Identification of impurities in polymyxin B and colistin bulk sample using liquid chromatography coupled to mass spectrometry

The European Pharmacopoeia (Ph. Eur.) describes liquid chromatography-ultraviolet (LC-UV) methods using C₁₈ stationary phases for the analysis of polymyxin B and colistin.Several unknown impurities were detected in commercial samples of those polypeptide complexes. However, the Ph. Eur. does not specify any related substances for polymyxin B and colistin. Since both methods use non-volatile buffers, the mobile phases were incompatible with mass spectrometry (MS). For the identification of related substances in bulk samples by LC/MS, volatile mobile phase systems were developed. However, the LC/MS methods (with volatile additives) showed inferior chromatographic separation compared to the LC-UV method (with non-volatile additives). Moreover, previously identified impurities by LC/MS could not be assigned in LC-UV methods as the separation in both systems was different.In this study, known impurities were traced in the LC-UV methods and new impurities present in polymyxin B and colistin bulk samples were characterized. To achieve this, each peak from the non-volatile system was collected separately and reinjected into an LC system with a volatile mobile phase coupled to MS. This way, collected impurity peaks were rechromatographed on a reversed phase column in order to separate the analyte from the buffer salts. Using this method, out of 39 peaks, five novel related substances were characterized in a polymyxin B bulk sample. Fourteen impurities, which were already reported in the literature were traced as good as possible in the LC-UV method. In the case of colistin, a total of 36 peaks were investigated, among which four new compounds. Additionally, 30 known impurities were traced in the LC-UV method.     

Simultaneous extraction and fate of linear alkylbenzene sulfonates, coconut diethanol amides, nonylphenol ethoxylates and their degradation products in wastewater treatment plants, receiving coastal waters and sediments in the Catalonian area (NE Spain)

A simple, isocratic liquid chromatographic (LC) method using volatile mobile phase constituents for the identification of related substances in erythromycin samples is described. For method development, evaporative light scattering detection (ELSD) was used. An XTerra RP18 column was used at 70 degrees C with a mobile phase consisting of acetonitrile-isopropanol-0.2M ammonium acetate pH 7.0-water (165:105:50:680). Mass spectral data were acquired on an ion trap mass spectrometer equipped with an electrospray interface operated in the positive ion mode. First, a library was created using MS/MS and MS(n) spectra of reference substances available in the laboratory. Using these reference spectra as interpretative templates, eight novel related substances in erythromycin samples were identified: N-demethylerythromycin E, erythromycin E N-oxide, anhydroerythromycin C, N-demethylerythromycin B, anhydro-N-demethylerythromycin A, pseudoerythromycin E enol ether, EF lacking the neutral sugar and EA lacking the neutral sugar.     

Mechanism for the degradation of erythromycin A and erythromycin A 2'-ethyl succinate in acidic aqueous solution

A major drawback of the antibiotic erythromycin A is its extreme acid sensitivity, leading to rapid inactivation in the stomach. The accepted model for degradation in aqueous acidic solution has erythromycin A in equilibrium with erythromycin A enol ether and degrading to anhydroerythromycin A. We report a detailed kinetic study of the acidic degradation of erythromycin A and of erythromycin A 2'-ethyl succinate (the market-leading pediatric prodrug), investigating the reaction rates and degradation products via NMR. This reveals that the accepted mechanism is incorrect and that both the enol ether and the anhydride are in equilibrium with the parent erythromycin. By implication, both the anhydride and enol ether are antibacterially inactive reservoirs for the parent erythromycin. The actual degradation pathway is the slow loss of cladinose from erythromycin A (or erythromycin A 2'-ethyl succinate), which is reported here for the first time in a kinetic study. The kinetic analysis is based on global, nonlinear, simultaneous least-squares fitting of time course concentrations for all species across multiple datasets to integrated rate expressions, to provide robust estimates of the rate constants.     

