A newly validated high-performance liquid chromatography method with diode array ultraviolet detection for analysis of the antimalarial drug primaquine in the blood plasma

Abstract INTRODUCTION: Primaquine (PQ) diphosphate is an 8-aminoquinoline antimalarial drug with unique therapeutic properties. It is the only drug that prevents relapses of Plasmodium vivax or Plasmodium ovale infections. In this study, a fast, sensitive, cost-effective, and robust method for the extraction and high-performance liquid chromatography with diode array ultraviolet detection (HPLC-DAD-UV ) analysis of PQ in the blood plasma was developed and validated. METHODS: After plasma protein precipitation, PQ was obtained by liquid-liquid extraction and analyzed by HPLC-DAD-UV with a modified-silica cyanopropyl column (250mm × 4.6mm i.d. × 5μm) as the stationary phase and a mixture of acetonitrile and 10mM ammonium acetate buffer (pH = 3.80) (45:55) as the mobile phase. The flow rate was 1.0mL·min-1, the oven temperature was 50OC, and absorbance was measured at 264nm. The method was validated for linearity, intra-day and inter-day precision, accuracy, recovery, and robustness. The detection (LOD) and quantification (LOQ) limits were 1.0 and 3.5ng·mL-1, respectively. The method was used to analyze the plasma of female DBA-2 mice treated with 20mg.kg-1 (oral) PQ diphosphate. RESULTS: By combining a simple, low-cost extraction procedure with a sensitive, precise, accurate, and robust method, it was possible to analyze PQ in small volumes of plasma. The new method presents lower LOD and LOQ limits and requires a shorter analysis time and smaller plasma volumes than those of previously reported HPLC methods with DAD-UV detection. CONCLUSIONS: The new validated method is suitable for kinetic studies of PQ in small rodents, including mouse models for the study of malaria.

Estudo da alteração do esvaziamento gástrico em roedores

Fatores que alteram os níveis plasmáticos de substâncias químicas e, por conseguinte, modificam a sua cinética, como por exemplo, a gravidez, podem ter impactos sobre a segurança e eficácia de medicamentos. Em estudo recente, realizado por Carmo (2015), no Laboratório de Toxicologia Ambiental do Departamento de Biologia da Escola Nacional de Saúde Pública da Fundação Oswaldo Cruz (ENSP/FIOCRUZ), foi observado que a concentração plasmática do antimalárico difosfato de primaquina em camundongos fêmeas grávidas DBA/2 era menor do que a concentração do fármaco registrada em igual intervalo de tempo pós-adminstração em camundongos fêmeas não grávidas. Vários estudos sugerem que a diminuição da concentração plasmática de fármacos na gestante pode se dever a um retardo no esvaziamento gástrico e/ou um aumento no volume de distribuição. Alterações do trânsito no trato gastrintestinal podem influenciar diretamente a absorção de fármacos, resultando em absorção mais rápida ou mais lenta. O fármaco analgésico e antipirético paracetamol é absorvido quase que exclusivamente no intestino. Assim a velocidade da sua absorção depende do tempo de esvaziamento gástrico. Fatores tais como alimentação, idade, gravidez e/ou o uso de fármacos que promovem aceleração (metoclopramida) ou o retardo (morfina) da motilidade gastrointestinal, podem influenciar em sua absorção. O objetivo desse trabalho foi desenvolver e padronizar uma metodologia de análise do paracetamol que permitisse investigar o efeito da gravidez sobre o esvaziamento gástrico sobre a cinética de fármacos administrados em pequenos roedores. O método empregado para determinar as concentrações plasmáticas de paracetamol foi a cromatografia em fase líquida de alta eficiência com detector por arranjo de diodos e visualização no ultravioleta (CLAE-DAD-UV), em equipamento Shimadzu Class-VP...

