The purpose of this paper is to describe the glycosylation of ambrisentan (AMB) by cultures of Cunninghamella elegans ATCC 9245. AMB is an endothelin receptor antagonist, which is used to treat pulmonary arterial hypertension. Filamentous fungi are morphologically complex and may exhibit different forms depending on the species and the nature of the culture medium. A biotransformation study was conducted to investigate the ability of C. elegans to metabolize AMB. Parameters were optimized by testing on different culture media and concentrations, pH, drug concentration, static and shaking conditions. Ambrisentan's metabolite, obtained after 240 h of incubation as a result of glycosylation pathway, was separated by HPLC and determined by high-resolution mass spectrometry. The method showed linearity over 300-1000 μg mL −1 (r = 0.998). Accuracy, precision, robustness and stability studies agree with international guidelines. Results are consistent in accordance with the principles of green chemistry as the experimental conditions had a low environmental impact, and used little solvent.

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Background Posaconazole is a triazole antifungal drug approved by FDA in 2006. No bioassay is available in literature or official codes for potency determination in bulk. Objective To conduct an analytical study focused on posaconazole in bulk. Methods An alternative microbiological assay was validated for drug quantitation, applying agar diffusion technics (3 x 3 design), using Saccharomyces cerevisiae ATCC MYA 1942 as test microorganism (2% inoculum). An isocratic HPLC-DAD method, with C8 Shim-pack column (250 x 4.6 mm, 5 μm) and methanol:water (75:25 v/v) mobile phase was used for stress stability by photolysis and oxidation, indicating the formation of degradation products, which were investigated by UPLC-QTOF-MS. Results The established conditions for the bioassay were satisfactory. It was linear in the range evaluated (2.5 – 10.0 µg/mL), as well as precise, accurate and robust. Stress tests showed drug susceptibility to the factors evaluated (60 % of degradation after 120 min). Kinetics curves for photolytic decomposition followed first-order. From photolytic and oxidative degraded matrix, three major degradation products were identified as being derivatives with modifications in the piperazine central ring and in the triazole and triazolone side chains, whose mass spectra results were m/z 683 (DP1), m/z 411 (DP2), m/z 465 (DP3). Conclusions Microbiological method was adequately validated and demonstrated to be equivalent to physico-chemical ones. Impurities found are described for the first time in studies with posaconazole raw material. Highlights a microbiological bioassay was developed for posaconazole; first-order kinetics was determined for photolytic degradation; structures for new degradation products were suggested.

Drug biotransformation studies appear as an alternative to pharmacological studies of metabolites, development of new drug candidates with reduced investment as well as the most efficient production of chemical structures involves and drug quality control studies. A wide range of reactions in biotransformations process is catalyzed by microorganisms. Fungi can be considered as a promising source of new biotransformation reactions. The aim of this study was to evaluate the capacity of metronidazole biotransformation through the filamentous fungus Cunninghamella elegans ATCC 9245. The monitoring of metabolite formation was performed by high-performance liquid chromatography (HPLC) coupled to ultraviolet (UV) spectrophotometry. The results of the biotransformation of metronidazole showed drug consumption in culture and the formation of four new chromatographic peaks of chemical structures not elucidated. The method showed it became linear over 10-70 μg/mL (r = 0.999953). Accuracy, precision and stability studies agree with international guidelines.  Results are consistent in accordance with the principles of green chemistry as the experimental conditions had a low environmental impact, and few solvents use.

This work describes the development and validation of a microbiological method using the cylinder-plate assay for quantitative determination of imipenem in powder for injection. The aim was to obtain a low-cost and suitable methodology that can be alternative to physicochemical techniques already described, contributing for the quality control of this antibiotic. Firstly, the analytical conditions were optimized, testing the microorganism, inoculum concentration and best range of sample and standard concentrations, in a way that provides the adequate measurement of the inhibition halos. Staphylococcus epidermidis ATCC 12228 was selected as test microorganism, as well as 2.0 % of inoculum concentration. The validation protocol followed the official guidelines, and the parameters evaluated were linearity, precision (intermediate precision and repeatability) and accuracy. All standard curves ranging 0.5-2.0 µg/mL showed r values higher than 0.999, and ANOVA confirmed that were no deviation from linearity (p-value > 0.05). The method also proved to be precise with RSD (relative standard deviation) values ranging 0.28-0.64 for repeatability and 2.49 for intermediate precision. It was performed three days of experiments, being three assays of eight plates a day. The drug mean content was 101.05%. Accuracy was assessed by recovery test, with standard recovery percentage of 101.70-107.90% (mean recovery = 104.86%), which was considered satisfactory. Therefore, the proposed microbiological method was considered validated and suitable for application in quantitative determination of this drug, being useful for quality control routine.