Thin-layer chromatographic study of the metabolites of erythromycins in the Wistar rat

The metabolites of erythromycin A, anhydroerythromycin A, N-demethylerythromycin A and erythromycin B in the Wistar rat were studied by thin-layer chromatography. In some experiments germ-free rats, rats with a cannulated bile duct and a gastrectomized rat were used. The erythromycins examined were shown to undergo two principal changes, N-demethylation and acid-catalysed degradation. It was demonstrated that the stomach and the liver are not the sole sites of acid degradation and demethylation of erythromycins, respectively. Erythromycin A gives three principal metabolites, anhydroerythromycin A, anhydro-N-demethylerythromycin A and N-demethylerythromycin A, and erythromycin A enol ether and N-demethylerythromycin A enol ether are present to a minor extent. 5-O-Desosaminylerythronolide A was also identified, suggesting the presence of an erythromycin glycosidase.     

Evaluation of silanol-deactivated silica-based reversed phases for liquid chromatography of erythromycin

The suitability of eleven silanol-deactivated reversed phases for the liquid chromatography of erythromycin was investigated. The selectivity and efficiency of each stationary phase were examined. The performance was compared to that of a non-deactivated C₁₈ silica-based reversed-phase material, Hypersil C₁₈ (5 μm). Two types of mobile phases were used, one containing no tetrabutylammonium (TBA) and the other containing TBA. Addition of TBA as a silanol-blocking agent improved the theoretical plate number and symmetry factor of the peaks corresponding to erythromycin A (EA) and erythromycin A enol ether for all the deactivated reversed phases. These results are an indication of the presence of some residual silanol activity in these phases. Separation of erythromycin E and EA was achieved on only two of the eleven phases. The selectivity was always poorer than that obtained in a previously described method using a poly(styrene-divinylbenzene) stationary phase.     

Characterization of dihydrostreptomycin‐related substances by liquid chromatography coupled to ion trap mass spectrometry

Dihydrostreptomycin sulphate (DHS) is a water‐soluble, broad‐spectrum aminoglycoside antibiotic. For quantitative analysis, the European Pharmacopoeia (Ph. Eur.) prescribes an ion‐pairing liquid chromatography/ultraviolet (LC/UV) method using a C18 stationary phase. Several unknown compounds were detected in commercial samples. Hence, for characterization of these unknown peaks in a commercial DHS sample, the Ph. Eur. method was coupled to mass spectrometry (MS). However, since the Ph. Eur. method uses a non‐volatile mobile phase, each peak eluted was collected and desalted before introduction into the mass spectrometer. The desalting procedure was applied to remove the non volatile salt, buffer and ion‐pairing reagent in the collected fraction. In total, 20 impurities were studied and 14 of them were newly characterized. Five impurities which are already reported in the literature were also traced in this LC/UV method. Copyright © 2009 John Wiley & Sons, Ltd.     

Discovery of a phosphine-mediated cycloisomerization of alkynyl hemiketals: access to spiroketals and dihydropyrazoles via tandem reactions

Reported here are details on the discovery of a phosphine-catalyzed isomerization of hemiketals and subsequent reactions of the cyclic keto enol ether products. The new cycloisomerization complements a previously reported amine-catalyzed process that gave oxepinones from the same hemiketal starting materials. In the absence of functionality (R(2)) on the cyclic keto enol ether, a rapid and facile dimerization occurs, giving spiroketal products. When the enone is substituted (i.e., R(2) = Ph), the cyclic keto enol ether is sufficiently stable so that it can be isolated; it can then be further reacted in the same pot to provide the corresponding dihydropyrazoles. Both the spiroketal and dihydropyrazole products arise by a tandem reaction that begins with the novel cycloisomerization. The method allows for the rapid introduction of complexity in the products from relatively simple starting materials. It should find application in the synthesis of natural product-like molecules.     

