Stress degradation of edaravone: Separation, isolation and characterization of major degradation products.

In the present study the International Conference on Harmonization-prescribed stress degradation was carried out to study the degradation profile of edaravone. To establish a Quality by Design (QbD)-assisted stability-indicating assay, the reaction solutions in which different degradation products were formed were mixed. Plackett Burman and central composite design were used to screen and optimize experimental variables to resolve edaravone and its impurities with good peak symmetry using an RP C18 column. The method was validated according to International Conference on Harmonization guidelines. Seven unknown and two known degradation products were identified and characterized by LC-MS/MS. Two major degradation products formed under thermal degradation were isolated and characterized as 4-(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl-4-(4,5-dihydro-5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one and 3-hydroxy-dihydro-thiazolo[1-(2-methyl-buta-1,3dienyl)-1-phenylhydrazine]5-one. The degradation pathways of degradants were proposed based on m/z values.

[Effect of different forms of inorganic nitrogen on the photodegradation of antipyrine in water].

The photodegradation performance and mechanisms of antipyrine (ANT, a member of anti-inflammatory analgesics) under simulated irradiation using a 300 W Xenon lamp were explored in this study. And the variation of the photodegradation of ANT in the presence of different forms of nitrogen and different pE values in the aqueous environment were also investigated. The results demonstrated that ANT photodegradation proceeds via pseudo first-order kinetics in all cases. Photodegradation of ANT in water occurs through direct photodegradation and self-sensitization via reactions with hydroxyl radical (*OH) and singlet oxygen (1O2). Presence of different forms of inorganic nitrogen (NH4+, NO2- and NO3-) showed rather different effects. At low pE value (< 5), NH4+ was the main form of inorganic nitrogen, and little effect was observed on ANT photodegradation. With the increase of pE value from 4.82 to 6.85, nitrogen form changed from NH4+ to NO2- accordingly, and the inhibiting effect increased gradually due to their competitive absorption for the irradiation and NO2- acting as a scavenger of *OH. At pE 6.85, NO2- was the main form of inorganic nitrogen, which showed a maximum inhibiting rate of 35.31%. When the pE value continued to increase from 6.85 to 8.15, the nitrogen form changed from NO2- to NO3-, and the suppression effect on ANT photodegradation decreased.

Green chromatography separation of analytes of greatly differing properties using a polyethylene glycol stationary phase and a low-toxic water-based mobile phase.

A simple, rapid, and environmentally friendly HPLC method was developed and validated for the separation of four compounds (4-aminophenol, caffeine, paracetamol, and propyphenazone) with different chemical properties. A "green" mobile phase, employing water as the major eluent, was proposed and applied to the separation of analytes with different polarity on polyethylene glycol (PEG) stationary phase. The chromatography separation of all compounds and internal standard benzoic acid was performed using isocratic elution with a low-toxicity mobile phase consisting of 0.04% (v/v) triethylamine and water. HPLC separation was carried out using a PEG reversed-phase stationary phase Supelco Discovery HS PEG column (15 × 4 mm; particle size 3 µm) at a temperature of 30 °C and flow rate at 1.0 mL min(-1). The UV detector was set at 210 nm. In this study, a PEG stationary phase was shown to be suitable for the efficient isocratic separation of compounds that differ widely in hydrophobicity and acid-base properties, particularly 4-aminophenol (log P, 0.30), caffeine (log P, -0.25), and propyphenazone (log P, 2.27). A polar PEG stationary phase provided specific selectivity which allowed traditional chromatographic problems related to the separation of analytes with different polarities to be solved. The retention properties of the group of structurally similar substances (aromatic amines, phenolic compounds, and xanthine derivatives) were tested with different mobile phases. The proposed green chromatography method was successfully applied to the analysis of active substances and one degradation impurity (4-aminophenol) in commercial preparation. Under the optimum chromatographic conditions, standard calibration was carried out with good linearity correlation coefficients for all compounds in the range (0.99914-0.99997, n = 6) between the peak areas and concentration of compounds. Recovery of the sample preparation was in the range 100 ± 5% for all compounds. The intraday method precision was determined as RSD, and the values were lower than 1.00%.

Solid-phase extraction of antipyrine dye for spectrophotometric determination of phenolic compounds in water.

In order to determine phenolic compounds in water, we propose a method based on the reaction of phenolic compounds with 4-aminoantipyrine in the presence of peroxodisulfate at pH 10 to form antipyrine dye and the solid-phase extraction of dye with a Varian Bond Elut Plexa cartridge. Dye collected on the cartridge is eluted with acetonitrile and the absorbance is measured at 475 nm. In our experiments, recovery ratios of >90% were obtained for phenol, o-aminophenol, m-aminophenol, o-methoxyphenol, m-methoxyphenol, p-methoxyphenol, o-cresol, m-cresol, o-chlorophenol, m-chlorophenol, p-chlorophenol, 2,5-dimethylphenol, and 2,4-dichlorophenol. The calibration curve obeyed Beer's law in the range 0 - 0.30 µg ml(-1) phenol. The precision of repeated tests (n = 4) was 1.7% of the phenol solution (0.10 µg ml(-1)); the detection limit was 0.0011 µg ml(-1). Recovery tests using river water, waste water, and sewage influent gave highly satisfactory results.

