Degradation of rifamycin from mycelial dreg by activated persulfate: Degradation efficiency and reaction kinetics.

Rifamycin mycelial dreg (RMD) is a biological waste, and its residual rifamycin (RIF) is potentially harmful to both the environment and human health. In this work, thermally activated persulfate (PDS) oxidative degradation of RIF in RMD was developed for the first time. The effects of reaction temperature, initial PDS concentration, and pH on RIF degradation in RMD were investigated, and the treatment conditions were optimized using response surface methodology (RSM). The results showed that 90 °C, 50 mg/g PDS, and pH = 5.3 were the optimal pretreatment conditions, and 100% degradation efficiency of RIF (734 mg/kg) was achieved. SEM and FTIR analyses confirmed that the RIF was destroyed and decomposed after the oxidation reaction. The possible degradation pathways of RIF in the thermally activated PDS system were discussed through HPLC/MS and ESR analyses. The intermediate product was identified, and the toxicity of the final product was predicted to be low or nontoxic. In this work, a degradation pathway of RMD was proposed by activating persulfate, which facilitates subsequent resource utilization and provides meaningful guidance for the practical treatment of antibiotic mycelium residue (AMR).

Status of Rifaximin: A Review of Characteristics, Uses and Analytical Methods.

Rifaximin, an oral antimicrobial, has many advantages because it is selective intestine, has minimal adverse effects and is used for the treatment of some diseases such as hepatic encephalopathy, irritable bowel syndrome, travelers' diarrhea, ulcerative colitis, Clostridium difficile and acute diarrhea. Rifaximin in the form of 200 mg tablets is commercially available. The crystalline α form is therapeutically safe and effective. In most of the official compendia, rifaximin has no monograph and in none of them is there a monograph for rifaximin tablets. The literature, however, contemplates this gap with varied methods. The literature presents some methods for evaluation of rifaximin in both biological fluid and pharmaceutical product. High performance liquid chromatography stands out for the evaluation of rifaximin. Most of the methods reported in the literature are for pharmaceuticals products. They use (1) toxic organic solvents, harmful to the operator and the environment, and/or (2) buffer solution, which has a shorter service life and requires time-consuming washes of the chromatographic system generating more waste. So, this work aims to discuss (i) properties; (ii) applications; (iii) polymorphism and (iv) analytical methods of rifaximin by the look of green chemistry. This review shows an extremely current topic of great importance to the chemical-pharmaceutical area and everything it involves, since the analytical process until the impact on the environment in which it is embedded.

Development of a sensitive and rapid method for rifampicin impurity analysis using supercritical fluid chromatography.

A novel simple, fast and efficient supercritical fluid chromatography (SFC) method was developed and compared with RPLC method for the separation and determination of impurities in rifampicin. The separation was performed using a packed diol column and a mobile phase B (modifier) consisting of methanol with 0.1% ammonium formate (w/v) and 2% water (v/v). Overall satisfactory resolutions and peak shapes for rifampicin quinone (RQ), rifampicin (RF), rifamycin SV (RSV), rifampicin N-oxide (RNO) and 3-formylrifamycinSV (3-FR) were obtained by optimization of the chromatography system. With gradient elution of mobile phase, all of the impurities and the active were separated within 4 min. Taking full advantage of features of SFC (such as particular selectivity, non-sloping baseline in gradient elution, and without injection solvent effects), the method was successfully used for determination of impurities in rifampicin, with more impurity peaks detected, better resolution achieved and much less analysis time needed compared with conventional reversed-phase liquid chromatography (RPLC) methods.

Impact of crystal polymorphism on the systemic bioavailability of rifaximin, an antibiotic acting locally in the gastrointestinal tract, in healthy volunteers.

