Identification of the major photodegradant in metronidazole by LC-PDA-MS and its reveal in compendial methods.

Metronidazole in aqueous solution is sensitive to light and UV irradiation, leading to the formation of N-(2-hydroxyethyl)-5-methyl-l,2,4-oxadiazole-3-carboxamide. This is revealed here by liquid chromatography with tandem photo diode array detection and mass spectrometry (LC-PDA-MS) and further verified by comparison with the corresponding reference substance and proton nuclear magnetic resonance (1H-NMR). However, in current compendial tests for related substances/organic impurities of metronidazole, the above photolytic degradant could not be detected. Thus, when photodegradation of metronidazole occurs, it could not be demonstrated. In our study, an improved LC method was developed and validated, which includes a detection at a wavelength of 230 nm and optimization of mobile phase composition thereby a better separation was obtained.

Evaluation of Different Light Conditions in the Working Environment for Handling Photosensitive and Thermolabile Compounds.

Lighting in the working environment plays a significant role on the degree of degradation of photosensitive, thermolabile compounds and on working efficiency. Light emitting diodes (LEDs) are semiconductor light emitting devices that are promising artificial light sources with easy modulation of light wave signals and are also known for low heat generation. Therefore, the effect of polychromatic LED light was tested in the working environment using the drug compounds montelukast, nifedipine, and clavulanic acid, which are known to be photosensitive or thermolabile. As a control, other lighting sources like a sodium lamp, a classic (incandescent, tungsten) lamp, and indirect sunlight were also used in this study. All the experiments were carried out with methanolic solutions at room temperature. An Acquity UPLC/MS/MS system was used for quantification of the main analytes and degradation products. Under the tested conditions, LED lighting proved to be more suitable for handling photosensitive and thermolabile compounds.

Characterization of the components of meleumycin by liquid chromatography with photo-diode array detection and electrospray ionization tandem mass spectrometry.

Reversed-phase liquid chromatography coupled with photo-diode array (PDA) detection and electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to characterize the components of meleumycin, a 16-membered macrolide antibiotic produced by fermentation. In total 31 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 others that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known components. Their ultraviolet spectra and chromatographic behavior were used to confirm the proposed structures: e.g. λmax shift from 232 nm to 282 nm would indicate the presence of an α-, ß-, γ-, δ-unsaturated ketone instead of a normal α-, ß-, γ-, δ-unsaturated alcohol in the 16-membered ring of the examined components. Compared to other methods, this LC/MS(n) method is particularly advantageous to characterize minor components at trace levels in multi-components antibiotics, in terms of sensitivity and efficiency.

Characterization of impurities in tylosin using dual liquid chromatography combined with ion trap mass spectrometry.

Investigation of unknown impurities in a tylosin sample was performed using liquid chromatography coupled to mass spectrometry (LC/MS). Separation was performed according to the recently described LC-UV method of Ashenafi et al. (2011) [14]. This method was reported to have a good selectivity as it was able to separate the four main components of tylosin from the already known and 23 unknown impurities. However, as this method uses a mobile phase with non-volatile constituents, direct characterization of these impurities using LC/MS was not possible. The impurity fractions were therefore first collected and then desalted before sending them to the MS. Identification of the impurities in the tylosin sample was performed with a quadruple ion trap (IT) MS, with an electrospray ionization (ESI) source in the positive ion mode. The structure of the impurities was deduced by comparing their fragmentation pattern with those of the main components of tylosin. As several peaks in the LC-UV method contained multiple compounds, using this method in total 41 new impurities were (partly) characterized.

Analysis of impurities in vertilmicin sulfate by liquid chromatography ion-trap mass spectrometry.

The characterization of impurities present in vertilmicin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed phase (RP)-LC method using a C18 column resistant to basic pH with an alkaline (pH 11) aqueous mobile phase was developed and coupled to MS with an electrospray ionization (ESI) source in the positive ion mode which provides MS(n) capability. In total, 18 impurities were detected in a commercial sample. Eleven impurities described in this work were newly identified.

Development and validation of a reversed phase liquid chromatographic method for analysis of oxytetracycline and related impurities.

