Triagem biológica de inibidores de sirtuína 2 (TcSir2rp1) candidatos a antichagásicos / Biological screening of sirtuin 2 inhibitors (TcSir2rp1) antichagasic candidates

A doença de Chagas é causada pelo Trypanosoma cruzi, e atualmente, acomete entre 6 a 7 milhões de pessoas em todo o mundo. A quimioterapia disponível para seu o tratamento se baseia apenas em dois fármacos, nifurtimox e benznidazol, com mais de 50 anos de descoberto. Estes fármacos apresentam eficácia limitada, pois são pouco efetivos na fase crônica e apresentam alta toxicidade, resultando em efeitos adversos graves. Esse panorama mostra a necessidade de novas abordagens terapêuticas contra essa doença. Nesse sentido, a inibição de vias bioquímicas essencias para o parasita se mostram como uma boa sugestão para identificação de compostos promissores candidatos a novos agentes quimioterápicos. A sirtuína 2 (Sir2) são enzimas reguladoras que participam de mecanismos epigenéticos em tripanossomatídeos, e no T. cruzi possuem um papel fundamental em todos os seus estágios evolutivos, devido a este fato, se apresentam como um alvo promissor na busca por novos fármacos contra a doença de Chagas. Neste sentido propomos a busca de inibidores da Sir2 proteína 1 do T. cruzi (TcSir2rp1) que é geneticamente validada como alvo farmacológico, por meio da estratégia de triagem biológica. Realizou-se a expressão da enzima recombinante por biologia molecular em um sistema de transformação utilizando cepa de Escherichia coli Artic Express (DE3). Foi feita a purificação e a confirmação da obtenção da proteína recombinante se deu por gel SDS-PAGE. Após a obtenção da enzima os parâmetros cinéticos foram determinados por experimentos de fluorimetria. A triagem foi realizada para um conjunto de 82 compostos, previamente sintetizados pelo nosso grupo de pesquisa, como inibidores da TcSir2p1 em dose única de 100 µM. Os ensaios foram realizados em triplicata e em experimentos independentes. Dentre os 82 compostos testados, 20 apresentaram inibições maior que 50% contra a enzima TcSir2rp1, na dose de 100 µM. Dentre estes, se destacaram 3 compostos derivados de chalconas, para os quais foi determinada a potência. O composto 1 foi o que mais potente, apresentando valor de IC50 de 11,65 µM, já os compostos 3 e 5 foram menos potentes (IC50= 38,50 µM e 19,85 µM, respectivamente). Diante dos resultados obtidos, pode-se concluir que a estratégia de triagem biológica é promissora na identificação de inibidores da TcSir2p1 candidatos a agentes anti- T. cruzi

Chagas disease is caused by Trypanosoma cruzi, and currently affects 6 to 7 million people worldwide. The chemotherapy available for its treatment is based on only two drugs, nifurtimox and benznidazole, with more than 50 years of discovery. These drugs have limited efficacy, as they are ineffective in the chronic phase and have high toxicity, resulting in serious adverse effects. This panorama shows the need for new therapeutic approaches against this disease. In this sense, the inhibition of essential biochemical pathways for the parasite proves to be a good suggestion for the identification of promising compounds candidates for new chemotherapeutic agents. Sirtuin 2 (Sir2) are regulatory enzymes that participate in epigenetic mechanisms in trypanosomatids, and in T. cruzi they have a fundamental role in all their evolutionary stages, due to this fact, they present themselves as a promising target in the search for new drugs against Chagas disease. In this sense, we propose the search for inhibitors of Sir2 protein 1 of T. cruzi (TcSir2rp1) which is genetically validated as a pharmacological target, through the biological screening strategy. The expression of the recombinant enzyme was performed by molecular biology in a transformation system using strain of Escherichia coli Artic Express (DE3). Purification was performed and confirmation of obtaining the recombinant protein was performed by SDS-PAGE gel. After obtaining the enzyme, the kinetic parameters were determined by fluorimetry experiments. Screening was performed for a set of 82 compounds, previously synthesized by our research group, as TcSir2p1 inhibitors in a single dose of 100 µM. Assays were performed in triplicate and in independent experiments. Among the 82 compounds tested, 20 showed inhibitions greater than 50% against the enzyme TcSir2rp1, at a dose of 100 µM. Among these, 3 compounds derived from chalcones stood out, for which the potency was determined. Compound 1 was the most potent, with an IC50 value of 11.65 µM, while compounds 3 and 5 were less potent (IC50= 38.50 µM and 19.88 µM, respectively). In view of the results obtained, it can be concluded that the biological screening strategy is promising in the identification of TcSir2p1 inhibitors candidates for anti-T. cruzi agents

