assuntos
Contaminação de Medicamentos/prevenção & controle , Isoquinolinas/análise , Inibidores de Proteases/análise , Sulfonamidas/análise , Tecnologia Farmacêutica/métodos , Acetonitrilos/química , Química Farmacêutica/métodos , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Interações Hidrofóbicas e Hidrofílicas , Polímeros/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Solventes/química , Ácido Trifluoracético/química
6.
Eur J Mass Spectrom (Chichester) ; 24(1): 145-156, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29232975

RESUMO

Selective androgen receptor modulators (SARMs) have been identified as a promising class of drug candidates potentially applicable to diverse pathological conditions commonly associated with significantly reduced muscle mass. Due to a suspected and meanwhile repeatedly proven misuse of SARMs in elite and amateur sport, sustaining constantly updated doping control analytical methods is critical for sports drug testing laboratories. These test methods predominantly utilize mass spectrometry-based instrumentations and, consequently, studies on the mass spectrometric behavior of new compounds and, where available, their metabolic products are vital for comprehensive doping controls. In this communication, the dissociation patterns of three new SARM drug candidates referred to as GSK2881078, PF-06260414, and TFM-4 AS-1 as observed under electron ionization as well as electrospray ionization/collision-induced dissociation are discussed. By means of high resolution/high accuracy tandem mass spectrometry employing quadrupole-orbitrap mass analyzers, information on precursor-product ion relationships and elemental compositions was obtained and subsequently utilized to suggest dissociation routes of the target compounds. This information can contribute to future studies concerning structure assignments of metabolites and accelerate the identification of related substances if distributed and/or illicitly used in the world of sport.


Assuntos
Androgênios/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Detecção do Abuso de Substâncias/métodos , Anabolizantes/análise , Doping nos Esportes , Humanos , Indóis/análise , Isoquinolinas/análise , Cinética
7.
Drug Test Anal ; 9(11-12): 1788-1793, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28801989

RESUMO

Higenamine is a key component of traditional Chinese herbal medicine. The fruit of Nandina domestica (which contains this component) is available as an ingredient in the so-called Nanten-nodo-ame throat lozenge found on the Japanese market, which is an over-the-counter pharmaceutical and is easy to purchase for Japanese athletes. However, higenamine is a non-selective ß2-agonist, which is exemplified in the prohibited list of the World Anti-Doping Agency (WADA). Therefore, some have raised a concern regarding the potential cause of increased unintentional higenamine doping cases in the Asian region. This study aimed to investigate components of throat lozenges and develop a mass-spectrometry method for the quantification of higenamine and coclaurine in human urine. Moreover, a population study of Japanese subjects (n = 246) and an excretion study (n = 4) of the corresponding throat-lozenge recipients were performed to test the applicability of the current reporting threshold (i.e., 10 ng/mL) of higenamine set by WADA. The estimates of higenamine and coclaurine were 2.2 ± 0.1 µg/drop (mean of n = 12) and 0.5 ± 0.01 µg/drop (mean of n = 12), respectively. The maximum concentrations of higenamine and coclaurine were 0.2-0.4 and 0.3-1.0 ng/mL, respectively, at 10-12 h after administration of higenamine (nine drops); however, the concentrations in all four volunteers did not reach the positivity criterion of 10 ng/mL. No higenamine and coclaurine could be detected in the Japanese subjects. Therefore, there is no risk of detecting unintentional higenamine doping when the WADA reporting threshold is used.


Assuntos
Alcaloides/análise , Doping nos Esportes , Isoquinolinas/análise , Tetra-Hidroisoquinolinas/análise , Frutas , Humanos , Espectrometria de Massas em Tandem
8.
Chem Commun (Camb) ; 53(43): 5806-5809, 2017 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-28470248

RESUMO

Crystallographic analyses of the VIM-5 metallo-ß-lactamase (MBL) with isoquinoline inhibitors reveal non zinc ion binding modes. Comparison with other MBL-inhibitor structures directed addition of a zinc-binding thiol enabling identification of potent B1 MBL inhibitors. The inhibitors potentiate meropenem activity against clinical isolates harboring MBLs.


