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Braz. J. Pharm. Sci. (Online) ; 55: e18328, 2019. tab, graf
Article in English | LILACS | ID: biblio-1039071


Recombinant human interferon beta 1b (rhIFNß-1b) is clinically used to treat multiple sclerosis. A reversed-phase liquid chromatography (RP-LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE-LC) method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 µg/mL (r2 = 0.9998) and 0.50-200 µg/mL (r2 = 0.9999), respectively, for RP-LC and SE-LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNß-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic

Biological Assay/methods , Humans , Chromatography, Reverse-Phase/methods , Interferon beta-1b/analysis , In Vitro Techniques , Biotechnology/classification , Validation Study
Braz. J. Pharm. Sci. (Online) ; 55: e18052, 2019. tab, graf
Article in English | LILACS | ID: biblio-1039069


A specific, precise, and accurate LC-UV method was developed and validated to assay raloxifene hydrochloride in rat plasma. Raloxifene was analyzed after liquid-liquid extraction and quantified by reversed phase liquid chromatography (C18 column) using acetonitrile and ammonium acetate buffer 0.05 M (pH 4.0) as mobile phase at a flow rate of 1 mL.min-1 and UV detection at 287 nm. Retention times of raloxifene and internal standard (dexamethasone) were approximately 11 min and 14 min, respectively. Linearity was checked for a concentration range between 25 ng.mL-1 and 1000 ng.mL-1. Intra- and inter-day precision had relative standard deviation lower than 10% and 15%, respectively. Recovery from plasma was higher than 90%. Accuracy values were 98.21%, 99.70%, and 102.70% for lower, medium, and upper limits of quantification, respectively. Limit of quantification was 25 ng.mL-1. Drug stability was analyzed at room temperature using plasma kept in a freezer at -80 °C for 45 days after processing for 6 h and three freeze-thaw cycles. The advantages of the method developed include stability under different conditions and low limit of quantification. Its applicability was confirmed by the analysis of raloxifene levels in plasma samples in a designed pharmacokinetic study in rats after intravenous administration (5

Animals , Male , Rats , Plasma/drug effects , Raloxifene Hydrochloride/pharmacokinetics , Chromatography, Reverse-Phase/methods , Biological Availability
Article in English | LILACS | ID: biblio-894164


Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis. Methods: P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. Results: The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896. 47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues. Conclusion: This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues.(AU)

Animals , Wasps , Receptors, Phospholipase A2/isolation & purification , Receptors, Phospholipase A2/chemistry , Poisoning , Mass Spectrometry/methods , Receptors, Phospholipase A2/chemistry , Chromatography, Reverse-Phase/methods
Braz. J. Pharm. Sci. (Online) ; 53(4): e17293, 2017. tab, graf, ilus
Article in English | LILACS | ID: biblio-889435


ABSTRACT A novel, accurate, precise and economical stability indicating Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method, was developed and validated for the quantitative determination of ubidecarenone (UDC) in bulk drug, UDC marketed formulation and UDC loaded cubosomes (CBMs) nanocarriers through Response surface methodology (RSM) design with three factors and three levels was performed to optimize the chromatographic variables followed by forced degradation studies of UDC were performed to detect degradation peak. RP-HPLC separation was achieved using mobile phase consisting of Acetonitrile:Tetrahydrofuran:Deionised water in the ratio 55:42:3 and a flow rate of 1.0 mL/min was optimized with a standard retention time (Rt) of 2.15 min, through experiment. The method was found linear in the concentration range of 5-100 µg/mL with a regression coefficient of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.04 µg/mL and 9.11 µg/mL, respectively.

Ubiquinone/analysis , Chromatography, Reverse-Phase/methods , Pharmaceutical Preparations/administration & dosage
Braz. J. Pharm. Sci. (Online) ; 53(1): e15177, 2017. tab, graf
Article in English | LILACS | ID: biblio-839449


Abstract In this study, conditions were optimized for development of a simple RP-HPLC method for simultaneous analysis of gatifloxacin and dexamethasone in different matrices like pharmaceuticals, human serum and urine. Good separation of gatifloxacin and dexamethasone from the induced degradation products was accomplished using C8 as stationary phase; 0.02 M phosphate buffer (pH 3.0) and methanol (42:58 v/v) as mobile phase. The concentration was measured with DAD at 270 nm. Linearity was observed in the range of 0.000040-0.000280 mol/L for gatifloxacin (r2≥0.999) and 0.000013-0.000091 mol/L for dexamethasone (r2≥0.999). Both the analyte peaks were completely separated from the peaks of induced degradation products as indicated by the peak purity index (≥0.9999 for both analytes). The optimized method is recommended to be used for concurrent analysis of gatifloxacin and dexamethasone in different matrices.