Autofluorescencia: un método innovador en la identificación intraoperatoria de adenomas paratiroideos / Autofluorescence: an innovative method in the intraoperative identification of parathyroid adenomas

RESUMEN Introducción El tratamiento definitivo del hiperparatiroidismo primario es la resección quirúrgica de la glándula paratiroidea anómala. Su identificación resulta un desafío aun para cirujanos expertos. Hasta el momento no se han descripto métodos inocuos y efectivos para la identificación intraoperatoria de las glándulas. Tenemos como objetivo reportar la experiencia del uso de autofluorescencia en la identificación de las glándulas paratiroideas. Método Se incluyeron pacientes con hiperparatiroidismo primario evaluados preoperatoriamente con laboratorio, ecografía cervical y centellografía con Tc-99 MIBI. Durante el acto operatorio se utilizó un método de autofluorescencia (VINFLUO-P) para identificar las glándulas paratiroides (GP). Se analizó la intensidad lumínica de las (GP) normales y anómalas (AP) y distintas covariables. Se dosó PTH ultra rápida post resección del AP y se evaluó la histopatología de la pieza intraoperatoriamente. Resultados Se incluyeron 59 pacientes. La ecografía preoperatoria predijo la ubicación correcta en el 68% y el centellograma Tc-99 MIBI el 75% de los AP. La localización más frecuente fue inferior derecha (29%). El VINFLUO-P facilitó la visualización de las GP y los AP en el 100% de los pacientes con un aumento del 27% respecto a la luz blanca. Se evidenció un descenso postoperatorio de PTH del 76,44% y de la calcemia en 1,8 mg/dl. La intensidad de la luz reflejada por los AP fue mayor que la de las GP normales (p <0,001). Se observó una relación lineal entre PTH e intensidad lumínica de AP. (CC = 0,448; p = 0,045). El patrón arquitectural sólido de los AP evidenció una asociación negativa (CC = -0,4709 p = 0,03). Conclusión La utilización del VINFLUO-P demostró ser efectivo para la identificación de las GP normales y patológicas. Las glándulas anómalas resultaron con mayor fluorescencia que los tejidos normales.

ABSTRACT Introduction The treatment of primary hyperparathyroidism consists on the resection of the abnormal parathyroid gland (PG). Identification of PGs is challenging even for expert surgeons. Currently, there are no effective and harmless methods for intraoperative identification of PGs. The aim of this study is to report our experience with the identification of PGs using autofluorescence. Materials and methods Patients with diagnosis of primary hyperparathyroidism were included in the study. Patients were preoperatively worked up with labs [parathyroid hormone (PTH), serum calcium], neck ultrasound (US) and Technetium (99mTc) sestamibi. The parathyroid gland Intraoperative fluorescent visualization (PG-IFV) method was used during the surgery to identify PGs. The fluorescent intensity ratio of normal PGs and parathyroid adenomas (PA) was analyzed and correlated to different variables. All patients underwent a post-resection rapid PTH analysis and frozen section. Results Fifty-nine patients were included in the study. The US accurately predicted the location of the PA in 68% of the cases, while 99mTc sestamibi was accurate in 75% of the cases. The most frequently reported localization of the adenoma was right inferior (29%). PG-IFV facilitated the visualization of the PGs in 100% of the cases, with a 27% increase in the visualization of the PGs when compared to white light. The postoperative PTH decreased 76.4% and the calcium 1.8 mg/dl. The fluorescent intensity ratio of the PAs was significantly higher than normal PGs (44.4 vs 27.2, p <0.001). There was positive correlation between the PTH and the fluorescent intensity ratio of the PAs [Spearman's correlation coefficient (SCC) = 0.448; p = 0.045]. The solid histoarchitectural pattern of the PAs presented a negative correlation with fluorescent intensity ratio (SCC = -0.4709, p = 0.03). Conclusion The use of PG-IFV is an effective method for intraoperative identification of normal and abnormal PGs. The fluorescent intensity ratio of abnormal PGs was significantly higher than normal PGs.