Assuntos
Isoquinolinas/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Enterobacter aerogenes/enzimologia , Escherichia coli/enzimologia , Isoquinolinas/análise , Klebsiella pneumoniae/enzimologia , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/análise
9.
J Sci Food Agric ; 97(12): 4190-4197, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28239868

RESUMO

BACKGROUND: We previously demonstrated that disruption of the pksCT gene of Monascus led to a greater than 98% decrease in its citrinin production capacity in Monascus (PHDS26). Two potentially toxic compounds, monascopyridine A (MPA) and monascopyridine B (MPB), were found in the fermentation products of the pksCT gene-disrupted Monascus. Moreover, a rapid and reliable high-performance liquid chromatography method was developed for the simultaneous determination of MPA and MPB. We studied the effects of various extraction parameters and designed an orthogonal experiment to investigate the importance of each factor. RESULTS: The optimal extraction conditions were: methanol concentration, 90%; extraction temperature, 40 °C; extraction time, 10 min; two extraction cycles; and a solid-liquid ratio of 1:25. Under the optimal chromatographic conditions, good linearity was reached over the concentration ranges 0.5-200 µg mL-1 and 0.5-300 µg mL-1 for MPA and MPB, respectively, and the corresponding determination coefficients were 0.9999 and 0.9997. The percentage relative standard deviation values of within-day and between-day precision for MPA were 2.0% and 2.1%, respectively; the corresponding values for MPB were 4.8% and 4.6%. The average recovery for MPA and MPB was 99.9% and 94%, respectively. CONCLUSION: Maximum MPA and MPB yields (2073.7 and 1961.7 µg g-1 , respectively) were observed after 16 days of cultivation. © 2017 Society of Chemical Industry.


Assuntos
4-Butirolactona/análogos & derivados , Proteínas Fúngicas/genética , Monascus/metabolismo , 4-Butirolactona/análise , 4-Butirolactona/biossíntese , 4-Butirolactona/toxicidade , Cromatografia Líquida de Alta Pressão , Fermentação , Proteínas Fúngicas/metabolismo , Inativação Gênica , Isoquinolinas/análise , Isoquinolinas/toxicidade , Monascus/química , Monascus/genética
10.
J Pharm Biomed Anal ; 134: 228-236, 2017 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-27918992

RESUMO

FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.


Assuntos
Cunninghamella/metabolismo , Doping nos Esportes , Glicina/análogos & derivados , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoquinolinas/metabolismo , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Cunninghamella/química , Doping nos Esportes/prevenção & controle , Glicina/análise , Glicina/metabolismo , Hepatócitos/química , Hepatócitos/metabolismo , Cavalos , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/análise , Isoquinolinas/análise , Extração Líquido-Líquido/métodos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo
11.
Electrophoresis ; 37(23-24): 3118-3125, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27699822

RESUMO

A simple, efficient, and green chitosan-assisted liquid-solid extraction method was developed for the sample preparation of isoquinoline derivative alkaloids followed by microemulsion LC. The optimized mobile phase consisted of 0.8% w/v of ethyl acetate, 1.0% w/v of SDS, 8.0% w/v of n-butanol, 0.1% v/v acetic acid, and 10% v/v ACN. Compared to pharmacopoeia method and organic solvent extraction, this new approach avoided the use of volatile organic solvents, replacing them with relatively small amounts of chitosan. Under the optimum conditions, good linearity (r2 > 0.9980) for all calibration curves and low detection limits between 0.05 and 0.10 µg/mL were achieved. The presented procedure was successfully applied to determine alkaloids in Rhizoma coptidis with satisfactory recoveries (81.3-106.4%).


Assuntos
Alcaloides/análise , Quitosana/química , Cromatografia Líquida/métodos , Isoquinolinas/análise , Microextração em Fase Líquida/métodos , Alcaloides/isolamento & purificação , Medicamentos de Ervas Chinesas/química , Isoquinolinas/isolamento & purificação , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
12.
Chem Biodivers ; 13(8): 990-7, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27286480

RESUMO

Annona crassiflora Mart. is a native tree from Brazilian savanna. Isoquinoline alkaloids are characteristic of species of Annonaceae. This work aimed to assess the magnitude of genetic diversity among different populations of A. crassiflora using AFLP markers, and verify the existence of any correlation between the AFLP data and previous reported alkaloid composition. A. crassiflora from eight populations in the states of São Paulo, Goiás, Minas Gerais, and Distrito Federal were analyzed. The data suggest a low, moderate, and high level of genetic diversity from different populations of A. crassiflora. Concentration of alkaloids was significantly correlated with AFLP data, suggesting interaction between chemical and molecular markers in A. crassiflora. The data of association between the chemical and genetic differentiation of A. crassiflora may be useful to establish cultivation areas allowing the definition of strategies to preserve their genetic diversity with an interest in specific chemotypes for genetic improvement programs focused on sustainable utilization of this specie.