BACKGROUND: Rifaximin is an antibiotic, acting locally in the gastrointestinal tract, which may exist in different crystal as well as amorphous forms. The current marketed rifaximin formulation contains polymorph alpha, the systemic bioavailability of which is very limited. This study compared the pharmacokinetics of this formulation with those of the amorphous form. METHODS: Amorphous rifaximin was specifically prepared for the study and formulated as the marketed product. Two doses (200 mg and 400 mg) of both formulations were given to two groups of 12 healthy volunteers of either sex according to a single-blind, randomized, two-treatment, single-dose, two-period, cross-over design. Plasma and urine samples were collected at preset times (for 24 hours or 48 hours, respectively) after dosing, and assayed for rifaximin concentrations by high-performance liquid chromatography-mass spectrometry. RESULTS: For both dose levels, peak plasma concentration, area under the concentration-time curve, and cumulative urinary excretion were significantly higher after administration of amorphous rifaximin than rifaximin-α. Ninety percent confidence intervals for peak plasma concentration, area under the concentration-time curve, and urinary excretion ratios were largely outside the upper limit of the accepted (0.80-1.25) range, indicating higher systemic bioavailability of the amorphous rifaximin. The few adverse events recorded were not serious and not related to the study medications. CONCLUSION: Rifaximin-α, a crystal polymorph, does differ from the amorphous form, the latter being systemically more bioavailable. In this regard, care must be taken when using - as a medicinal product - a formulation containing even small amounts of amorphous form, which may alter the peculiar pharmacologic properties of this poorly absorbed antibiotic.

13C and 15N CP/MAS, 1H-15N SCT CP/MAS and FTIR spectroscopy as tools for qualitative detection of the presence of zwitterionic and non-ionic forms of ansa-macrolide 3-formylrifamycin SV and its derivatives in solid state.

(13)C, (15)N CP/MAS, including (1)H-(13)C and (1)H-(15)N short contact time CP/MAS experiments, and FTIR methods were applied for detailed structural characterization of ansa-macrolides as 3-formylrifamycin SV (1) and its derivatives (2-6) in crystal and in powder forms. Although HPLC chromatograms for 2/CH3 OH and 2/CH3 CCl3 were the same for rifampicin crystals dissolved in respective solvents, the UV-vis data recorded for them were different in 300-375 nm region. Detailed solid state (13)C and (15)N CP/MAS NMR and FTIR studies revealed that rifampicin (2), in contrast to 3-formylrifamycin SV (1) and its amino derivatives (3-6), can occur in pure non-ionic or zwitterionic forms in crystal and in pure these forms or a mixture of them in a powder. Multinuclear CP/MAS and FTIR studies demonstrated also that 3-6 derivatives were present exclusively in pure zwitterionic forms, both in powder and in crystal. On the basis of the solid state NMR and FTIR studies, two conformers of 3-formylrifamycin SV were detected in powder form due to the different orientations of carbonyl group of amide moiety. The PM6 molecular modeling at the semi-empirical level of theory, allowed visualization the most energetically favorable non-ionic and zwitterionic forms of 1-6 antibiotics, strongly stabilized via intramolecular H-bonds. FTIR studies indicated that the originally adopted forms of these type antibiotics in crystal or in powder are stable in standard laboratory conditions in time. The results presented point to the fact that because of a possible presence of two forms of rifampicin (compound 2), quantification of the content of this antibiotic in relevant pharmaceuticals needs caution.

Impurity profile of rifaximin produced in China.

Impurity profiles of rifaximin produced in China were investigated systematically by LCMS methods. Eleven impurities from the raw materials of rifaximin produced in China were detected. We adopted the Diagnostic fragment-ion-based extension strategy (DFIBES) for deducing the structure of unknown impurities. Impurity 1 was the 30-hydroxylated product of rifaximin. Impurity 2 was the 25-deacetyled rifaximin. Impurity 6 was the isomeride of rifaximin. Impurity 9 was rifamycin-O.

Variability in the proportion of components of iodoform-based Guedes-Pinto paste mixed by dental students and pediatric dentists.