Serum Fluorescent Advanced Glycation End (F-AGE) products in gestational diabetes patients

ABSTRACT Objectives Advanced glycation end products (AGEs) are involved in the pathogenesis and complications of diabetes mellitus (DM). Gestational DM (GDM) is characterized by increased glycemia and oxidative stress, which are factors associated with high serum AGE concentrations. The aim of this study was to evaluate the utility of a serum fluorescence AGE (F-AGE) method as a screening tool for gestational diabetes. Subjects and methods Serum samples from 225 GDM patients and 217 healthy pregnant women (healthy controls) were diluted 50-fold in phosphate-buffered saline, and the AGEs were estimated by fluorometric analysis (λEx 350 nm/ λEm 440 nm). Results No significant (P > 0.05) differences in AGE concentrations, expressed in Arbitrary Units (UA/mL × 104), were observed in the women with GDM or in the healthy controls. Furthermore, F-AGE concentrations did not change significantly during the pregnancy (12-32 weeks of gestation). Only the GDM group had a positive correlation (r = 0.421; P < 0.001) between F-AGEs and serum creatinine concentrations. Conclusion It was not possible to distinguish women with gestational diabetes from the healthy controls on the basis of serum F-AGE concentrations.

Desenvolvimento de uma metodologia semi-automatizada para busca de novas drogas utilizando Leishmania amazonensis fluorescente

As Leishmanioses são doenças negligenciadas. O tratamento dos pacientes é a principal medida de controle, porém a quimioterapia das Leishmanioses apresenta dificuldades incluindo: toxicidade dos medicamentos disponíveis, via de administração parenteral e resistência natural de algumas cepas. O método de teste de drogas clássico in vitro baseado na contgem microscópica de macrófagos infectados apresenta limitações por ser laborioso, não automatizado e sujeito a variações do observador. Deste modo, a busca por novos métodos de triagem de drogas faz-se necessário. O estabelecimento de métodos semi-automatizados contribuiria para aumentar a eficiência da busca de novas drogas contra Leishmania. Neste trabalho, a cepa de Leishmania amazonensis (PH8) foi transfectada com a proteína vermelha fluorescente (RFP). Os parasitos transfectados foram avaliados segundo parâmetros celulares e de susceptibilidade aos fármacos leishmanicidas tradicionais e derivados do propranolol. Os parasitos selvagens (WT) e transfectados (RFP) foram analisados pela Citometria de Fluxo e Microscopia de Fluorescência sendo facilmente discriminados por estas técnicas. Em geral,não foram observadas diferenças na susceptibilidade entre as cepas WT e RFP frente às moléculas testadas.

Os parasitos RFPs foram submetidos ao teste de drogas com posterior leitura no fluorímetro para a padronização do método fluorimétrico. O método fluorimétrico se mostrou bastante reprodutível em relação ao método clássico. Entretanto, durante a padronização do método, mudanças na concentração das drogas foram necessárias bem como a determinação de um ponto de corte nos valores de IC50. Este trabalho também avaliou a atividade leishmanicida dos derivados das poliaminas e complexos metálicos de lapachol utilizando-se o método clássico. Não só os derivados do propranolol, mas também os das poliaminas e lapachol se mostraram tóxicos para células de Hepatoma humano (HepG2). Este trabalho possibilitou pela primeira vez a utilização de parasitos RFPs como modelo de um teste de drogas semi-automatizado

Desenvolvimento de uma metodologia semi-automatizada para busca de novas drogas utilizando Leishmania amazonensis fluorescente / Development of a methodology for semi-automated search for new drugs using fluorescent Leishmania amazonensis