Chromatography, High Pressure Liquid/methods , Drug Stability , Chromatography, Reverse-Phase/methods , Anti-Bacterial Agents/analysis , Dexamethasone/analysis , Validation Study
São Paulo; s.n; s.n; 2017. 121 p. tab, ilus, graf.
Thesis in Portuguese | LILACS | ID: biblio-884207


Danos em biomoléculas podem ocorrer a partir de uma interação direta entre as biomoléculas e espécies reativas de oxigênio e nitrogênio como também, pela reação de produtos secundários dessas espécies como eletrófilos gerados na peroxidação lipídica. Alguns desses produtos secundários possuem estabilidade química maior que as espécies reativas das quais foram derivadas e podem se ligar covalentemente as biomoléculas comprometendo o funcionamento normal das mesmas. Portanto, modificações em proteínas por aldeídos gerados na lipoperoxidação têm sido investigadas por suas implicações com desordens patológicas relacionadas à agregação proteica, e modificações em diversas vias de sinalização amplificando os efeitos deletérios em sistemas biológicos. Os objetivos desse trabalho foi contribuir na elucidação dos mecanismos moleculares associados ao desenvolvimento da esclerose lateral amiotrófica (ELA) através da identificação, caracterização e quantificação de modificações póstraducionais em proteínas pelos aldeídos 4-hidroxi-2-hexenal (HHE) e trans-4-hidroxi-2-nonenal (HNE) in vitro (citocromo c) e in vivo (modelo ELA) a partir de técnicas de Western blot, imunoprecipitação e espectrometria de massa com abordagem proteômica de "shotgun" em ratosSOD1G93A modelo de esclerose lateral amiotrófica (ELA). Estudos com citocromo c mostraram a ligação dos aldeídos ao citocromo c e mecanismos de reação foram propostos. Foram encontrados seis peptídeos modificados por HHE e um para o HNE, e o peptídeo TGPNLHGLFGR se mostrou modificado pelos dois aldeídos paralelamente. Foi demonstrado que a histidina 33 é um "hot spot" frente as adições pelos aldeídos. Nas análises por western blot das proteínas ligadas a aldeídos foi possível observar uma tendência de aumento na concentração de proteínas ligadas ao HNE nos animais ELA, mais acentuada nas amostras de 70 dias comparadas ao controle. Com relação aos resultados obtidos com HHE tanto os animais pré-sintomáticos quanto os sintomáticos não apresentaram diferenças de HHE-proteína, tantonos controles quanto nos animais ELA. Nas amostras dos animais sintomáticos não detectamos diferença significativa na concentração de aldeído-proteína entre os grupos. Já as análises proteômicas revelaram 24 proteínas diferencialmente expressas, com destaque para proteínas com os maiores valores de significância (p-value), como a ubiquitina no grupo dos pré- sintomáticos e a neurogranina, no grupo dos animais sintomáticos e várias proteínas de metabolismo energético, de neurofilamentos, proteínas de processos redox e proteínas ligadas o metabolismo de cálcio (fundamentais na fisiopatologia em ELA). Algumas proteínas importantes foram encontradas com exclusividades nos grupos pré-sintomáticos e sintomáticos pelo diagrama de Venn. Com relação a proteínas modificadas pelos aldeídos, foram encontradas algumas relevantes como a proteína 2 de interação com a polimerase delta que foi modificada por HNE via adição de Michael encontrada nos animais ELA pré-sintomáticos e sintomáticos, a catalase que foi encontrada modificada por HNE via base de Schiff apenas nos ELA pré- sintomáticos, e a tiol redutase induzível por interferon gama no grupo dos animais ELA sintomáticos. Com relação a proteínas modificadas por HHE, foram encontradas a Janus quinase e proteína 3 de interação com microtúbulo, modificadas tanto por adição de Michael quanto via base de Schiff nos animais ELA sintomáticos. É interessante ressaltar que algumas modificações encontradas em proteínas não caracterizadas podem indicar proteínas novas ainda não descritas como modificadas por esses aldeídos. Os resultados mostram que algumas das proteínas modificadas por HNE e HHE encontradas neste trabalho, estão relacionadas ao estresse redox, vias metabólicas energéticas, proteínas envolvidas na resposta a danos oxidativos, e processos inflamatórios. Tais modificações ocorrem não só no modelo de neurodegeneração, mas foram previamente descritas em outros processos patológicos, como doença cardiovascular, lesão hepática por uso crônico de álcool