A simple, robust and fast high-performance liquid chromatographic method is described for the analysis of oxytetracycline and its related impurities. The principal peak and impurities are all baseline separated in 20 min using an Inertsil C8 (150 mm × 4.6 mm, 5 µm) column kept at 50 °C. The mobile phase consists of a gradient mixture of mobile phases A (0.05% trifluoroacetic acid in water) and B (acetonitrile-methanol-tetrahydrofuran, 80:15:5, v/v/v) pumped at a flow rate of 1.3 ml/min. UV detection was performed at 254 nm. The developed method was validated for its robustness, sensitivity, precision and linearity in the range from limit of quantification (LOQ) to 120%. The limits of detection (LOD) and LOQ were found to be 0.08 µg/ml and 0.32 µg/ml, respectively. This method allows the separation of oxytetracycline from all known and 5 unknown impurities, which is better than previously reported in the literature. Moreover, the simple mobile phase composition devoid of non-volatile buffers made the method suitable to interface with mass spectrometry for further characterization of unknown impurities. The developed method has been applied for determination of related substances in oxytetracycline bulk samples available from four manufacturers. The validation results demonstrate that the method is reliable for quantification of oxytetracycline and its impurities.

Impurity profiling of micronomicin sulfate injection by liquid chromatography-ion trap mass spectrometry.

The characterization of impurities present in micronomicin sulfate injection by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed phase (RP)-LC method using a C18 column resistant to an alkaline (pH 11) aqueous mobile phase was developed and coupled to MS with an electrospray ionization (ESI) source in the positive ion mode which provides MS(n) capability. A total of thirty six impurities were detected in commercial samples: five impurities were identified by comparison of their fragmentation patterns with those of available related substances, eleven of them were identified in accordance with relevant literature, while the other twenty impurities were newly identified using the MS/MS spectra of the available related reference substances as interpretative templates combined with knowledge of the nature of functional group fragmentation behaviors. This work was applied to evaluate the quality of micronomicin sulfate injection from different manufacturers.

Characterization of impurities in josamycin using dual liquid chromatography combined with mass spectrometry.

The European Pharmacopoeia (Ph. Eur.) prescribes a selective and sensitive liquid chromatography/ultraviolet (LC-UV) method for the separation of the 16-membered ring macrolide josamycin and its related compounds. Since josamycin is obtained by fermentation, several closely related substances can be found in the sample. Several impurities have already been identified using reference substances. However, many peaks in the chromatogram cannot be correlated with known compounds or correspond to structures which were not described previously. The hyphenation of LC to mass spectrometry (MS) is a very useful tool for the characterization of impurities. The existing LC-UV method however uses non-volatile buffers, while for LC/MS a volatile mobile phase is required. In this study, each peak from the non-volatile system was collected separately and reinjected into a LC system using volatile mobile phase constituents. This way, the analyte could be separated from the buffer salts. Mass spectral data of this macrolide antibiotic were acquired on a LCQ ion trap mass spectrometer, equipped with an electrospray ionization (ESI) probe operating in the positive ion mode. The identity of the unknown compounds was deduced using the MS/MS and MS(n) collision-induced dissociation spectra of reference substances, combined with knowledge about the nature of functional group fragmentation behavior. The impurity profiling was done on 30 peaks in a josamycin bulk sample. This way, 12 compounds reported in the literature and Ph. Eur. were found in the bulk sample. Furthermore, 12 novel related substances were characterized and 18 compounds were partially characterized.

A Novel Depsipeptide Produced by Paenibacillus alvei 32 Isolated from a Cystic fibrosis Patient.