As Leishmanioses são doenças negligenciadas. O tratamento dos pacientes é a principal medida de controle, porém a quimioterapia das Leishmanioses apresenta dificuldades incluindo: toxicidade dos medicamentos disponíveis, via de administração parenteral e resistência natural de algumas cepas. O método de teste de drogas clássico in vitro baseado na contgem microscópica de macrófagos infectados apresenta limitações por ser laborioso, não automatizado e sujeito a variações do observador. Deste modo, a busca por novos métodos de triagem de drogas faz-se necessário. O estabelecimento de métodos semi-automatizados contribuiria para aumentar a eficiência da busca de novas drogas contra Leishmania. Neste trabalho, a cepa de Leishmania amazonensis (PH8) foi transfectada com a proteína vermelha fluorescente (RFP). Os parasitos transfectados foram avaliados segundo parâmetros celulares e de susceptibilidade aos fármacos leishmanicidas tradicionais e derivados do propranolol. Os parasitos selvagens (WT) e transfectados (RFP) foram analisados pela Citometria de Fluxo e Microscopia de Fluorescência sendo facilmente discriminados por estas técnicas. Em geral,não foram observadas diferenças na susceptibilidade entre as cepas WT e RFP frente às moléculas testadas. Os parasitos RFPs foram submetidos ao teste de drogas com posterior leitura no fluorímetro para a padronização do método fluorimétrico. O método fluorimétrico se mostrou bastante reprodutível em relação ao método clássico. Entretanto, durante a padronização do método, mudanças na concentração das drogas foram necessárias bem como a determinação de um ponto de corte nos valores de IC50. Este trabalho também avaliou a atividade leishmanicida dos derivados das poliaminas e complexos metálicos de lapachol utilizando-se o método clássico. Não só os derivados do propranolol, mas também os das poliaminas e lapachol se mostraram tóxicos para células de Hepatoma humano (HepG2). Este trabalho possibilitou pela primeira vez a utilização de parasitos RFPs como modelo de um teste de drogas semi-automatizado.

Fatty acid and phospholipase A2 plasma levels in children with autism / Níveis plasmáticos de ácidos graxos e fosfolipase A2 em crianças com autismo

OBJECTIVE: To evaluate fatty acid plasma levels, phospholipase A2 activity, and the developmental profiles of children with autism vs. control subjects. METHODS: Twenty four children with autism underwent laboratory analysis for fatty acid quantification using gas chromatography and PLA2 activity determination by fluorometric assay. RESULTS: No correlation was observed between the developmental quotient and fatty acid plasma levels. Phospholipase A2 activity was significantly higher among autistic children compared with controls. CONCLUSION: The study did not show a correlation between fatty acid and phospholipase A2 plasma levels and the developmental profile of children with autism

OBJETIVO: Avaliar os níveis plasmáticos de ácidos graxos, a atividade da fosfolipase A2 e o perfil de desenvolvimento de crianças com autismo versus controles. MÉTODOS: Vinte e quatro crianças com autismo foram submetidas a exames laboratoriais para quantificação plasmática de ácidos graxos por cromatografia gasosa e para determinação da atividade de fosfolipase A2 por ensaio fluorimétrico. RESULTADOS: Nenhuma correlação foi observada entre o coeficiente de desenvolvimento e os níveis plasmáticos dos ácidos graxos quantificados. A atividade da fosfolipase A2 foi significativamente maior no grupo de crianças com autismo quando comparado ao grupo controle. CONCLUSÃO: O estudo não demonstrou correlação entre os níveis plasmáticos de ácidos graxos e fosfolipase A2 e o perfil de desenvolvimento de crianças com autismo

Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.

Spectrophotometric and spectrofluorimetric determination of some selective serotonin reuptake inhibitors [SSRIs] through ternary complex formation with eosin and copper [II] in pharmaceuticals

