Determination of rifampicin in human plasma by high-performance liquid chromatography coupled with ultraviolet detection after automatized solid-liquid extraction.

A precise and accurate high-performance liquid chromatography (HPLC) quantification method of rifampicin in human plasma was developed and validated using ultraviolet detection after an automatized solid-phase extraction. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision, accuracy, lower limit of quantification and stability. Chromatographic separation was performed on a Chromolith RP8 column using a mixture of 0.05 m acetate buffer pH 5.7-acetonitrile (35:65, v/v) as mobile phase. The compounds were detected at a wavelength of 335 nm with a lower limit of quantification of 0.05 mg/L in human plasma. Retention times for rifampicin and 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline used as internal standard were respectively 3.77 and 4.81 min. This robust and exact method was successfully applied in routine for therapeutic drug monitoring in patients treated with rifampicin.

Biophysical activity of animal-derived exogenous surfactants mixed with rifampicin.

Exogenous pulmonary surfactant is a potential delivery system for topical medications via the conducting airways. Due to the sensitivity to inactivation of surfactant, mutual interaction with the shipped drug should be evaluated. Little is known about the interactions between surfactant and antimicrobial drugs. The aim of the present study was to evaluate whether biophysical properties of animal-derived surfactants are modified by the bactericidal antibiotic rifampicin. An intracellular activity and a broad antimicrobiotic spectrum toward Gram-negative and Gram-positive bacteria make rifampicin an interesting substance against pulmonary infections. Curosurf® (porcine surfactant from minced lungs) and Survanta® (bovine surfactant extract) were diluted to 2.5-5.0 mg/ml of phospholipids in 0.9 % NaCl and rifampicin (RIF) was added at 1, 5, and 10 % (w/w). Minimum (γ(min)) and maximum (γ(max)) surface tension of a cyclically compressed bubble in the mixture was assessed with a pulsating bubble surfactometer. After 5 min, γ(min) of Survanta at a concentration of 3 mg/ml was significantly increased after addition of 5 and 10 % RIF (both p < 0.001). At 1 % RIF, the γ(min) of Survanta was ≈10 mN/m and this value was not significantly different to that of Survanta alone. The γ(min) of Curosurf at 3 mg/ml was increased with 10 % RIF (p < 0.001), but not with 1 and 5 %. At 5 mg/ml Survanta was inhibited by 10 % RIF (p < 0.05), while γ(min) of Curosurf was low (<5 mN/m) in all mixtures. In conclusion, Curosurf and Survanta interfere with RIF in a concentration-dependent manner. At the appropriate phospholipid concentration, especially porcine-derived surfactant is able to retain good surface activity when mixed with antibiotics.

Optimization of a reversed-phase-high-performance thin-layer chromatography method for the separation of isoniazid, ethambutol, rifampicin and pyrazinamide in fixed-dose combination antituberculosis tablets.

This paper presents the development of a new RP-HPTLC method for the separation of pyrazinamide, isoniazid, rifampicin and ethambutol in a four fixed-dose combination (4 FDC) tablet formulation. It is a single method with two steps in which after plate development pyrazinamide, isoniazid and rifampicin are detected at an UV wavelength of 280 nm. Then ethambutol is derivatized and detected at a VIS wavelength of 450 nm. Methanol, ethanol and propan-1-ol were evaluated modifiers to form alcohol-water mobile phases. Systematic optimization of the composition of each alcohol in the mobile phase was carried out using the window diagramming concept to obtain the best separation. Examination of the Rf distribution of the separated compounds showed that separation of the compounds with the mobile phase containing ethanol at the optimal fraction was almost situated within the optimal Rf-values region of 0.20-0.80. Therefore, ethanol was selected as organic modifier and the optimal mobile phase composition was found to be ethanol, water, glacial acetic acid (>99% acetic acid) and 37% ammonia solution (70/30/5/1, v/v/v/v). The method is new, quick and cheap compared to the actual method in the International Pharmacopoeia for the assay of the 4 FDC tablets, which involves the use of two separate HPLC methods.

Automated determination of rifampicin in plasma samples by in-tube solid-phase microextraction coupled with liquid chromatography.