Removal of selected pharmaceuticals by chlorination, coagulation-sedimentation and powdered activated carbon treatment.

Removal property of nine pharmaceuticals (clofibric acid, diclofenac, fenoprofen, gemfibrozil, ibuprofen, indomethacin, ketoprofen, naproxen and propyphenazone) by chlorination, coagulation-sedimentation and powdered activated carbon treatment was examined by laboratory-scale experiments under the conditions close to actual drinking water treatment processes. Indomethacin and propyphenazone were completely degraded by chlorination within 30 minutes, but others remained around 30% (naproxen and diclofenac) or more than 80% of the initial concentration after 24 hours. A couple of unidentified peaks in a chromatogram of the chlorinated samples suggested the formation of unknown chlorination by-products. Competitive adsorption was observed when the mixed solution of the target pharmaceuticals was subjected to batch adsorption test with powdered activated carbon. Clofibric acid and ibuprofen, which were relatively less hydrophobic among the nine compounds, persisted around 60% of the initial concentration after 3 hours of contact time. Removal performance in actual drinking water treatment would become lower due to existence of other competitive substances in raw water (e.g. natural organic matter). Coagulation-sedimentation using polyaluminium chloride hardly removed most of the pharmaceuticals even under its optimal dose for turbidity removal. It is suggested that the most part of pharmaceuticals in raw water might persist in the course of conventional drinking water treatments.

Behaviour and redox sensitivity of pharmaceutical residues during bank filtration - Investigation of residues of phenazone-type analgesics.

The behaviour of residues of phenazone-type pharmaceuticals during bank filtration was investigated at a field site in Berlin, Germany, where bank-filtered water is used for drinking water production. The concentrations of the pharmaceutical residues in the shallow, young bank filtrate (travel times

Use of elevated flow rates in preparative subcritical fluid chromatography.

The benefits of using high flow rates in preparative subcritical fluid chromatography are explored. It is demonstrated that chromatograms loaded to onset of peak coalescence do not deteriorate as flow increases. This allows separation of material in very short time periods leading to dramatically increased production rates. A key factor to accessing elevated flows is the use of shorter columns and the resulting decrease in pressure drop.

Silicone rod extraction of pharmaceuticals from water.

Silicone elastomer (in the form of a flexible rod) is one of the materials currently tested for use in analytical extraction techniques and passive sampling devices. We investigated the applicability of this material for the enrichment of selected polar pharmaceuticals (antipyrine, carbamazepine, diclofenac, ibuprofen, bezafibrate) from water. Time-resolved batch extraction tests (up to five weeks in duration) were performed in stirred water samples ( approximately 0.5 L) with 2-cm-long silicone rod pieces. After re-extraction of trapped compounds with methanol, analysis was performed using liquid chromatography coupled with mass spectrometry. Distribution equilibrium was reached after a test duration of seven days. The silicone rod/water partition coefficients of the investigated pharmaceuticals range from 4 (antipyrine) to 1250 (diclofenac), and are related to the hydrophobicity of the compound. Enrichment of these pharmaceuticals in the silicone rod is lower than for other compound classes (chlorinated and polycyclic hydrocarbons).

Identification and significance of phenazone drugs and their metabolites in ground- and drinking water.

Residues of three phenazone-type pharmaceuticals have been identified in routine analyses of groundwater samples from selected areas in the north-western districts of Berlin, Germany. Phenazone, propiphenazone, and dimethylaminophenazone have been detected in some wells at concentrations up to the low microg/l-level. Additionally, three phenazone-type metabolites namely 1-acetyl-1-methyl-2-dimethyl-oxamoyl-2-phenylhydrazide (AMDOPH), 1-acetyl-1-methyl-2-phenylhydrazide, and dimethyloxalamide acid-(N'-methyl-N-phenyl)-hydrazide have also been identified in these groundwater samples. The residues are suspected to originate from former production spills of a pharmaceutical plant located in a city north of Berlin. It was observed that with the exception of AMDOPH all other residues were efficiently removed during conventional drinking water treatment. The drug metabolite AMDOPH deriving from dimethylaminophenazone residues was found at concentrations of 0.9 microg/l in finished drinking water. However, a following study on the toxicological relevance of the AMDOPH residues has shown that there is no toxicological harm for humans at the low concentrations of AMDOPH observed in Berlin drinking water.

[Optimal conditions for extraction of "caffetin" and "saridon" tablet components from aqueous solutions]. / Izuchenie optimal'nykh uslovii ékstraktsii iz vodnykh rastvorov komponentov tabletok "kaffetin" i "saridon".