OBJECTIVE: Guedes-Pinto paste (GPP) is an iodoform paste used in most dental schools in Brazil. The paste is a composite of medicines (Rifocort , camphorated paramonochlorophenol [PMCC], and iodoform) used for endodontic treatment of primary teeth. The aim of this study was to evaluate the proportion variability of GPP components when mixed by undergraduate dentistry students and pediatric dentists. MATERIALS AND METHODS: The study was divided into 4 groups: G1 (15 undergraduate students), G2 (15 specialists in Pediatric Dentistry), G3 (15 professors with clinical activity), and G4 (7 professors-researchers). All volunteers prepared GPP according to the original specifications: the same visual proportion for each component. The components were weighed using an analytical balance and the percentage was calculated. RESULT: After normality (Kolmogorov-Smirnov) and homogeneity tests (Levene test), the data were submitted to analysis of variance and intraclass correlation coefficient tests (P<0.05). The percentage means of each respective group were as follows: Rifocort 20.2%, 20.8%, 26.7%, 27.3%; camphorated PMCC 9.2%, 8.1%, 6.7%, 5.1%; and the iodoform 70.6%, 71.1%, 64.7%, 67.6%. There were no significant differences between groups for the component percentages. There was a high intraclass correlation coefficient (G1 0.945; G2 0.951; G3 0.921; and G4 0.870). CONCLUSION: The proportion of GPP was similar in all the groups, allowing us to conclude that ideal GPP proportion, based on the entire group mean, was 23.8% of Rifocort® ; 7.0% of camphorated PMCC; and 69.2% of iodoform.

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.

Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability.

We are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (FRET) measurements. In this article, we consider the problem of docking small-molecule ligands within macromolecular complexes. Using simulated FRET data, we have generated a series of benchmarks that permit estimation of model accuracy based on the quantity and quality of FRET-derived distance restraints, including the number, random error, systematic error, distance distribution, and radial distribution of FRET-derived distance restraints. We find that expected model accuracy is 10 A or better for models based on: i), > or =20 restraints with up to 15% random error and no systematic error, or ii), > or =20 restraints with up to 15% random error, up to 10% systematic error, and a symmetric radial distribution of restraints. Model accuracies can be improved to 5 A or better by increasing the number of restraints to > or =40 and/or by optimizing the distance distribution of restraints. Using experimental FRET data, we have defined the positions of the binding sites within bacterial RNA polymerase of the small-molecule inhibitors rifampicin (Rif) and rifamycin SV (Rif SV). The inferred binding sites for Rif and Rif SV were located with accuracies of, respectively, 7 and 10 A relative to the crystallographically defined binding site for Rif. These accuracies agree with expectations from the benchmark simulations and suffice to indicate that the binding sites for Rif and Rif SV are located within the RNA polymerase active-center cleft, overlapping the binding site for the RNA-DNA hybrid.

Resolution of binary mixtures of rifamycin SV and rifampicin by UV/VIS spectroscopy and partial least-squares method (PLS).

A procedure is proposed for the joint determination of rifamycin SV and rifampicin by UV/VIS spectroscopy. A partial least-squares regression (PLS) was used for the resolution of the overlapping spectrophotometric signals from mixtures of the two drugs. The application of a genetic algorithm to select some of the predictor variables allows one to considerably reduce the number of experimental variables, as well as to improve the prediction capacity of the PLS model constructed with these selected potentials. Finally, the methods were applied to the determination of these drugs in biological samples.

Stability of rifampicin in dissolution medium in presence of isoniazid.