Two simple, and sensitive, spectrophotometric and spectrofluorimetric procedures have been developed and validated for analysis of selective serotonin reuptake inhibitors [SSRIs] namely, fluoxetine hydrochloride [FX], fluvoxamine maleate [FV] and sertraline hydrochloride [SE]. Both methods were based on the formation of ternary complex between the cited drugs, eosin and copper sulphate, Spectrophotometrically, the complex was estimated by two procedures, the first procedure depends on the extraction of the ternary complex with chloroform. The second spectrophotometric one depends on the direct measurement of the complex after addition of sodium lauryl sulphat. The ternary complexes showed an absorption maximum at 530 nm for the three cited drugs. Different variables and parameters affecting the reactions were studied and optimized. The formed complexes obey Beer's law in concentration range 1-18, 1-16, 0.25-10 micro gml[-1] using the extractive method and 0.4-12, 0.5-14, 0.1-8 micro gml-1 using surfactant with good correlation coefficients. A fluorescence quenching method for the determination of the [SSRIs] through the formed ternary complexes was also investigated to enhance the sensitivity of the analysis. The formed fluorophores were measured at lambda E[x] 310 nm and lambda E[m]510nm for the three drugs. Regression analysis showed good correlation coefficients [0.9996-0.9999] over the concentration ranges 0.1-25, 0.1-15 and 0.1-12 micro gml-1 for FX, FV and SE, respectively. The proposed methods were validated and successfully applied to the analysis of the three drugs in drug substances and drug products with good accuracy. No interference was observed from common pharmaceutical excipients. The results were favorably in good agreement with those obtained by official methods

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37°C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 æM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 æM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 æM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

A micromethod for quantitation of debrisoquine and 4-hydroxydebrisoquine in urine by liquid chromatography

We describe a new simple, selective and sensitive micromethod based on HPLC and fluorescence detection to measure debrisoquine (D) and 4-hydroxydebrisoquine (4-OHD) in urine for the investigation of xenobiotic metabolism by debrisoquine hydroxylase (CYP2D6). Four hundred µl of urine was required for the analysis of D and 4-OHD. Peaks were eluted at 8.3 min (4-OHD), 14.0 min (D) and 16.6 min for the internal standard, metoprolol (20 µg/ml). The 5-µm CN-reverse-phase column (Shimpack, 250 x 4.6 mm) was eluted with a mobile phase consisting of 0.25 M acetate buffer, pH 5.0, and acetonitrile (9:1, v/v) at 0.7 ml/min with detection at lexcitation = 210 nm and lemission = 290 nm. The method, validated on the basis of measurements of spiked urine, presented 3 ng/ml (D) and 6 ng/ml (4-OHD) sensitivity, 390-6240 ng/ml (D) and 750-12000 ng/ml (4-OHD) linearity, and 5.7/8.2 percent (D) and 5.3/8.2 percent (4-OHD) intra/interassay precision. The method was validated using urine of a healthy Caucasian volunteer who received one 10-mg tablet of Declinax®, po, in the morning after an overnight fast. Urine samples (diuresis of 4 or 6 h) were collected from zero to 24 h. The urinary excretion of D and 4-OHD, Fel (0-24 h), i.e., fraction of dose administered and excreted into urine, was 6.4 percent and 31.9 percent, respectively. The hydroxylation capacity index reported as metabolic ratio was 0.18 (D/4-OHD) for the person investigated and can be compared to reference limits of < 12.5 for poor metabolizers (PM) and < 12.5 for extensive metabolizers (EM). In parallel, the recovery ratio (RR), another hydroxylation capacity index, was 0.85 (4-OHD: SD + 4-OHD) versus reference limits of RR < 0.12 for PM and RR > 0.12 for EM. The healthy volunteer was considered to be an extensive metabolizer on the basis of the debrisoquine test.