Damage to biomolecules can occur from a direct interaction between biomolecules and reactive of oxygen and nitrogen species as well as from the reaction of secondary products of these species as electrophiles generated in lipid peroxidation. Some of these by-products have greater chemical stability than the derived reactive species and can bind to biomolecules compromising their normal function. Therefore, protein modifications by aldehydes generated during lipoperoxidation have been investigated for their implications with pathological disorders related to protein aggregation and modifications in signaling pathways amplifying the deleterious effects in biological systems. The aim of this work was to contribute to the elucidation of the molecular mechanisms associated with the development of amyotrophic lateral sclerosis (ALS) through the identification, characterization and quantification of posttranslational modifications in proteins by 4-hydroxy-2-hexenal (HHE) and trans-4-hydroxy-2- nonenal (HNE) in vitro, cytochrome c, and in vivo, rat model (SOD1G93A) of amyotrophic lateral sclerosis (ALS), throught Western blot techniques, and mass spectrometry with shotgun proteomics approach. The results showed the binding of aldehydes to cytochrome c. Six peptides were modified by HHE and one by HNE. The peptide TGPNLHGLFGR was modified by the two aldehydes. Histidine 33 has been shown to be a hot spot against aldehydes additions. By western blot analysis of the aldehyde-bound proteins, it was possible to observe a tendency of increase in the concentration of HNE-bound proteins in the ALS animals, more pronounced in the samples of 70 days compared to control samples. Regarding the results obtained with HHE, both pre-symptomatic and symptomatic animals did not show HHE-protein differences, both in controls and in ALS animals. We did not detect a significant difference in the aldehyde-protein concentration between the groups in the samples of the symptomatic animals. Proteomic analysis revealed 24 differentially expressed proteins, with emphasis on proteins with thehighest values of significance (p-value), such as the ubiquitin in the pre-symptomatic group and neurogranin in the group of the symptomatic animals and several proteins of the energetic metabolism pathways, neurofilaments, proteins of redox processes and proteins linked to calcium metabolism (fundamental in the pathophysiology of ALS). Some important proteins were found exclusivity in the pre-symptomatic and symptomatic groups by the Venn diagram. With regard to aldehyde-modified proteins, some relevant ones such as Delta-2 polymerase interaction protein, that was modified by HNE via the addition of Michael found in presymptomatic and symptomatic ELA animals, catalase that was found to be modified by HNE via Schiff's base only in pre-symptomatic ALS, and gamma interferon-inducible thiol reductase in the group of symptomatic ALS animals. Janus kinase and microtubule interaction protein 3, were found to be modified by Michael addition and Schiff base pathway respectively in symptomatic ALS animals. It is interesting to note that some modifications found in uncharacterized proteins may indicate new proteins not yet described as modified by these aldehydes. The results show that some of the proteins modified by HNE and HHE found in this work are related to redox stress, energetic metabolic pathways, proteins involved in the response to oxidative damage, and inflammatory processes. Such modifications occur not only in the neurodegeneration model, but were previously described in other pathological processes, such as cardiovascular disease, liver injury due to chronic alcohol use

Animals , Female , Rats , Amyotrophic Lateral Sclerosis/physiopathology , Proteins/analysis , Aldehydes/analysis , Biomarkers/metabolism , Blotting, Western/methods , Chromatography, Reverse-Phase/methods , Cytochromes c , Genotyping Techniques/instrumentation , Mass Spectrometry/methods , Protein Modification, Translational , Proteomics/instrumentation , Small Ubiquitin-Related Modifier Proteins
Arab Journal of Pharmaceutical Sciences. 2013; 9 (4): 30-40
in Arabic | IMEMR | ID: emr-142816


A reverse phase high performance liquid chromatography method was developed for the determination of diloxanide furoate and metronidazole in tablets. The separation was achieved using C8 column [4.6 mm x 250 mm] 5 micro m and mobile phase acetonitrite: water [65:35] v/v, at a flow rate of 1ml/min. Detection was carried out using UV at 230 nm. The retention times of metronidazole and diloxanide furoate were 3.17 and 5.18 min. the detection limits for metronidazole and diloxanide furoate were 0.03, 0.04 micro g/ml and the Quantitation limits were 0.11, 0.13 micro g/ml respectively. The method has validated for accuracy, precision, selectivity and robustness, Linearity was in the range of 32 - 48 micro g/ml for metronidazole, and 40 -60 micro g/ml for diloxanide furoate. The recovery values of Accuracy were 99.08 - 100.52% for metronidazole and diloxanide furoate respectively