Assuntos
Annona/química , Annona/genética , Isoquinolinas/análise , Brasil
13.
Invest Ophthalmol Vis Sci ; 57(4): 2264-76, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-27124322

RESUMO

PURPOSE: In this study, we investigated the therapeutic potential of a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia. METHODS: In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17. RESULTS: Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 µmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 µmol/L and 6.8 to 14.8 µmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion. CONCLUSIONS: Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization.


Assuntos
Endotélio Vascular/citologia , Isoquinolinas/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Sulfonamidas/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Endotélio Vascular/efeitos dos fármacos , Humanos , Hipóxia/tratamento farmacológico , Técnicas In Vitro , Isoquinolinas/administração & dosagem , Isoquinolinas/análise , Camundongos , Camundongos Endogâmicos C57BL , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Soluções Oftálmicas , Fosforilação/efeitos dos fármacos , Retina/química , Sulfonamidas/administração & dosagem , Sulfonamidas/análise , Espectrometria de Massas em Tandem , Fator A de Crescimento do Endotélio Vascular/farmacologia
14.
J Pharm Biomed Anal ; 124: 267-273, 2016 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-26970596

RESUMO

An HPLC method for the assay of a DNA topoisomerase inhibitor, LMP776 (NSC 725776), has been developed and validated. The stress testing of LMP776 was carried out in accordance with International Conference on Harmonization (ICH) guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of LMP776 from its impurities and degradation products was achieved within 40 min on a Supelco Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 µm) with a gradient mobile phase comprising 38-80% acetonitrile in water, with 0.1% trifluoroacetic acid in both phases. LC/MS was used to obtain mass data for characterization of impurities and degradation products. One major impurity was isolated through chloroform extraction and identified by NMR. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25-0.75 mg/mL, r = 0.9999), accuracy (recovery 98.6-100.4%), precision (RSD ≤ 1.4%), and sensitivity (LOD 0.13 µg/mL). The validated method was used in the stability study of the LMP776 drug substance in conformance with the ICH Q1A (R2) guideline.


Assuntos
Antineoplásicos/análise , Benzodioxóis/análise , Cromatografia Líquida de Alta Pressão/métodos , Isoquinolinas/análise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Reprodutibilidade dos Testes
15.
Bioanalysis ; 8(4): 265-74, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26807991

RESUMO

BACKGROUND: A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. CONCLUSION: AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.


Assuntos
Análise Química do Sangue/métodos , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Isoquinolinas/análise , Limite de Detecção , Oligopeptídeos/análise , Bibliotecas de Moléculas Pequenas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Cães , Feminino , Humanos , Proteínas Imobilizadas/antagonistas & inibidores , Proteínas Imobilizadas/química , Proteínas Inibidoras de Apoptose/química , Isoquinolinas/química , Isoquinolinas/farmacologia , Masculino , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
16.
Talanta ; 142: 90-6, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26003696

RESUMO

A new method has been developed for separation of chelerythrine and sanguinarine in medicinal plants used in traditional Chinese medicine (TCM). The separation is achieved by microchip electrophoresis (CE) using laser-induced fluorescence detection. The CE separation is achieved by using a hydro-organic medium as the electrolyte buffer. The experimental results are consistent with the prediction by theory in terms of resolution and migration speed because of the low Joule heat generated in microchip CE. In addition, formamide was found to have a potential for separation of molecules with similar chemical structures. Based on these findings, a run buffer containing 50% formamide was used to separate chelerythrine (CHE) and sanguinarine (SAN). The influencing factors, such as solvent of run buffer, pH of buffer, separation distance, and separation voltage, were optimized. Baseline separation of chelerythrine and sanguinarine was achieved within 120 s under an electrical voltage of 1.8 kV. Good linearity was observed in the concentration range of 0.15-550 µg mL(-1) (r=0.9993) for CHE and in the range of 0.3-600 µg mL(-1) (r=0.9998) for SAN. A low limit of detection (LOD) was achieved because of the high sensitivity achieved by laser-induced fluorescence detection (i.e. 5.0 ng mL(-1) and 2.0 ng mL(-1) for CHE and SAN, respectively). The contents of CHE are found to be 641.8±7.5 and 134.0±2.3 mg/kg in extracts of Macleaya cordata and Chelidonium majus, respectively, with good recovery of above 99%. The corresponding values for SAN found in these Chinese herbal extracts are 681.8±7.9 mg/kg and 890.5±8.9 mg/kg, respectively.


