Development of a rapid HPLC method for quantification of the copper tetramibi tetraflourborate in a kit for preparation of Technetium 99 m Sestamibi Injection.

Radiopharmaceuticals are radioactive compounds that can be used for diagnostic and therapeutic purposes. Technetium (99mTc) Sestamibi is the most commercialized radiopharmaceutical in the world. It includes a coordination complex consisting of the radioisotope 99 m technetium bound to six copper tetramibi tetrafluorborate ligands, and is mainly used to image the myocardium via scintigraphy. As radiopharmaceuticals are regarded as drugs, they are subject to the same regulations; therefore, the objective of this study was to develop a quantification method for the active pharmaceutical ingredient before their complexation with the radioisotope by employing high-performance liquid chromatography (HPLC) methodology. A simple and efficient method (retention time = 2.5 min) was developed and validated for copper tetramibi tetrafluorborate in the final product using a buffer and organic solvent mixtures (ACN-methanol-ammonium sulfate buffer) and a C18 column. The analytical protocol was fast, taking around 30 min until evaluation of results. The validation parameters were evaluated with satisfactory results: in terms of linearity r > 0.99 (160-240 µg/mL) and no deviation was observed. The RSD of precision was <5%, and an average recovery of 99% was observed for accuracy. The proposed method was thus considered adequate for routine analysis in the pharmaceutical industries.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES: Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS: A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton's medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS: Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton's medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION: The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv.

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease's causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli's nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG(#), ΔS(#), ΔH(#)) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.

Evaluation of the exhaled carbon monoxide levels in smokers with COPD.

OBJECTIVE: To measure exhaled carbon monoxide (COex) levels in smokers with and without COPD. METHODS: Smokers treated at outpatient clinics of São Lucas Hospital in the city of Porto Alegre, Brazil, between September of 2007 and March of 2009 were invited to participate in this study. The participants completed a questionnaire regarding demographic and epidemiologic characteristics and were submitted to spirometry, as well as to determination of COex and urinary cotinine levels. The participants were divided into two groups: those with COPD and those without COPD. RESULTS: The study involved 294 smokers, of whom 174 (59.18%) had been diagnosed with COPD. All of the participants presented with urinary cotinine levels > 50 ng/mL. Smokers with COPD presented significantly higher median values for age and pack-years than did those without COPD (p < 0.001 and p = 0.026, respectively). No other statistically significant differences were found. When adjusted for gender, age at smoking onset, number of cigarettes/day and urinary cotinine level, the mean values of COex were higher, but not statistically so, in the COPD group than in the non-COPD group (17.8 +/- 0.6 ppm and 16.6 +/- 0.7 ppm, respectively; p = 0.200). The differences remained nonsignificant when plotted logarithmically. A wide dispersion of COex values was found when the participants were classified by FEV1 level (r = -0.06; p = 0.53) or by Global Initiative for Chronic Obstructive Lung Disease classification (r = 0.08; p = 0.34). The proportions of false-negative results for smoking were 18.4% and 6.7%, respectively, in the COPD and non-COPD groups (p = 0.007). CONCLUSIONS: Since COex values did not differ significantly between smokers with COPD and those without, there seem to be no major contraindications to their use in smokers with COPD.

Avaliação da concentração de monóxido de carbono no ar exalado em tabagistas com DPOC / Evaluation of the exhaled carbon monoxide levels in smokers with COPD

OBJETIVO: Medir os níveis de monóxido de carbono no ar exalado (COex) em tabagistas com e sem DPOC. MÉTODOS: Tabagistas frequentadores dos ambulatórios do Hospital São Lucas em Porto Alegre (RS) entre setembro de 2007 e março de 2009 foram convidados a participar do estudo. Os participantes responderam a um questionário com características demográficas e epidemiológicas e realizaram espirometria, medição de cotinina urinária e de COex. Os participantes foram agrupados conforme a presença de DPOC. RESULTADOS: Foram incluídos 294 tabagistas, 174 (59,18 por cento) diagnosticados com DPOC. Todos os participantes apresentavam níveis de cotinina urinária > 50 ng/mL. Os fumantes com DPOC apresentaram medianas significativamente superiores as do grupo sem DPOC para as variáveis idade e maços-ano (p < 0,001 e p = 0,026, respectivamente). Não houve diferença significativa nas demais variáveis. Quando ajustados para sexo, início do tabagismo, cigarros/dia e cotinina urinária, os valores médios de COex foram mais altos no grupo DPOC que no grupo sem DPOC, mas sem significância estatística (17,8 ± 0,6 ppm e 16,6 ± 0,7 ppm, respectivamente; p = 0,200). As diferenças permaneceram não significativas quando o método de base logarítmica foi usado. Uma ampla dispersão dos valores de COex foi encontrada quando os participantes foram classificados conforme os valores de VEF1 (r = -0,06; p = 0,53) ou o sistema de classificação de Global Initiative for Chronic Obstructive Lung Disease (r = 0,08; p = 0,34). As proporções de resultados falso-negativos para tabagismo foram de 18,4 por cento e 6,7 por cento, respectivamente, nos grupos com e sem DPOC (p = 0,007). CONCLUÕES: Esse estudo mostrou que os valores de COex não apresentaram diferenças significativas em fumantes com ou sem DPOC. Desse modo, parece não haver nenhuma restrição relevante para a sua aplicabilidade em fumantes com DPOC.