Rifampicin (RIF) hydrolyzes in acidic medium to form insoluble and poorly absorbed 3-Formyl rifamycin SV (3-FRSV). This study describes development of two principally different methods, Dual Wavelength UV-Vis. spectrophotometry (DW spectrophotometry) and HPTLC, to determine 3-FRSV in presence of RIF. Using DW spectrophotometry, RIF was estimated by using wavelengths 475.0 and 507.0 nm and 3-FRSV was estimated using 457.0 and 492.0 nm. In HPTLC method, a mixture of chloroform:methanol:water (80:20:2.5 v/v) was used as the mobile phase to resolve 3-FRSV from RIF and 3-FRSV was quantified at 333 nm. The linearity range for 3-FRSV was 2-10 microg/ml and 50-250 ng/spot for DW spectrophotometric method and HPTLC method, respectively, and 5-50 microg/ml for RIF using DW spectrophotometric method. Both the methods were found to be specific, accurate and reproducible. The proposed methods were successfully applied to determine the rate of degradation of RIF to 3-FRSV in dissolution medium (0.1 N HCl) and also in presence of isoniazid (INH). The rate of degradation of RIF in presence of INH was almost two times more than that of RIF alone. These methods were utilized to study the stability of RIF in market formulations of RIF and RIF with INH in dissolution medium. It has been observed that RIF degrades by 12.4% to form 3-FRSV (RIF formulations) while in presence of INH the degradation is catalyzed to about 21.5% (RIF+INH formulations), in 45 min. Thus, lower concentration of RIF may be available for absorption leading to poor bioavailability of RIF from combination dosage forms (RIF+INH) as compared to formulations containing only RIF. It is proposed that specific analytical method should be used to measure RIF in presence of 3-FRSV in a dissolution study.

High-performance liquid chromatographic determination of 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) and its deacetyl metabolite in plasma, whole blood, urine and tissue samples in rats.

A reversed-phase high-performance liquid chromatographic method was developed for the determination of 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648, I), a new rifamycin derivative, and its 25-deacetyl metabolite (KRM-1671, II) in plasma, whole blood, tissues and urine from rats. I and II were coextracted with an internal standard from each sample matrix by solid-phase extraction (Bond Elut). Plasma and urine were directly loaded onto Bond Elut, while whole blood and tissues were homogenized and extracted with methanol or dichloromethane-chloroform prior to Bond Elut extraction. The extracts were chromatographed on Shim-pack CLC-ODS(M) using acetonitrile-0.02 M citrate buffer containing 0.1 M sodium perchlorate (2:1, v/v), and peaks were detected at 643 nm. The validation data showed that the assays for I and II in plasma, whole blood, tissues and urine were selective, accurate and reproducible.

Azithromycin, rifabutin, and rifapentine for treatment and prophylaxis of Mycobacterium avium complex in rats treated with cyclosporine.

Azithromycin, rifabutin, and rifapentine were used to treat or prevent disseminated Mycobacterium avium complex (MAC) infections produced in rats immunosuppressed with cyclosporine. Animals with bacteremic infections were treated 1 week after intravenous inoculation with 10(7) CFU of MAC with azithromycin, 100 mg/kg of body weight administered subcutaneously for 5 days and then 75 mg/kg on Monday, Wednesday, and Friday, or with rifabutin or rifapentine, 20 mg/kg administered intraperitoneally on Monday through Friday. All three drugs showed efficacy after 1 and 2 months. Rifabutin cleared the organisms from tissues more rapidly than azithromycin or rifapentine. To approximate prophylaxis, treatment was started 2 weeks before intravenous inoculation with 10(4) organisms. MAC infections were undetectable in treated animals after 4 months, while control animals had disseminated infections. These findings support the rationale for clinical trials of treatment and prophylaxis with these agents. The cyclosporine-treated rat appears to be a useful model in which to evaluate compounds for the treatment and prophylaxis of disseminated MAC infections.

High-performance liquid chromatographic assay for two rifamycin-derived hypocholesterolemic agents in liver and biological fluids.