The important viscosity of the respiratory tract mucus of Cystic fibrosis (CF) patients impairs the mucociliary transport system and allows the growth of numerous micro-organisms. Among them, Pseudomonas aeruginosa and Staphylococcus aureus are known to be responsible for pulmonary infections. We imagined that CF microflora could also harbour micro-organisms naturally equipped to compete with these pathogens. A method was developed to recover these antibiotic-producing strains within 20 CF sputum. Using this approach, we have isolated an unusual Gram-positive bacterium identified as Paenibacillus alvei by Api galleries and 16S rRNA gene sequence analysis. This strain has inhibited the growth of P. aeruginosa, S. aureus and Klebsiella pneumoniae, in co-cultures. A liquid mineral medium named MODT50 was designed and optimised for the production and the recovery of the antimicrobial compounds. The supernatant has inhibited the growth of all Gram-positive strains tested, even Methicillin-resistant S. aureus. One antimicrobial compound with a peptide structure (mainly active against S. aureus, Micrococcus luteus, and Pseudomonas stutzeri) has been purified and characterised by liquid chromatography-mass spectrometry. The new active molecule (m/z 786.6) named depsipeptide L possesses a 15-guanidino-3-hydroxypentadecanoic acid side chain (m/z 298) linked on a cyclic part of four amino acids residues (Ser, two Leu/Ile, Arg). This work reports for the first time the production of such a molecule by a P. alvei strain in a mineral medium. The CF lung microflora might represent a valuable source for the discovery of new antimicrobial-producing strains.

Improved reversed phase liquid chromatographic method with pulsed electrochemical detection for tobramycin in bulk and pharmaceutical formulation.

Tobramycin is one of the aminoglycoside antibiotics that lack a UV absorbing chromophore. However, the application of pulsed electrochemical detection (PED) has been used successfully for the analysis of this and similar antibiotics. This work describes an improved liquid chromatographic (LC) method combined with PED, which is able to separate much more impurities than before. Using a Discovery C-18 RP column (250 mm×4.6 mm i.d., 5 µm), isocratic elution was carried out with a mobile phase, containing sodium sulfate (35 g/L), sodium octanesulphonic acid (1 g/L), tetrahydrofuran (14 mL/L) and 0.2 M phosphate buffer pH 3.0 (50 mL/L). Using these experimental conditions, the limit of quantification (LOQ, S/N=10) was 5 ng. The linearity was examined in the range LOQ-60 µg/mL and the coefficient of determination was 0.998. The method also proved to be repeatable and the recovery was close to 100%. The influence of the different chromatographic parameters on the separation was investigated by means of an experimental design. The proposed method is useful in quality control of tobramycin drug substances and drug products.

Impurity profiling of capreomycin using dual liquid chromatography coupled to mass spectrometry.

The characterization of unknown (UNK) impurities in capreomycin (CMN) using liquid chromatography coupled to mass spectrometry (LC/MS) has been described. The ion-pair liquid chromatography method coupled to ultraviolet detection (LC-UV) described by Mallampati et al. was used for the separation of CMN from its related substances. As the method uses non-volatile reagents it could not be directly coupled to mass spectrometry (MS) for impurity characterization. So, these UNK impurities were collected and desalted before sending to MS for structural characterization. As no information about the fragmentation of the main components of CMN, except for CMN IB, was available in the literature, they were studied first. Next, the structures of the impurities were deduced by comparing their fragmentation to that of the main components of CMN. Fourteen UNK impurities that were never described before, were (partly) characterized.

Impurity profiling of etimicin sulfate by liquid chromatography ion-trap mass spectrometry.

A reversed phase (RP)-LC method using a C(18) column resistant to basic pH and an alkaline (pH 10) aqueous mobile phase was developed and coupled to MS with an electrospray ionization (ESI) source in the positive ion mode which provides MS(n) capability. In total, 26 impurities were detected in a commercial sample. The structures of the impurities were proposed based on comparison of their fragmentation patterns with those of the available reference substances, the synthetic route and literature data. Starting material and its residual impurities, intermediates, synthetic by-products and degradation products were the main sources of those impurities. 14 impurities described in this work were newly identified.

Development and validation of a reversed-phase liquid chromatographic method for analysis of demeclocycline and related impurities.

A simple, robust, and rapid reversedphase high-performance liquid chromatographic method for the analysis of demeclocycline and its impurities is described. Chromatographic separations were achieved on a Symmetry Shield RP8 (75 mm × 4.6 mm, 3.5 µm) column kept at 40°C. The mobile phase was a gradient mixture of acetonitrile, 0.06 M sodium edetate (pH 7.5), 0.06 M tetrapropylammonium hydrogen sulphate (pH 7.5) and water, A (2:35:35:28 v/v/v/v) and B (30:35:35:0 v/v/v/v) pumped at a flow rate of 1 mL/min. UV detection was performed at 280 nm. The developed method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantification, linearity, precision, and robustness. An experimental design was applied for robustness study. Results show that the peak shape, chromatographic resolution between the impurities, and the total analysis time are satisfactory and better than previous methods. The method has been applied for the analysis of commercial demeclocycline bulk samples available on the market.

