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A stability indicating simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method was developed and validated for the combined tablet formulation of pyrimethamine & sulphadoxine. Chromatographic separation was optimized by gradient HPLC on a C18 column [Inertsil Silica, 250 x 4.6 mm, 5μ] utilizing a mobile phase of potassium dihydrogen phosphate and acetonitrile taken in the ratio 70:30 at a flow rate of 1.0 ml/min with UV detection at 221nm. The retention time of pyrimethamine and sulphadoxine was 2.77 and 6.57 min respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, robustness and stress degradation studies. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated method can be recommended for the routine laboratory analysis.
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This paper presents the development of a new LC-MS method for the determination of pyrimethamine, sulphadoxine and artesunate in human plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate. The analytes and internal standard artemether were separated on a Phenomenex C18 column using gradient elution with 20mM ammonium acetate (mobile phase A) and methanol (mobile phase B) both containing 0.5% acetic acid at a flow rate of 0.8 ml/min. The calibration curves were constructed over the range of 5-30 ng/ml for pyrimethamine, 50-300 ng/ml for sulphadoxine and 25-150 ng/ml for artesunate respectively. Within day and between-day precision and accuracy did not exceed 8%. All the three analytes were found to be stable in plasma samples with no evidence of degradation during three freeze-thaw cycles and three months storage in -20 °C. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the simultaneous quantitative analysis of pyrimethamine, sulphadoxine and artesunate in human plasma.
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Background and Aims of Study: Malaria in pregnancy remains one of the infectious diseases threatening the health of pregnant women and the unborn child in Africa. The use of Sulphadoxine-Pyrimethamine (SP) as intermittent preventive treatment of malaria in pregnancy (IPTp) has been shown to greatly reduce the impact of malaria in pregnancy and its complications when administered appropriately according to set protocol. The aim of this study is to ascertain the appropriate use of SP among pregnant women who received ante-natal care (ANC) and determine its relationship with feto-maternal outcome. Place and Duration of Study: Daughters of charity Primary Health Center, Kubwa, Abuja, between January 2010 and June 2011. Methodology: A retrospective study of 200 pregnant women, who attended ANC, delivered and followed-up to post-natal clinic at Daughters of Charity primary health centre and was administered SP between January 2010 and June 2011. Ante-natal records were retrieved and socio-demographic variable, number of doses of SP received as well as feto-maternal outcome were collected and analyzed. Results: The use of IPTp among pregnant women in this facility is low, accounting for only 6% of the study population, while 79% were not administered IPTp throughout their pregnancy. Majority of the primigravida (61 out of 70) who were more vulnerable to malaria in pregnancy did not receive any dose of IPTp. Conclusion: More effort is required to increase IPTp coverage in the community. This may be achieved by improving the awareness of IPTp among health care workers, pregnant women and the entire community at large.
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Objective: to validate an analytical method for simultaneous determination and quantification of sulphadoxine and pyrimethamine in human blood dried onto filter paper, whose cost and analysis time can be reduced. Methods: whole blood spotted on filter paper of a healthy volunteer and solutions of sulphadoxine-pyrimethamine standard mixture were used. HPLC separations were carried out on Agilent equipment using a LiChrospher® column C18 with a mobile phase acetonitrile/0.1 M potassium phosphate buffer at pH 3.0 (1:1) for eight minutes under isocratic conditions. A flow rate of 0.7 mL/min, and a 20 mL volume injection were used. External standard method for quantitation of analytes was used. Results: the HPLC method described for the simultaneous determination of sulphadoxine and pyrimethamine in 100 mL of whole blood spotted on filter paper has been found to be linear, precise, accurate and selective. In this method, the sample preparation is simple using liquid-liquid extraction, and HPLC with ultraviolet detection is used. Conclusions: a simple, fast and sensitive method for determination of sulphadoxine and pyrimethamine in human blood dried onto filter paper was validated. This method can be used for the monitoring of both metabolites in pharmacokinetic and clinical studies.