Versatile route to centro-substituted triquinacene derivatives: synthesis of 10-phenyltriquinacene

[reaction: see text] A versatile route to prepare centro-substituted triquinacene derivatives (1, R = various substituents), as exemplified by the preparation of 10-phenyltriquinacene (1, R = Ph), is reported. The quaternary, centro substituent (C-10) was installed by a trimethylsilyl chloride-promoted conjugate addition reaction of an organocuprate, derived from phenylmagnesium bromide, and the protected bicyclic enone (11). The resultant trimethylsilyl enol ether was then converted regioselectively to the C-2-allylated conjugate addition products (13, R = Ph). The allyl moiety, following oxidative cleavage of the carbon-carbon double bond, was used to elaborate the tricyclic ring system by an intramolecular aldol/acetal deprotection reaction. The product of this reaction was then converted to the target compound using a standard series of functional group transformation reactions.     

Structure elucidation of a novel analog of sildenafil detected as an adulterant in a dietary supplement using LC-UV and LC/MS

A sildenafil-related compound was detected in a dietary supplement marketed as an aphrodisiac. The compound was detected during analysis of the dietary supplement using LC-UV and LC/electrospray ionization-MS. The structure of the compound was established using high resolution MS, NMR spectrometry, and X-ray crystal structure analysis. The compound was identified as 5-(5-((3,5-dimethylpiperazin-1-yl)sulfonyl)-2-ethoxyphenyl)-l-methyl-7-((1-methyl-4-nitro-1H-imidazol-5-yl)thio)-3-propyl-1H-pyrazolo[4,3-d] pyrimidine. Based on this structure, the compound was named nitroprodenafil. The dietary supplement was found to contain 90 mg nitroprodenafil/capsule. This article describes the structural characterization of a new sildenafil-related compound. The compound was detected during analysis of a dietary supplement using LC-UV and LC/electrospray ionization (ESI)-MS. The structure was established using high resolution MS (HRMS), NMR spectrometry, and X-ray crystal structure analysis. The structures of methisosildenafil, thiomethisosildenafil, and this new analog, named nitroprodenafil (21), are shown in Figure 1. In the Demizu et al. report, the compound is named mutaprodenafil instead ofnitroprodenafil. Considering the naming right, the authors of this paper think the use of mutaprodenafil is appropriate as the compound name, although nitroprodenafil is used.     

New approaches toward the synthesis of (D-homo) steroid skeletons using Mukaiyama reactions

New, short, and flexible procedures have been developed for syntheses of steroid and D-homo steroid skeletons. A Mukaiyama reaction between the silyl enol ether of 6-methoxytetralone and 2-methyl-2-cyclopentenone or carvone, with transfer of the silyl group to the receiving enone, gave a second silyl enol ether. Addition of a carbocation, generated under Lewis acid conditions from 3-methoxy-2-butenol, 3-ethoxy-3-phenyl-2-propenol or 3-methoxy-2-propenol to this second silyl enol ether gave adducts, which could not be cyclized by aldol condensation to (D-homo) steroid skeletons. The Mukaiyama-Michael reaction of the silyl enol ether of 6-methoxy tetralone with 2-methyl-2-cylopentenone gave a second silyl enol ether, which reacted in high yield with a carbocation generated from 3-hydroxy-3-(4-methoxyphenyl)propene. Ozonolysis of the double bond in this adduct gave a tricarbonyl compound (Zieglers triketone), which has been used before in the synthesis of 9,11-dehydroestrone methyl ether. A second synthesis of C17 substituted CD-trans coupled (D-homo) steroid skeletons has been developed via addition of a carbocation, generated with ZnBr₂ from a Torgov reagent, to a silyl enol ether containing ring D precursor. The obtained seco steroids have been cyclized under formation of the 8-14 bond by treatment with acid. The double bonds in one of the cyclized products have been reduced to a C17-substituted all trans steroid skeleton.     

