Presence of benznidazole conjugated metabolites in urine identified by β-glucuronidase treatment

Chagas disease is a serious public health problem in Latin America and, due to migration, in other non-endemic regions. Benznidazole (BNZ) is first choice drug in pediatric therapeutics. However, little is known regarding its metabolism in humans. The aim of the study was to isolate and identify products of human BZN metabolism in urine samples obtained from a pediatric Chagas patient and a healthy adult volunteer both treated with BZN. Urine samples were collected after dose of BNZ. Urine was treated with β-glucuronidase followed by an extraction procedure under two different pH conditions and a HPLC/UV and MS/MS identification of BZN and its metabolites. BZN (m/z 260.09847) was identified in all urine extracts. Peaks from each extracted chromatograms were selected for MS and MS/MS identification. Three compounds structurally related to BZN were identified: BZN-Na+ (m/z 283.08009), N-amine-BZN (m/z 230.12307) and N-hydroxi-amine-BZN (m/z 246.11702). BNZ-Na+ was identified in all extracts, but N-amine-BZN and N-hydroxi-amine-BZN were only observed in those extracts treated with β-glucuronidase. This is the first experimental report showing elimination of BZN N-reduced metabolites in urine. As they were released after treatment with β-glucuronidase it can be suggested that glucuronization plays a role in BNZ metabolism and renal elimination.

Application of quality by design approach in RP-HPLC method development for simultaneous estimation of saxagliptin and dapagliflozin in tablet dosage form

A simple, sensitive, precise, accurate and robust high performance liquid chromatographic method has been developed for simultaneous estimation of saxagliptin (SAXA) and dapagliflozin (DAPA) in pharmaceutical formulation. Design of experiments (DoE) was applied for multivariate optimization of the experimental conditions of RP-HPLC method. Risk assessment was performed to identify the critical method parameters. Three independent factors; mobile phase composition, flow rate and column temperature were used to design mathematical models. Central composite design (CCD) was used to study the response surface methodology and to study in depth the effects of these independent factors. Desirability function was used to simultaneously optimize the retention time and resolution of SAXA and DAPA. The optimized and predicted data from contour diagram consisted of acetonitrile and ortho phosphoric acid (0.1%) in the ratio of 50:50 respectively, at a flow rate of 0.98 ml/min and column temperature 31.4 °C. Using these optimum conditions baseline separation of both drugs with good resolution and run time of less than 6 min were achieved. The optimized assay conditions were validated according to ICH guidelines. Hence, the results clearly showed that Quality by design approach could be successfully applied to optimize RP-HPLC method for simultaneous estimation of SAXA and DAPA.

Validated analytical study of the effect of Lycopene on the pharmacokinetics of Paracetamol and Chlorzoxazone in rats

Lycopene was reported to influence some cytochrome P450 enzymes activity. The present study investigates the effect of lycopene on the pharmacokinetics of paracetamol and chlorzoxazone. Lycopene (20 mg/kg) was intra-peritoneally administered to two groups of rats for eight consecutive days and two other groups were given vehicle. On the eighth day, chlorzoxazone and paracetamol were separately intravenously administered to a lycopene group and a control group. Blood samples were collected at different time intervals, treated and analyzed using HPLC. The HPLC method used for paracetamol analysis was based on isocratic elution using a mobile phase consisting of water: methanol, (77:23 v/v) at a flow rate 1 mL min−1, Kromasil C18 column, and UV detection at 254 nm using caffeine as internal standard. About chlorzoxazone, separation was carried out using water: acetonitrile (60: 40, v/v) as the mobile phase at a flow rate 1 mL min−1, Inertsil ODS-3 C18 column, UV detection at 283 nm and esomeprazole as internal standard. Statistical analysis of the pharmacokinetic data using student t test showed a significant increase in AUC 0-t , AUC 0-Inf and t1/2 of paracetamol (P<0.05) and of chlorzoxazone (P<0.05) in the groups pretreated with lycopene (20 mg/kg), significant increase in the volume of distribution of paracetamol (P < 0.05), but no significant difference in that of chlorzoxazone. In other words, paracetamol and chlorzoxazone showed significant decrease (P < 0.05), respectively. These results demonstrate that treatment of rats with Lycopene (20mg/kg, ip) has a significant effect on the metabolic clearance and the pharmacokinetics of both drugs

Midazolam and haloperidol for palliative sedation: physicochemical stability and compatibility of parenteral admixtures

This study was done to determine the time while the binary admixtures with midazolam and haloperidol drugs are administered by perfusion to the patients in the clinical routine. Samples with different concentrations of both drugs were prepared following the usual clinical practice. Solvents used were 0.9 % sodium chloride solution and 5% dextrose, and viaflo plastic bags were used as the containers of the admixtures. Samples were not protected from light and were stored at 20 ºC or at 4 ºC. Compatibility and physicochemical stability were studied by visual inspection, turbidity measurement, pH determination and ultraviolet detection high performance liquid chromatography (UV-HPLC) was used to determine midazolam and haloperidol concentrations. The assay was validated following the FDA and EMA guidelines. Darunavir was used as internal standard (IS). For the studied admixtures, turbidity measurements and pH determinations showed little changes in function of the time. Haloperidol and midazolam concentrations determined by HPLC are within the acceptable range of drug concentrations, which are considered stable for four days in case of admixtures stored at 20 ºC and for seven days for refrigerated admixtures. Taking into account the microbiological risk matrix, the compatibility and the chemical and microbiological stability of the midazolam and haloperidol in the co-administered admixtures in viaflo plastic bags with 0.9 % sodium chloride solution and 5% dextrose can be set as 48 hours when samples are stored at 20 ºC and one week if they are refrigerated.