A sensitive and automated method is described for determination of rifampicin in plasma samples for therapeutic drug monitoring by in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC). Important factors in the optimization of in-tube SPME are discussed, such as coating type, sample pH, sample draw/eject volume, number of draw/eject cycles, and draw/eject flow rate. Analyte pre-concentrated in the polyethylene glycol phase was directly transferred to the liquid chromatographic column by percolation of the mobile phase, without carryover. The method was linear over the 0.1-100 µg/mL range, with a linear coefficient value (r(2)) of 0.998. The inter-assay precision presented coefficient of variation ≤ 1.7%. The effectiveness and practicability of the proposed method are proven by analysis of plasma samples from ageing patients undergoing therapy with rifampicin.

The elution and binding characteristics of rifampicin for three commercially available protein-sealed vascular grafts.

Rifampicin was absorbed onto gelatin-sealed (Gelsoft and Unigraft) or collagen-sealed (Hemashield) vascular grafts by soaking for 15 min in a 1000 mg/L solution. Bound drug was then eluted from the grafts by incubation in phosphate buffered saline (PBS) at 37 degrees C and at timed intervals the concentration of rifampicin remaining in the grafts was determined. Although all three grafts contained high concentrations of rifampicin immediately after absorption of drug, rifampicin concentrations rapidly fell during elution with PBS to approximately 1.25 mg/kg of graft after 5 h incubation in PBS, indicating that most of the rifampicin absorbed to the grafts was only loosely bound. However, once this loosely bound fraction had been removed there was a much slower elution of the remaining rifampicin from the grafts, suggesting a second and much more tightly bound fraction. The tightly bound fraction eluted with an apparent half-life of 47-76 h, depending on the graft, and extrapolation back to time zero from this phase suggests that only a very small amount of the rifampicin is tightly bound to the graft after initial soaking (0.6-1.3 mg/kg).

[Stability of rifampicin in eyedrops]. / Rifampicin tartalmú szemcseppek stabilitásának vizsgálata.

The best composition of rifampicin eye-drops for extemporaneous preparation in pharmacies was developed by the authors. The stability of the eye-drops has been studied and storage condition and shelf-life were determined. The preparation, may be stored at minus 12 degrees C for one month and may be subsequently used when stored between 2 and 8 degrees C after the first opening for five days. Rifampicin and its degradation products have been separated by TLC using silicagel layer and chloroform-methanol (42 : 58) eluent. After separation of rifampicin and its degradation products their quantity have been determined by densitometry at 475 wavelength (540 nm for rifampicin quinone). This rifampicin eye-drops have been recommended for inclusion into the 7th Edition of the Official Model Prescriptions (Formulae Normales).

Resistencia de Mycobacterium tuberculosis en pacientes mexicanos. I. Características clínicas y factores de riesgo / Drug resistence of M. tuberculosis in Mexican patients: I. clinical manifestations and risk factors

Objetivo. Determinar las manifestaciones asociadas con la infección por M. tuberculosis resistente y la susceptibilidad antimicrobiana de aislados de pacientes mexicanos. Diseño. Vigilancia epidemiológica. Sitio. Centro de tercer nivel. Pacientes. Casos de tuberculosis confirmada. Mediciones. Resistencia primaria: cuando no hubo antecedente de tratamiento, y secundaria cuando lo hubo. Se emplearon las siguientes concentraciones críticas (µg/mL): son isoniacida 0.2 y 1; rifampicina 1 y 5; etambutol 5 y 10; estreptomicina 2 y 10; etionamida 5; kanamicina 6; y ácido paraaminosalicílio (PAS) 2 y 10. Resultados. Se incluyeron 84 pacientes con edad promedio de 44.7 años (6-80 años); 54 hombres (64 por ciento) y 30 mujeres (36 por ciento). La mayoría (35/62) del Distrito Federal y del Estado de México. Sólo en 34 pacientes se obtuvo información clínica: 26 presentaron fiebre y pérdida de peso, y ocho síntomas respiratorios. Se encontraron 59 pacientes (70 por ciento) con M. tuberculosis sensible y 25 resistente (30 por ciento). En 17 (68 por ciento) hubo resistencia a dos drogas y 16 (64 por ciento) de ellos a isoniacida y rifampicina. La tasa de resistencia global fue: isoniacida 24 por ciento, rifampicina 19 por ciento, estreptomicina 12 por ciento, etambutol 10 por ciento, PAS 9 por ciento, etianamida 7 por ciento y kanamicina 6 por ciento. En 47 pacientes sin tratamiento previo, ocho tuvieron infección por organismos resistentes (17 por ciento), y la tasa de resistencia primaria fue: isoniacida 9 por ciento, rifampicina 6 por ciento, estreptomicina 2 por ciento, etambutol 2 por ciento, PAS 6 por ciento y multirresistencia 6 por ciento. De 37 pacientes con tratamiento previo, 17 (46 por ciento) tuvieron un organismo resistente, y la tasa de resistencia secundaria fue: isoniacida 44 por ciento, rifampicina 35 por ciento, estreptomicina 24 por ciento, etambutol 19 por ciento, PAS 12 por ciento y multirresistencia 35 por ciento. Incluimos cuatro médicos con M. tuberculosis mono o multirresistente y siete pacientes con SIDA, uno de ellos con multirresistencia y otro con resistenia a isoniacida. Conclusión. Los resultados muestran tasas elevadas de resistencia a isoniacida y rifampicina, y de multirresistencia en M. tuberculosis aislado de pacientes mexicanos