Identification of the oxidation products of cysteamine and cystamine by CE-MS interfaced by a noncommercial electrospray ionization source.

Sodium cysteamine phosphate is a prodrug derivative of cysteamine that can be used in cystinosis treatment. Although titrimetric assays are very well established and precise, iodimetric determination of sodium cysteamine phosphate requires considerably more carefulness and time from the analyst than usual. The possibility to assess sodium cysteamine phosphate by CE was evaluated by means of the quantification of its oxidation product, cystamine, which is a more suitable substance to be used as primary standard than sodium cysteamine phosphate. Apparently, this approach should be straightforward, but systematic differences between the results obtained with CE and titrimetric assays were noticed. MS and CE-MS were employed to aid in the investigation of the possible causes of imprecision of the sodium cysteamine phosphate titration and CE determination. For this purpose, a simple and inexpensive ESI source was constructed. It was observed that cystamine is not the final product of the cysteamine and/or sodium cysteamine phosphate iodine-oxidation and other species besides cystamine may be formed depending on the reaction conditions, which explains the difficulties observed in the sodium cysteamine phosphate quantification.

Identification of the components of bitespiramycin by liquid chromatography-mass spectrometry.

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to characterize the components of bitespiramycin, a group of 4"-acylated spiramycins produced by bioengineered strains. In total 38 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 other that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known major components. Their ultraviolet spectra were used to confirm the presence of an α-, ß-, γ-, δ-unsaturated butadiene in the macrocyclic lactone. Compared with the classical method, LC/ESI-MS/MS is particularly advantageous in terms of sensitivity and efficiency to characterize minor components at trace levels in multi-component antibiotics.

Optimization of capillary electrophoresis method with contactless conductivity detection for the analysis of tobramycin and its related substances.

A method was validated and optimized to determine tobramycin (TOB) and its related substances. TOB is an aminoglycoside antibiotic which lacks a strong UV absorbing chromophore or fluorophore. Due to the physicochemical properties of TOB, capillary electrophoresis (CE) in combination with Capacitively Coupled Contactless Conductivity Detection (C(4)D) was chosen. The optimized separation method uses a background electrolyte (BGE) composed of 25 mM morpholinoethane-sulphonic acid (MES) adjusted to pH 6.4 by L-histidine (l-His). 0.3 mM cetyltrimethyl ammonium bromide (CTAB) was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg L(-1) was used as internal standard (IS). 30 kV was applied in reverse polarity (cathode at the injection capillary end) on a fused silica capillary (65/43 cm; 75 µm id). The optimized separation was obtained in less than 7 min with good linearity (R(2)=0.9995) for tobramycin. It shows a good precision expressed as RSD on relative peak areas equal to 0.2% and 0.7% for intraday and interday respectively. The LOD and LOQ are 0.4 and 1.3 mg L(-1) corresponding to 9 pg and 31 pg respectively.

Optimization and validation of liquid chromatography and headspace-gas chromatography based methods for the quantitative determination of capsaicinoids, salicylic acid, glycol monosalicylate, methyl salicylate, ethyl salicylate, camphor and l-menthol in a topical formulation.

Capsaicinoids, salicylic acid, methyl and ethyl salicylate, glycol monosalicylate, camphor and l-menthol are widely used in topical formulations to relieve local pain. For each separate compound or simple mixtures, quantitative analysis methods are reported. However, for a mixture containing all above mentioned active compounds, no assay methods were found. Due to the differing physicochemical characteristics, two methods were developed and optimized simultaneously. The non-volatile capsaicinoids, salicylic acid and glycol monosalicylate were analyzed with liquid chromatography following liquid-liquid extraction, whereas the volatile compounds were analyzed with static headspace-gas chromatography. For the latter method, liquid paraffin was selected as compatible dilution solvent. The optimized methods were validated in terms of specificity, linearity, accuracy and precision in a range of 80% to 120% of the expected concentrations. For both methods, peaks were well separated without interference of other compounds. Linear relationships were demonstrated with R² values higher than 0.996 for all compounds. Accuracy was assessed by performing replicate recovery experiments with spiked blank samples. Mean recovery values were all between 98% and 102%. Precision was checked at three levels: system repeatability, method precision and intermediate precision. Both methods were found to be acceptably precise at all three levels. Finally, the method was successfully applied to the analysis of some real samples (cutaneous sticks).