Objetivo: validar un método de análisis para la determinación y cuantificación simultánea de sulfadoxina y pirimetamina en sangre humana secada sobre papel de filtro que sea rápido y barato. Métodos: se usó sangre de un voluntario sano impregnada sobre papel de filtro y soluciones estándar de la mezcla sulfadoxina y pririmetamina. Las separaciones por cromatografía líquida de alta resolución (CLAR) se hicieron en un equipo Agilent sobre una columna C18 LiChrospher® con acetonitrilo/buffer fosfato de potasio 0,1 M a pH 3,0 como fase móvil, usando condiciones isocráticas durante 8 min. Se usó un flujo de 0,7 mL/min y un volumen de inyección de 20 mL. Para la cuantificación de los analitos se utilizó el método del estándar externo. Resultados: el método CLAR descrito para la determinación simultánea de sulfadoxina y pirimetamina en 100 mL de sangre impregnada sobre el papel de filtro mostró linealidad, precisión, exactitud y selectividad. En este método la preparación de la muestra es simple ya que usa extracción líquido-líquido y detección ultravioleta. Conclusión: se obtuvo un método validado que es simple, rápido y sensible para la determinación de sulfadoxina y pirimetamina en sangre humana impregnada sobre papel de filtro, que puede ser usado para el monitoreo de ambos metabolitos en estudios farmacocinéticos y clínicos.
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Introduction: Surveillance of the genetic characteristics of dhps and dhfr can be useful to outline guidelines for application of intermittent preventive therapy in Northwest Colombia and to define the future use of antifolates in artemisinin-based combination therapy schemes. Objective: To evaluate the frequency of mutations in dhps and dhfr and to characterize parasite populations using msp-1, msp-2 and glurp in historic samples before artemisinin-based therapy was implemented in the country. Methods: A controlled clinical study was carried out on randomly selected Plasmodium falciparum infected volunteers of Northwest Colombia (Turbo and Zaragoza). A sample size of 25 subjects per region was calculated. Treatment efficacy to antifolates was assessed. Molecular analyses included P. falciparum genotypes by msp-1, msp-2 and glurp and evaluation of the status of codons 16, 51, 59, 108 and 164 of dhfr and 436, 437, 540, 581 and 613 of dhps. Results: In total 78 subjects were recruited. A maximum number of 4 genotypes were detected by msp-1, msp-2 and glurp. Codons 16, 59 and 164 of the dhfr gene exhibited the wild-type form, while codons 51 and 108 were mutant. In the dhps gene, the mutant 437 glycine was detected in 85% on day 0, while codons 436, 540, 581 and 613 were wild-type. Conclusions: Plasmodium falciparum populations were very homogeneous in this region of Colombia, and the triple mutants of dhfr and dhps Asn108, Ile51 and Gly437 were predominant in clinical isolates.
Introducción. La vigilancia de las características genéticas de dhps y dhfr puede utilizarse para delinear guías de aplicación de terapia preventiva intermitente en el nordeste de Colombia y para definir el uso futuro de los antifolatos en esquemas terapéuticos basados en artemisinina. Objetivo. Evaluar la frecuencia de mutaciones en dhps y dhfr, y caracterizar las poblaciones parasitarias usando msp-1, msp-2 y glurp, en muestras históricas obtenidas antes de la implementación en el país de la terapia basada en artemisinina. Métodos. Se llevó a cabo un estudio clínico controlado en voluntarios infectados con Plasmodium falciparum seleccionados aleatoriamente y provenientes del nordeste de Colombia (Turbo y Zaragoza). Se calculó una muestra de 25 sujetos por región. Se evaluó la eficacia al tratamiento con antifolatos. Los análisis moleculares incluyeron la obtención de genotipos de msp-1, msp-2 y glurp y el estado de los codones 16, 51, 59, 108 y 164 de dhfr, y 436, 437, 5540, 581 y 613 de dhps. Resultados. Se estudiaron 78 sujetos. Se detectó un número máximo de 4 genotipos con msp-1, msp-2 y glurp. Los codones 16, 59 y 164 del gen dhfr se encontraron en su forma silvestre, mientras que los codones 51 y 108 estaban mutados. En el gen dhps, la forma mutante (glicina) en el codón 437, se detectó en 85% el día 0, mientras que los codones 436, 540, 581 y 613 se encontraron silvestres. Conclusiones. Las poblaciones de P. falciparum son muy homogéneas en esta región de Colombia y las triple mutantes de dhfr y dhps Asn108, Ile51 and Gly437, predominaron en los aislamientos clínicos.