CGP 43371 (compound I), a mono-pivaloyl oxazole derivative of a 3-piperazino-rifamycin, has been in clinical trials as a potential hypocholesterolemic agent. A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed using a C18 column and a gradient solvent system of methanol-0.1 M sodium acetate, pH 4.5, at a flow-rate of 1 ml/min. The compound and internal standard (rifampicin) were detected by their ultraviolet absorption at 254 nm. Isolation of the compounds from plasma and liver homogenates was accomplished by precipitation of proteins with acetonitrile, followed by evaporation under nitrogen and reconstitution in methanol. Bile, lymph and urine were injected onto the HPLC column without pretreatment. Calibration curves were linear (r > 0.999) over the concentration range 0.25-20.0 micrograms/ml. The assay procedure was also applicable to other rifamycin derivatives and was able to distinguish between molecular species containing small differences in functionality.

Application of derivative resolution of UV spectra to the quality control of rifaximine and its possible impurities.

An UV spectrophotometric method with derivative resolution was developed for the analysis of rifaximine (a synthetic rifamycin) in the presence of potential impurities of synthesis and oxidation. The method was particularly suitable for the qualitative identification of the set of compounds and had good detection limits.

A sensitive method for quantitation of rifabutin and its desacetyl metabolite in human biological fluids by high-performance liquid chromatography (HPLC).

Sensitive HPLC-UV methodology has been developed and validated for quantitating rifabutin, an antimycobacterial, and its 25-desacetyl metabolite, LM-565, in human plasma and urine. The HPLC separation for both plasma and urine samples was performed on an ODS, 5-microns, reverse-phase column (25 cm x 4.6-cm ID) using a mobile phase of acetonitrile/0.05 M potassium phosphate, pH 4.2, with triethylamine, (38:61.5:0.5, v/v), at a flow rate of 1.0 ml/min. The separation eluate was monitored by absorbance at 275 nm. Plasma samples (1 ml) were spiked with an internal standard (medazepam), buffered at pH 7.4 and extracted with 80:20 (v/v) hexane:ethyl acetate, and then back extracted with acidified water (0.05 M H3PO4). Linearity was established between 5.0-800 and 2.5-400 ng/ml for rifabutin and LM-565, respectively. Intraday imprecision for rifabutin and LM-565 plasma quality controls prepared at 7.3 and 3.2 ng/ml, respectively, was less than 15% relative standard deviation (RSD). Absolute recovery for parent drug and metabolite, from plasma, was greater than 90% throughout the respective dynamic ranges and greater than 70% for medazepam. Urine samples (1 ml) were acidified with 50 microliters of 3.6 M H2SO4 and diluted with 0.1 M ammonium acetate. Linearity was established between 100 and 5000 ng/ml for both rifabutin and LM-565. Intraday imprecision for a urine control at 200 ng/ml was less than or equal to 12% RSD for either component. The method is currently being used to support Phase I kinetics program for rifabutin in prophylaxis of MAC infection of AIDS patients. Application of this method to a bioavailability assessment is presented.

Liquid chromatographic-mass spectrometric studies on rifamycin antibiotics.

The use of a direct liquid introduction type liquid chromatographic-mass spectrometric interface to study highly thermally labile rifamycin antibiotics is described. Using negative ionization, abundant molecular ions were observed, and the spectra, also contained structurally significant fragments. Variation of the high-performance liquid chromatographic parameters did not change the spectra, thus making it easy to change chromatographic conditions. In quantitative studies, a surprising correlation was found, indicating that the mass spectrometric signal was proportional to the square of the sample concentration.

Laser-induced vaporization mass spectrometry of rifamycins.

The mass spectra of many rifamycins cannot be obtained by electron ionization (EI) owing to their thermal decomposition. When a laser beam is used to vaporize the sample through an optic fibre inserted in a hollow probe which reaches the sample cup, decomposition is minimized and the EI spectra show abundant molecular ions and fragments of structurally high diagnostic value. These ionic species are easily observed owing to the lack of chemical noise often present in soft ionization methods, such as direct liquid chemical ionization and fast atom bombardment.