A simple, rapid fluorometric assay for the determination of glucose 6-phosphate dehydrogenase activity in dried blood spot specimens.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy of human beings and is the most common cause of jaundice and acute hemolytic anemia in South East Asia. The deficiency causes acute hemolytic anemia following ingestion of 6-amino quinoline antimalarials, phenacetin, and other substances. The rapid identification of infants or patients with this deficiency would help to prevent their exposure to these substances and subsequent risk to health. The assay is relatively simple. A 3mm punch from a dried blood spot sample is placed in a well of a black fluorescent microtiter plate containing calibrators and controls in duplicate. 100 microl of reagent is added and the sample is allowed to react for 30 minutes at ambient temperature after which 200 microl of stop reagent is added. The plate may be read immediately or up to one hour in a fluorescent reader (ex 355 nm: em 460 nm). Glutathione. ascorbate and bilirubin do not affect the assay. hemoglobin does quench the fluorescence by about 1.1 fluorescence units/g/dHb. This would not cause any false negatives and deficients would not be missed. G6PD activity in whole blood normal samples was examined at -20, 6 and 37 degrees C over 14 days. The samples lost about 20% activity after 48 hours and 31% by the end of 14 days. The samples stored at -20 degrees C and 6 degrees C remained relatively stable over this period. In a preliminary study eight diagnosed G6PD deficient samples had a mean value of 2.0 U/gHb (range 0.8 to 4.4) and fell within 3 SD units of the mean. Forty one normal samples had a mean of 6.6 micromol/min/gHb. Only one sample with a low hemoglobin level fell outside of 3 SD units of the mean. The Wallac assay was compared to the Sigma G6PD assay and although the values appeared lower at normal levels, the deficient samples compared well.

Fluorometric determination of amiodarone. amlodipine and propafenone

Three fluorimetric methods have been developed for the determination of some cardiovascular drugs. Amiodarone hydrochloride in 0.1 N sulphuric acid showed native fluorescence with excitation at 270 nm and emission at 540 nm. Methanolic solution of amlodipine besylate exhibits maximum excitation and emission at 365 nm and 450 nm, respectively. Propafenone hydrochloride forms fluorescent ion-pair with eosine at pH 5, which is extractable in chloroform, the excitation and emission at 318 nm and 550 nm, respectively. Linearity is obtained upon determining authentic samples in concentration range of 40 - 200 mug/ml, 0.4-2.8 mug/ml and 7-24.5 mug/ml for amiodarone, amlodipine and propafenone, respectively. The precision of the proposed method is checked by analyzing different samples of bulk powder and mean percentage recoveries are 100.26 +/- 1, 99.83 +/- 1.03 and 100.2 +/- 0.91 for amiodarone, amlodipine and propafenone, respectively. The pharmaceutical formulations are also estimated applying the proposed fluorimetric methods using st and ard addition technique, showing accurate results having great agreement with those of the reference methods

Spectrofluorometric determination of the two antilipemic drugs lovastatin and simvastatin in spiked human plasma and in dosage forms

A new spectrofluorimetric method for the determination of the two antilipemic drugs, lovastatin and simvastatin, was described. The method is based on heating drug solution with 2-aminoethane sulfonic acid [taurine] 0.025% w/v solution in phosphate-borate buffer of pH 7.4 at 70C for 30 minutes. The resulting reaction product exhibits strong fluorescence at 388 nm after excitation at 318 nm. Different assay parameters have been optimized to achieve maximum sensitivity and reliability of the method for the determination of the 2 drugs in spiked human plasma as well as in their dosage forms. The intensity of the resulting fluorescence showed linear relation with concentrations of both drugs in the range of 0.2 to 0.8 mug/L. The mean percentage recoveries of lovastatin and simvastatin were 99.851 SD +/- 2.101 and 99.806 SD +/- 1.805, respectively, in case of six concentrations of human plasma. The limit of detection was found to be 0.2 mug/L. The method is recommended for drug monitoring in biological fluids. The method was successfully applied for the determination of lovastatin and simvastatin in dosage forms. The results were in agreement with those of some other reported methods

Utilización de un micrométodo sencillo para la determinación de protoporfirina eritrocitaria libre por fluorimetría / Use of a simple micromethod for the determination of free erythrocyte protoporphyrin by fluorimetry

En 80 muestras de sangre heparinizada, procedentes de 40 sujetos sanos y de 40 que presentaban deficiencia de hierro, se determinó la concentración de protoporfirina eritrocitaria libre (PEL) por el micrométodo fluorimétrico descrito por Piomelli y por la técnica espectrofotométrica de Heller empleada en nuestro laboratorio. No se encontró diferencia significativa entre los valores obtenidos por los 2 métodos al aplicar la prueba t de Student para series apareadas (t = 0,90, p > 0,05) y se obtuvo un coeficiente de correlación de 0,99 (p < 0,001) al realizar el análisis de regresión lineal. El micrométodo fluorimétrico mostró un coeficiente de variación del 2,7