An improved liquid chromatographic method for the analysis of tylosin and its impurities.

A selective reversed-phase (RP) liquid chromatographic (LC) method coupled with UV for the determination of tylosin and its related substances is described. The gradient method uses a Capcell pak C18 ACR column (25 cm×4.6 mm id, 5 µm) maintained at a temperature of 60°C. The mobile phases consist of acetonitrile, phosphate buffer pH 5.5 and water: (A; 27.5:10:62.5 v/v/v) and (B; 50:10:40 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. It allows the separation of all known and 22 other unknown related substances (≥0.02%) from the main compound and from one another. The method shows good precision, sensitivity, linearity (between 0.2 µg/mL and 1.25 mg/mL) and robustness. The limit of quantification is 0.2 µg/mL, corresponding to 0.020%. Seven bulk tylosin samples containing a large number of impurities were examined using this method.

Capacitively coupled contactless conductivity detection as an alternative detection mode in CE for the analysis of kanamycin sulphate and its related substances.

A method was developed to determine simultaneously kanamycin, its related substances and sulphate in kanamycin sulphate using capacitively coupled contactless conductivity detection. Kanamycin is an aminoglycoside antibiotic that lacks a strong UV-absorbing chromophore. Due to its physicochemical properties, CE in combination with capacitively coupled contactless conductivity detection was chosen. The separation method uses a BGE composed of 40 mM 2-(N-morpholino)ethanesulphonic acid monohydrate and 40 mM L-histidine, pH 6.35. A 0.6 mM N-cetyltrimethyl ammonium bromide (CTAB) solution was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg/L was used as internal standard. In total, 30 kV was applied in reverse polarity on a fused-silica capillary (65/41 cm; 75 µm id). The optimized separation was obtained in less than 6 min with good linearity (R(2)=0.9999) for kanamycin. It shows a good precision expressed as RSD on the relative peak areas equal to 0.3 and 1.1% for intra-day and inter-day precision, respectively. The LOD and LOQ are 0.7 and 2.3 mg/L, respectively. Similarly, for sulphate, a good linearity (R(2)=0.9996) and precision (RSD 0.4 and 0.6% for intra-day and inter-day, respectively) were obtained.

Improving quantitative gas chromatography-electron ionization mass spectrometry results using a modified ion source: demonstration for a pharmaceutical application.

Gas chromatography-mass spectrometry is a well established analytical technique. However, mass spectrometers with electron ionization sources may suffer from signal drifts, hereby negatively influencing quantitative performance. To demonstrate this phenomenon for a real application, a static headspace-gas chromatography method in combination with electron ionization-quadrupole mass spectrometry was optimized for the determination of residual dichloromethane in coronary stent coatings. Validating the method, the quantitative performance of an original stainless steel ion source was compared to that of a modified ion source. Ion source modification included the application of a gold coating on the repeller and exit plate. Several validation aspects such as limit of detection, limit of quantification, linearity and precision were evaluated using both ion sources. It was found that, as expected, the stainless steel ion source suffered from signal drift. As a consequence, non-linearity and high RSD values for repeated analyses were obtained. An additional experiment was performed to check whether an internal standard compound would lead to better results. It was found that the signal drift patterns of the analyte and internal standard were different, consequently leading to high RSD values for the response factor. With the modified ion source however, a more stable signal was observed resulting in acceptable linearity and precision. Moreover, it was also found that sensitivity improved compared to the stainless steel ion source. Finally, the optimized method with the modified ion source was applied to determine residual dichloromethane in the coating of coronary stents. The solvent was detected but found to be below the limit of quantification.