Factors that affect plasma levels of chemicals, and consequently their kinetics, such as pregnancy, can impact on the safety and efficacy of medicines. In a recent study, conducted by Carmo (2015) at the laboratory of Environmental Toxicology (Department of Biological Sciences, National School Public Health, Oswaldo Cruz Foundation -ENSP / FIOCRUZ), it was shown that plasma concentrations of the anti-malarial drug primaquine diphosphate in pregnant female DBA/2 mice were lower than levels found in non pregnant female mice. During pregnancy a delayed gastric emptying and/or an increased volume of distribution may result in lower drug plasma concentrations. Pregnancy-produced changes in the gastrointestinal transit may influence drug absorption. Depending on whether gastric emptying is accelerated or slowed and on the place where drug absorption takes place (stomach or intestines) absorption can be accelerated or slowed. Paracetamol, an analgesic and antipyretic drug, is absorbed almost exclusively in the intestines and is used to investigate the effects of treatment on the gastric emptying rate. Factors such as diet, age, pregnancy or the administration of drugs which accelerate (metoclopramide) or delay (morphine) gastric emptying influence the absorption of paracetamol. The aim of this study was to develop and standardize a methodology to investigate the effect of gastric emptying on the kinetics of drugs administered in small rodents. The methodology used in the analysis of plasma concentrations of paracetamol was High Performance Liquid Chromatography coupled to diode-array detector and visualization on ultraviolet range (HPLC-DAD-UV), using a Shimadzu Class-VP equipment...

Evaluation of the in vitro and in vivo anticancer properties of Moroccan propolis extracts

This investigation aimed to evaluate the in vitro and in vivo antitumor potential of a Moroccan propolis extracts. For in vitro assays, three mammalian tumor cell lines were used: BSR (hamster renal adenocarcinoma), Hep-2 (human laryngeal carcinoma) and P815 (murin mastocytoma). The propolis ethanolic extract as well as the ethyl acetate extract, exert an in vitro cytotoxic activity in dose-dependent manner. The IC50 values were ranging from 15 µg/mL to 38 µg/mL. This activity depends not only on the extract's chemical composition (analysed by HPLC/ESI-MS), but also on the target tumor cells. Interestingly, the cytotoxic effect of these extracts on the normal human peripheral blood mononuclear cells (PBMC) was weak when compared to that induced on tumor cells. On the other hand, oral route treatment of P815 tumor-bearing mice (DBA2/P815) with propolis ethanolic extract (5 mg per mouse every fourth day, five times for group A, and 2.5 mg per mouse every fourth day, five times for group B) significantly reduced the tumor volume (1.2 cm³ for group A and 2.7 cm³ for group B at the 22nd day after tumor graft). These effects are statistically significant as compared to those obtained with the control untreated mice (tumor volume 3.5 cm³ at day 22).

Study on Protective Immunity against Infection of Plasmodium yoelii 17XL in DBA/2 Mice / 中国寄生虫学与寄生虫病杂志

Objective To investigate the development and dynamic changes of host immune response in DBA/2 mice infected with Plasmodium yoelii 17XL. Methods Female DBA/2 mice were infected by intraperitoneal ( i. p. ) injection of 106 P. yoelii 17XL parasitized erythrocytes ( PRBC). Levels of IL-12, IFN-γ, IL-4, IL-10 and P. yoelii 17XL-specific antibody in sera were measured by ELISA. Concentrations of NO in cell supernatants were measured by the Griess reaction. Parasitemia,percentage of mononuclear-macrophages of individual mice were monitored daily, and phagocytosis of mononuclear macrophages was also observed. Results Primary parasitemia in vein blood was developed on day 3 postinfection, which peaked with a level of 46. 9% on day 9. Most mice cleared the infection and survived by day 20 postinfection. From day 6 to day 16, the phagocytosis of PRBC by rodent macrophages was observed on the blood smear. Infected mice had a continuously increased level of IL-12 in serum from day 1 postinfection. Accordingly, high level of IFN-γ was also detected in sera from day 1 postinfection,which peaked on day 6. Infected mice produced higher level of IL-4 and IL-10 in serum on day 6 postinfection, which peaked on day 9 and day 15 postinfection respectively. In addition, splenocytes from infected mice produced significantly higher level of NO on day 6 and 20 postinfection. Level of P. yoelii 17XL-specific IgG was determined in the sera of infected mice with a steadily increased trend after infection, which peaked on day 70 postinfection. Conclusions Effective polarizing of Thl cells is significant in inhibition of parasitemia and eventual clearance of the Plasmodium parasites. Activated mononuclear-macrophages play a key role in inhibiting parasitemia in the early phase of infection with P. yoelii 17XL.