Isocratic separation of erythromycin, related substances and degradation products by liquid chromatography on XTerra RP18

Summary A simple, sensitive, selective and robust isocratic LC method is described for the analysis of erythromycin on XTerra RP₁₈. The main component, erythromycin A, is separated from all known related substances and degradation products. Several unknown impurities are also separated. Acetonitrile-0.2 MK₂HPO₄pH7.0-water, (35∶5∶60, v/v) was used as a mobile phase at 1.0 mL min⁻¹. UV detection was at 215 nm. The robustness of the method was evaluated by a full-factorial experimental design.     

Determination of sulfonylurea degradation products in soil by liquid chromatography-ultraviolet detection followed by confirmatory liquid chromatography-tandem mass spectrometry.

A method based on liquid extraction followed by sample enrichment on reversed-phase solid-phase extraction was developed for the extraction of five degradation products of four sulfonylurea herbicides (chlorsulfuron, metsulfuron-methyl, thifensulfuron-methyl and tribenuron-methyl) from soil. The compounds have been quantified by LC-UV and identified by tandem LC-MS with electrospray ionization or atmospheric pressure chemical ionization. The limits of detection for the five compounds were between 10 and 50 micrograms kg-1. The method has been applied to the extraction of soil samples after microbial degradation of sulfonylurea herbicides.     

Palladium‐Catalyzed Hydrolytic Cleavage of Aromatic C−O Bonds

Metallic palladium surfaces are highly selective in promoting the reductive hydrolysis of aromatic ethers in aqueous phase at relatively mild temperatures and pressures of H₂. At quantitative conversions, the selectivity to hydrolysis products of PhOR ethers was observed to range from 50 % (R=Ph) to greater than 90 % (R=n ‐C₄H₉, cyclohexyl, and PhCH₂CH₂). By analysis of the evolution of products with and without incorporation of H₂¹⁸O, the pathway was concluded to be initiated by palladium metal catalyzed partial hydrogenation of the phenyl group to an enol ether. Water then rapidly adds to the enol ether to form a hemiacetal, which then undergoes elimination to cyclohexanone and phenol/alkanol products. A remarkable feature of the reaction is that the stronger Ph−O bond is cleaved rather than the weaker aliphatic O−R bond.     

Separation of erythromycin and related substances by high-performance liquid chromatography on poly(styrene-divinylbenzene) packing materials

A comparative evaluation of three brands of poly(styrene-divinylbenzene) copolymers, Hamilton PRP-1 (10 micron), Rogel (8 micron) and TSK-Gel (10 micron), as column packing materials for high-performance liquid chromatographic separation of erythromycins is presented. Erythromycins A, B and C, anhydroerythromycin A, erythromycin A enol ether, N-demethylerythromycin A, anhydro N-demethylerythromycin A and N-demethylerythromycin A enol ether were chromatographed. The effects of column temperature, concentration of organic modifier in the mobile phase, concentration of phosphate buffer, the addition of quaternary ammonium salts and pH are described. The best separations were obtained on TSK-Gel with the mobile phase acetonitrile-methanol-0.2 M tetramethylammonium hydroxide pH 8.0-0.2 M phosphate buffer pH 8.0-water (30:15:25:5:25). PRP-1 and Rogel gave equally good separations but with higher retention volumes.     

An integrated method for degradation products detection and characterization using hybrid ion trap/time-of-flight mass spectrometry and data processing techniques: Application to study of the degradation products of danofloxacin under stressed conditions

A new strategy using hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography and post-acquisition data mining techniques was developed and applied to the detection and characterization of degradation products of danofloxacin. The degradation products formed under different forced conditions were separated using an ODS-C18 column with gradient elution. Accurate full-scan MS data were acquired in the first run and processed with the combination of extracted ion chromatograms and LC-UV chromatograms. These processes were able to find accurate molecular masses of possible degradation products. Then, the accurate MS/MS data acquired through data-dependent analysis mode in another run facilitated the structural elucidations of degradation products. As a result, a total of 11 degradation products of danofloxacin were detected and characterized using the developed method. Overall, this analytical strategy enables the acquisition of accurate-mass LC/MS data, search of a variety of degradation products through the post-acquisition processes, and effective structural characterization based on elemental compositions of degradation product molecules and their product ions. The ability to measure degradation products via tandem mass spectrometry coupled with accurate mass measurement, all in only two experimental runs, is one of the most attractive features of this methodology. The results demonstrate that use of the LC/MS-IT-TOF approach appears to be rapid, efficient and reliable in structural characterization of drug degradation products.     