Using a mathematical method of experiment planning (Latin square), the authors suggest the optimal conditions for extraction of propifenasone and paracetamol, the basic components of caffeine and saridon tablets, from water solutions: extraction with ethylacetate (pH 2) for 5 min in the presence of an electrolyte (sodium chloride or ammonium sulfate) quantum satis. The possibility of extraction of caffeine and codeine under these conditions was tested. When extracting the components of caffeine and saridon tablets, paracetamol, propifenasone, and caffeine should be extracted with ethylacetate at pH 2 and codeine by chloroform at pH 10.

The development of a semi-preparatory scale supercritical-fluid chromatograph for high-throughput purification of 'combi-chem' libraries.

A unique separator was developed which allowed automatic separation and peak collection using semi-preparative supercritical fluid chromatography (SFC). A peak detector switched the effluent between waste and special collection cassettes. Up to 50 mg of various solutes were injected onto a 21-mm I.D. Cyano column. The entire flow path was contained and no aerosols were generated. Collection efficiency was as high as 95%. Peak purity was often greater than 99. 9%. Typical run times were less than 10 min. An analytical SFC was used to screen the performance of a wide range of mobile and stationary phases for the elution of more than 60 miscellaneous small drug compounds. The best 'universal' gradient employed 0.4% isobutyl or isopropylamine dissolved in methanol, then mixed from 5 to 55% into carbon dioxide at 10%/min. Flow rate was 50 ml/min. The analytical SFC was shown to be a good predictor of the semi-prep instrumental performance.

Separation of disaccharides by affinity capillary electrophoresis in lectin-containing electrophoretic solutions.

Separation of the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of simple disaccharides (maltose, cellobiose, gentiobiose, lactose, and melibiose) by affinity capillary electrophoresis was investigated using lectin-containing neutral phosphate buffers, filled in a linear polyacrylamide-coated capillary. When Lens culinaris agglutinin (LCA) was added, the derivatives of glucobioses were retarded with varying magnitudes depending on the amount of LCA and were well separated from each other and from galactosyl glucose under optimized conditions. Addition of Ricinus communis 60 kDa agglutinin (RCA60) to the phosphate buffer gave a different migration profile, in which the derivatives of galactosyl glucoses were more retarded than those of glucobioses. However, addition of either lectin did not accomplish complete separation of the derivatives of all these disaccharides even under optimum conditions. The addition of two kinds of lectins in appropriate proportions improved separation. Thus, the binary system composed of LCA and RCA60, as well as LCA and soybean agglutinin from Glycine max (SBA), gave better separation of these derivatives, giving peak tops for all derivatives.

Separation of 1-phenyl-3-methyl-5-pyrazolone derivatives of monosaccharides by capillary electrochromatography.

1-Phenyl-3-methyl-5-pyrazolone (PMP) derivatives of component monosaccharides in glycoproteins (fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine) and epimeric aldopentoses (arabinose, lyxose, ribose and xylose) were well separated from each other by capillary electrochromatography on a Hypersil ODS column with a mixture of 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer, pH 6.0 to approximately 6.3, and acetonitrile (2.2:1 v/v) as eluent. The elution of these compounds showed relatively strong dependence on the pH and concentration of the buffer salts contained in the eluent, as compared to the elution by pressure-driven high-performance liquid chromatography (HPLC) on the same stationary phase, but separation of PMP-monosaccharides was better than that by HPLC. Retention times of PMP-monosaccharides were highly reproducible with a relative standard deviation (RSD) of approximately 0.6%, and quantification with an RSD less than 5% could be achieved using 3-O-methylglucose as an internal standard.

Comparison of radio-labeled butanol and iodoantipyrine as cerebral blood flow markers.

Cerebral blood flow (CBF) techniques based on the principle of indicator fractionation rely upon free diffusibility of the blood flow indicator into brain (i.e. complete cerebral extraction). Extraction of two commonly-used indicators, iodoantipyrine and n-butanol, was evaluated in rats by measuring torcular venous efflux after systemic injection of the indicator under conditions of normal and high CBF. The extraction of n-butanol was found to be virtually complete at all blood flows examined; iodoantipyrine, on the other hand, was completely extracted only at flows under 180 ml/100 g/min, despite the fact that the oil: water partition coefficient for iodoantipyrine exceeds that for n-butanol. Brain uptake indices for the two indicators were also measured: brain uptake of n-butanol was greater than that of iodoantipyrine, and the difference was more marked if the indicator entered brain mixed with blood than if it entered as a bloodless bolus. Blood components may thus interact with iodoantipyrine to retard its movement across the blood-brain barrier and thereby limit extraction of this lipid-soluble substance. Inasmuch as iodoantipyrine is diffusion-limited at blood flows above 180 ml/100 g/min, butanol is a more accurate CBF indicator above the normal flow range in the rat.