Validación del método analítico para el control de la calidad y estudio de estabilidad de ribavirina inyectable 100 mg/mL / Validation of a analytical method for quality control and stability study of 100 mg mL injectable Ribavirin

El inyectable de ribavirina 100 mg/mL se emplea en la práctica médica como antiviral de amplio espectro, combinado de forma efectiva con el interferón alfa-2 beta, contra una gran variedad de virus DNA y RNA. En este trabajo se desarrolló y validó un método analítico por cromatografía líquida de alta resolución, para el control de la calidad y los estudios de estabilidad del inyectable de ribavirina 100 mg/mL. El método se basó en la separación del principio activo a través una columna cromatográfica Lichrospher RP-18 (5 µm) (250 x 4 mm), con detección ultravioleta a 207 nm, para lo cual se empleó una fase móvil compuesta por una solución de dihidrógeno fosfato de potasio 0,01 M a pH 4,5, con una velocidad de flujo de 1,0 mL/min. La curva de calibración se realizó en el intervalo de 60 al 140 por ciento, donde fue lineal con un coeficiente de correlación igual a 0,9996; la prueba estadística para el intercepto y la pendiente se consideró no significativa. Se obtuvo un recobrado del 99,87 por ciento en el intervalo de concentraciones estudiados y las pruebas de Cochranï(G) y Studentïs (t) resultaron no significativas. El coeficiente de variación en el estudio de la repetibilidad fue igual a 0,79 por ciento para las 6 réplicas ensayadas, mientras que en los análisis de la precisión intermedia las pruebas de Fischer y Student fueron no significativas. El método analítico resultó lineal, preciso, específico y exacto en el intervalo de concentraciones estudiadas

The 100 mg/mL Ribavirin injectable is used in medical practice as a broad spectrum antiviral combined in an effective way with 2 â-á-Interferon against a great variety of ADN and ARN viruses. En present paper an analytical method by a high ressolution liquid chromatography to quality control and stability studies of 100l mg/ml Ribavirin injectable. Method was based on separation of active principle through a Lichrospher RP-18 (5 µm) (250 x 4 mm) chromatography column with UV detection at 207 nm using a mobile phase composed by a dihydrogen phosphate of 0,01 Ma pH 4,5 with a flow speed of 1,0 mL/min. Calibration curve was carried out in the 60-140 percent interval where it was linear with a correlation coefficient similar to 0,9996, statistical test for the interceptive and slope weren't significant. A recovery of 99,87 percent was achieved in the interval of study concentrations and the Cochran's and Students's tests (G). Variation coeficient in repetition study was similar to 0,79 percent for the six assayed replicas whereas in intermdiate accuracy analysis the Fischer's and Student's tests weren't sinificant. Nalytical method was linear, accuracy, specific and exact in the interval of study concentrations

Study on the Identification and Potency Determination of L-asparaginases from 2 Kinds of Strain / 中国药房

OBJECTIVE: To discuss the identification method of L-asparaginases prepared from E.coliASI.357 and Erwinia carotovora and the optimal enzymatic reaction conditions of potency determination.METHODS: HPLC method and isoelectric focusing electrophoresis were applied for the identification of L-asparaginase from 2 kinds of strain.The effects of category of buffer solution and pH value on enzymatic reaction of potency determination of L-asparaginase were investigated.RESULTS: HPLC chromatogram of L-asparaginases from E.coli ASI.357 was different from that from Erwinia carotovora.The retention time of the peaks were 11.0 min and 11.8 min.The isoelectric point (PI) of L-asparaginase produced from E.coliASI.357 was within 4.65～5.1 and that produced from Erwinia carotovora was within 7.1～8.20.The optimal enzymatic reaction conditions of potency determination of L-asparaginase produced from E.coliASI.35 were Tris-HCl (pH=9.0) as buffer and that produced from Erwinia carotovora was 0.2 mol?L-1 phosphate (pH=8.0) as buffer.CONCLUSION: The isoelectric point (PI) of L-asparaginases produced from 2 kinds of strain is different from each other as well as their optimal enzymatic reaction conditions of potency determination.The L-asparaginases from 2 kinds of strains should be controlled as 2 different categories.

Study on Absorption of Troxerutin onto Infusion Systems of Polyvinyl Chloride / 中国药房

0.05).CONCLUSION:In the study,PVC infusion systems have no adsorption effect on troxerutin.