Combination of a liquid chromatography-ultraviolet method with a non-volatile eluent, peak trapping and a liquid chromatography-mass spectrometry method with a volatile eluent to characterise erythromycin related substances

The selectivity and sensitivity obtained with volatile liquid chromatographic (LC) methods are often inferior compared to non-volatile ones. However, the buffers often used in the non-volatile system are incompatible to mass spectrometry (MS). So, the characterisation of unknown peaks in a non-volatile system, based on data obtained from a volatile LC-MS method, is problematic. In this study, the unknown peaks in a non-volatile liquid chromatography coupled with ultraviolet detection (LC-UV) system were directly characterised by a volatile LC-MS system using a peak trapping technique. Each peak eluted from the non-volatile system was trapped by a switching valve and sent to a LC-MS system using a volatile mobile phase. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with electrospray ionisation (ESI) operated in the positive ion mode. Using this technique, the fragmentation behaviour of erythromycin and its related substances was studied and the components occurring in commercial samples were investigated. In total 25 compounds mentioned in the literature were traced. Fourteen more unknown impurities were also studied.     

Synteesis of 2-Adamantane Carboxylic Acid

Despite the fact that there are two well-described routes to 2-adamantane carboxylic acid (IV)^1,2 it is not an easily accessible compound. In connection with our work in the field of adamantane ketenes and other 2-substituted adamantane derivatives^5,6 on adamantanone (I), followed by conversion of the enol ether (II) to the aldehyde (III) and oxidation of the latter to give 2-adanabtabe carvixtkuc acud (IV). The yield of this one-pot procedure is 70%-75% (about 10 runs).     

Oxidative C-C bond-forming reaction of electron-rich alkylbenzyl ether with trimethylvinyloxysilane

Treatment of an electron-rich benzyl ether with DDQ at ambient temperature followed by addition of a silyl enol ether undergoes a C-C bond-forming reaction to afford 3-alkoxy-3-phenyl-propionyl compound. This is a general reaction and works well with a variety of silyl enol ethers to give carbonyl products in yields ranging from 10 to 85%.     

Chimie organometallique : IX. Reaction du tungstadiphenylcarbene avec les ethers d'enols cycliques

Treating (CO)₅WC(Ph)Ph with cyclic enol ethers in n-hexane, leads, in the case of the cyclopentanone derivative, to ring-opened product (CO)₅WC(OEt)[(CH₂)₃CHC(Ph)Ph] which has been isolated and characterized. The ring size in the enol ether is the determining factor.     

Determination of erythromycin and related substances in commercial samples using liquid chromatography/ion trap mass spectrometry

A sensitive, precise and accurate quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the measurement of erythromycin A (EA) and related substances in commercial samples was developed and validated. The samples were chromatographed on a reversed-phase column with a polar endcapping and analyzed by ion trap tandem mass spectrometry in the multiple reaction monitoring (MRM) mode using positive electrospray ionization. The method showed high recovery (>or=98.82%), high sensitivity (lower limit of quantitation of 0.25 ng/mL for EA and less than 7.3 ng/mL for the related substances) and high precision (or=0.991) with a run time of only 13 min. The method was successfully applied to the determination of EA and related substances in commercial samples. Moreover, using the advanced data-dependent acquisition capability of the ion trap software two new unexpected EA related substances could be detected and possible structures for these substances were postulated.