Assuntos
Benzofenantridinas/análise , Isoquinolinas/análise , Papaveraceae/química , Benzofenantridinas/química , Eletroforese em Microchip , Fluoresceína/química , Fluorescência , Isoquinolinas/química , Plantas Medicinais/química , Rodamina 123/química , Sementes/química , Solventes/química
17.
J Sep Sci ; 38(13): 2332-9, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25872750

RESUMO

Capillary electrophoresis with electrochemiluminescence detection for the simultaneous analysis of cisatracurium besylate and its degradation products (laudanosine, quaternary monoacrylate) in pharmaceutical preparation was developed and fully validated. The significant parameters that influence capillary electrophoresis separation and electrochemiluminescence detection were optimized. The total analysis time of the analytes was 15 min. The linearities of the method were 0.1∼40.0 µg/mL for cisatracurium besylate and 0.04∼8.00 µg/mL for laudanosine, with correlation coefficients (r) of 0.999 and 0.998, respectively. The detection limits (S/N = 3) were 83.0 ng/mL for cisatracurium besylate and 32.0 ng/mL for laudanosine. The intraday relative standard deviations of the analytes were <3.0%, and the interday relative standard deviations were <8.0%. The developed method was cost-effective, sensitive, fast, and resource-saving, which was suitable for the ingredient analysis in pharmaceutical preparation.


Assuntos
Atracúrio/análogos & derivados , Eletroforese Capilar/métodos , Preparações Farmacêuticas/química , Atracúrio/análise , Atracúrio/química , Isoquinolinas/análise , Isoquinolinas/química , Limite de Detecção , Luminescência , Reprodutibilidade dos Testes
18.
Electrophoresis ; 36(4): 502-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25363636

RESUMO

A Tee configuration sheath flow cuvette with square cross-section channels has been produced in PDMS for CE detection. The output of a 1.4 W laser diode operating at 450 nm was focused onto the 300 µm core of a 370 µm od fiber optic whose end was inserted into one arm of the Tee for LIF. The optimal configuration had the fiber optic positioned 500 µm downstream from the intersection and the end of the 35 cm 50 µm id 365 µm od capillary just outside the intersection and in the leg of the Tee, resulting in a 90° configuration. Detection limits of 50 and 3 pM and linear calibrations of at least three orders of magnitude were obtained for Lucifer Yellow and fluorescein, respectively.


Assuntos
Dimetilpolisiloxanos/química , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Tecnologia de Fibra Óptica/instrumentação , Líquido Amniótico/química , Calibragem , Desenho de Equipamento , Fluoresceína/análise , Fluorescência , Corantes Fluorescentes/análise , Isoquinolinas/análise , Lasers Semicondutores , Limite de Detecção
19.
J Sep Sci ; 38(1): 9-17, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25330407

RESUMO

A simple and efficient method was developed for the simultaneous determination of eight isoquinoline alkaloids in methanol extracts of Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids of D. leptopodum by high-performance liquid chromatography with diode array detection. The chromatographic conditions were optimized on a SinoChrom ODS-BP column to obtain a good separation of the four types of alkaloid analytes, including two aporphines (isocorydine, corydine), two protopines (protopine and allocryptopine), a morphine (sinoacutine), and three quaternary protoberberine alkaloids (berberrubine, 5-hydroxycoptisine, and berberine). The separation of these alkaloids was significantly affected by the composition of the mobile phase, and particularly by its pH value. Acetonitrile (A) and 0.2% phosphoric acid solution adjusted to pH 6.32 with triethylamine (B) were selected as the mobile phase with a gradient elution. With this method, a new quaternary protoberberine alkaloid was isolated and the two structural isomers (isocorydine and corydine) were baseline separated. The appropriate harvest period for D. leptopodum was also recommended based on our analysis. The method for the effective fraction of the alkaloids of D. leptopodum was optimized under this method with regard to the varying significant pharmacological activities of the alkaloids.


Assuntos
Alcaloides/análise , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/isolamento & purificação , Isoquinolinas/análise , Isoquinolinas/isolamento & purificação , Papaveraceae/química , Alcaloides/isolamento & purificação , Cromatografia Líquida de Alta Pressão/instrumentação
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