OBJECTIVE: To measure exhaled carbon monoxide (COex) levels in smokers with and without COPD. METHODS: Smokers treated at outpatient clinics of São Lucas Hospital in the city of Porto Alegre, Brazil, between September of 2007 and March of 2009 were invited to participate in this study. The participants completed a questionnaire regarding demographic and epidemiologic characteristics and were submitted to spirometry, as well as to determination of COex and urinary cotinine levels. The participants were divided into two groups: those with COPD and those without COPD. RESULTS: The study involved 294 smokers, of whom 174 (59.18 percent) had been diagnosed with COPD. All of the participants presented with urinary cotinine levels > 50 ng/mL. Smokers with COPD presented significantly higher median values for age and pack-years than did those without COPD (p < 0.001 and p = 0.026, respectively). No other statistically significant differences were found. When adjusted for gender, age at smoking onset, number of cigarettes/day and urinary cotinine level, the mean values of COex were higher, but not statistically so, in the COPD group than in the non-COPD group (17.8 ± 0.6 ppm and 16.6 ± 0.7 ppm, respectively; p = 0.200). The differences remained nonsignificant when plotted logarithmically. A wide dispersion of COex values was found when the participants were classified by FEV1 level (r = -0.06; p = 0.53) or by Global Initiative for Chronic Obstructive Lung Disease classification (r = 0.08; p = 0.34). The proportions of false-negative results for smoking were 18.4 percent and 6.7 percent, respectively, in the COPD and non-COPD groups (p = 0.007). CONCLUSIONS: Since COex values did not differ significantly between smokers with COPD and those without, there seem to be no major contraindications to their use in smokers with COPD.

Cotinine as a biomarker of tobacco exposure: development of a HPLC method and comparison of matrices.

Tobacco dependence reaches one-third of the world population, and is the second leading cause of death around the world. Cotinine, a major metabolite of nicotine, is the most appropriate parameter to evaluate tobacco exposure and smoking status due to its higher stability and half-life when compared to nicotine. The procedure involves liquid-liquid extraction, separation on a RP column (Zorbax XDB C(8)), isocratic pump (0.5 mL/min of water-methanol-sodium acetate (0.1 M)-ACN (50:15:25:10, v/v/v/v), 1.0 mL of citric acid (0.034 M) and 5.0 mL of triethylamine for each liter) and HPLC-UV detection (261 nm). The analytical procedure proved to be sensitive, selective, precise, accurate and linear (r>0.99) in the range of 5-500.0 ng/mL for cotinine. 2-Phenylimidazole was used as the internal standard. The LOD was 0.18 ng/mL and the LOQ was 5.0 ng/mL. All samples from smoking volunteers were collected simultaneously to establish a comparison between serum, plasma, and urine. The urinary cotinine levels were normalized by the creatinine and urine density. A significant correlation was found (p<0.01) between all matrices. Results indicate that the urine normalization by creatinine or density is unnecessary. This method is considered reliable for determining cotinine in serum and plasma of smokers and in environmental tobacco smoke exposure.

A high-performance liquid chromatography method for the determination of carbamazepine and carbamazepine-10,11-epoxide and its comparison with chemiluminescent immunoassay.

BACKGROUND: Carbamazepine is a first-choice antiepileptic drug for the treatment of simple and complex partial seizures. The use of an established therapeutic range for carbamazepine concentration is limited by the presence of carbamazepine-10,11-epoxide, its active metabolite that significantly contributes to the efficacy and toxicity and is not routinely measured and accounted for. This article describes the development of a HPLC method for determination of carbamazepine and carbamazepine-10,11-epoxide in serum, and compares it with chemiluminescence immunoassay to evaluate the importance of considering the active metabolite in therapeutic strategies. METHODS: The procedure involves protein precipitation, separation on a reverse-phase column and ultraviolet detection. The analytical procedure proved to be sensitive, selective, precise, accurate and linear (regression coefficients >0.999) in the range of 0.5-25.0 microg/mL and 0.1-10.0 microg/mL for quantification of carbamazepine and carbamazepine-10,11-epoxide, respectively. For the comparison between methods, serum samples of 75 patients using the medication were evaluated. RESULTS: The Pearson correlation coefficient showed that the carbamazepine concentrations measured by HPLC are significantly higher than those obtained by immunoassay (mean difference of 1.07 microg/mL, 95% limits of agreement from -0.65 to 2.80 microg/mL). CONCLUSIONS: This difference may be decisive for the therapy. In some cases, this may affect the individual dosage adjustment and subsequent treatment.

Validação do método para determinação de cotinina em urina por cromatografia líquida de alta eficiência / Validation of a high-performance liquid chromatography method for the determination of cotinine in urine

Tobacco dependence reaches nearly one third of the world population, and is the second leading cause of deth around the world. Cotinine, a major metabolite of nicotine, is conssidered the most appropriate parameter to evaluate tobacco exposure HLPC (High Performance Liquid Chromatography) was choses to analyze cotinine concentrations in urine. Aiming at validating this method of analysis, urine samples with added cotinine were submitted to liquid-liquid extractions using2-phenylimidazole as the internal standard. After evaporation, residue was injected in HLPC with ultraviolet detection and C8 column. The following parameters were stabilishe: linearity, precision, detection and qualification limit, accuracy, ans stability. The calibration curve, determined ian a interval of 10ng/mL and 1000ng/mL, presented linearity (R2 = 0,09979). The method limit of detection was of 5ng/mL, and the limit of quantification was of 10ng/mL. In 50, 500, and 1000ng/mL concentrations, the precision was of 11.2; 12.3, and 8.1 per cent, accuracy found was of 107.0; 101.7, and 97.0 per cent, and loss during the stability test was of 8.1...