[Comparative study of methods of drying mycelial waste]. / Sravnitel'noe izuchenie sposobov sushki mitselialnykh otkhodov.

Two types of apparatus recommended for drying paste-like products, i.e. a continuous-belt film drier and a spouting-bed drier were tested to choose a process for drying mycelial waste. The laboratory studies with the use of the continuous-belt film drier confirmed that a dry moulded product with the moisture content of less than 5 to 6 per cent could be obtained in such an apparatus. In the laboratory and pilot plant studies with the use of the spouting-bed drier equipped with a specially constructed granulator the technological conditions of the drying were defined. The conditions provided stable hydrodynamic drying and obtaining of a dry product with the moisture content of 5 to 6 per cent. Preliminary dried and ground mycelial waste of the tetracycline and rifampicin manufacture is useful as a dry additive in the preparation of the paste with the required moisture content.

Nitrite reductase gene cloning of Amycolatopsis mediterranei U-32.

Southern blot analysis showed great homology existed between niaD (NR gene) of Aspergillus nidulans and A. mediterranei U-32 chromosome DNA. A 5.0kb PstI fragment from A. mediterranei U-32 complementary to A. nidulans niaD gene was cloned in E. coli NM522 using niaD as a probe. An identical DNA band was observed through back-hybridization of the cloned DNA fragment to PstI digest of A. mediterranei U-32 chromosome DNA. Its 2.1 kb SmaI-EcoRV fragment can only hybridize with total RNA from nitrate-cultured mycelium but not with that from ammonia-cultured mycelium. These data suggested that the cloned DNA fragment contains NR gene of A. mediterranei U-32. This is the first report on NR gene cloning from a aerobic bacterium. It was deduced from the molecular weight of the nitrate reductase that the coding sequence of NR gene is almost 1.5 kb in size. Further hybridization analysis indicated that the cloned DNA fragment covers the full-length NR gene of A. mediterranei U-32. We also constructed the physical map of the recombinant plasmid pJL1 with various restriction endonucleases, among them ten with no restriction site, six with unique site and two with double sites on the insert.

Use of electrophoresis in the identification and quantitation of antibiotics administered in combinations.

The investigation presents a method of electrophoretic separation of antibacterial drugs which are used in combinations in clinical medicine. Subsequent to electrophoresis in agarose gel, a microbiological assay was performed. This technique permitted the determination of the concentrations of beta-lactam antibiotics, rifampicin, and clindamycin in the presence of aminoglycosides. In therapeutic combinations of fusidic acid and clindamycin, the concentrations of each drug could be determined.

Assay of rifampicin in serum.

Two methods for the assay of rifampicin in serum are described. The first is a conventional plate diffusion method, measuring concentrations down to 0.02 mug/ml, and the second a chemical extraction followed by measurement of the inhibition of uptake of (14)C-uridine by Staphylococcus aureus, which estimates in the range of 0.02 to 0.001 mug/ml. The methods were used to measure serum concentrations in man following doses of about 1050 mg and 75 mg rifampicin.