Furans/isolation & purification , Chromatography, Reverse-Phase/methods , Chromatography, High Pressure Liquid/methods , Tablets/chemistry , Limit of Detection
Braz. j. pharm. sci ; 48(2): 315-323, Apr.-June 2012. graf, tab
Article in English | LILACS | ID: lil-643024


A new, simple, fast, reproducible and sensitive reversed phase HPLC method, using a new stationary phase containing embedded urea polar groups, has been developed and validated for the simultaneous determination of clobutinol hydrochloride (CLO) and doxylamine succinate (DOX) in syrups. The determination was carried out on a C8 urea column (125 mm x 3.9 mm i.d., 5 µm particle size) synthetized at the Liquid Chomatography Laboratory (LabCrom) of the Chemistry Institute of Unicamp. The mobile phase consisted of a mixture of acetonitrile:methanol:phosphate buffer (pH 2.5) in the gradient mode. The diode array detector (DAD) was operated at 230 nm for CLO and 262 nm for DOX. The method showed adequate precision, with relative standard deviations (RSD) less than 1%. The presence of the excipients did not interfere in the results of the analysis. Accuracy was determined by adding standards of the drugs to a placebo and good recovery values were obtained. The analytical curves were linear (r² 0.9999 for CLO and 0.9998 for DOX) over a wide concentration range (2.4-336 µg mL-1 for CLO and 2.3-63 µg mL-1 for DOX). The solutions were stable for at least 72 hours at room temperature. The criteria for validation using the ICH guidelines were fulfilled.

Um novo método simples, fácil e reprodutível, de fase reversa para CLAE, usando uma fase estacionária contendo um grupo polar, uréia, embutido, foi desenvolvido e validado para determinação simultânea de cloridrato de clobutinol (CLO) e succinato de doxilamina (DOX) em xarope. A determinação foi realizada em uma coluna C8 uréia (125 mm x 3,9 mm d.i., 5 µm tamanho de partícula) sintetizada em nosso laboratório (LabCrom). A fase móvel consistiu de mistura de acetonitrila:metanol:tampão fosfato pH 2,5, em eluição por gradiente. O detector por arranjos de diodo (DAD) foi utilizado a 230 nm para CLO e a 262 nm para DOX. O método apresentou precisão adequada, com desvio padrão relativo menor que 1%. A presença de excipientes não interferiu nos resultados obtidos. A exatidão foi realizada pela adição dos padrões dos fármacos ao placebo e valores de recuperação aceitáveis foram obtidos. As curvas analíticas mostraram-se lineares (r² 0,9999 para CLO e 0,9998 para DOX) em uma ampla faixa de concentração (2,4-336 µg mL-1 para CLO e 2,3-63 µg mL-1 para DOX). A solução padrão foi estável por 72 horas a temperatura ambiente. Os parâmetros de validação foram realizados conforme o guia ICH.

Chromatography, Reverse-Phase/methods , Urea/classification , Cough , Doxylamine/classification , Evaluation Studies as Topic/analysis
Pakistan Journal of Pharmaceutical Sciences. 2011; 24 (3): 303-313
in English | IMEMR | ID: emr-129856


The effect of temperature stresses on Cefaclor suspensions under different storage conditions for a duration of 14 days was tested. The degradation of Cefaclor was determined on the 2[nd], 7[th] and 14[th] day after reconstitution using a sensitive and precise Reversed phase High Performance Liquid Chromatographic [RP-HPLC] method. The RSD values for Forticef, Midocef, Ceclor, Cefabac and Cloracef, indicated a good precision of the RP-HPLC method. The limit of detection [LOD] and the limit of quantification [LOQ] were found 0.008 mg/ml and 0.03mg/ml respectively. The antimicrobial effect of Cefaclor suspension was also tested against pathogenic bacteria using the cylinder diffusion method. The RSD values range of the antimicrobial assay for all the Cefaclor compounds were 1.47-3.7%. The LOD and LOQ were 0.2mg/ml and Img/ml respectively. During the normal use of Ceclor, Midocef, and Forticef the loss of activity and the degradation were less than 5% on the 14[th] day of preservation at 4°C. However, the percentage of degradation for Cefabac and Cloracef on the 14th day reached 5 and 6%, respectively. Statistical multiple comparison between the effect of 4°C and 25°C indicated non significant mean differences [P >/= 0.05] for Forticef, Cefabac, Ceclor and Cloraf and significant effect for Midocef [P

Humans , Cefaclor/pharmacology , Cefaclor/administration & dosage , Chromatography, Reverse-Phase/methods , Disk Diffusion Antimicrobial Tests/methods , Drug Stability , Administration, Oral , Suspensions , Temperature , Time Factors , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Drug Storage/statistics & numerical data