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Concentrated solutions of doxycycline are often added to drinking water of animals for oral antibiotic therapy. However, stability concerns of doxycycline in solution involve an accurate selection of the solvent system to ensure that the active substance will remain within the acceptance range during the product shelf-life and to avoid sub-therapeutic dosage. Different solvent systems have been evaluated in order to determine their influence on the stability of concentrated doxycycline solutions. The results showed differences in the degradation kinetics of doxycycline depending on the co-solvent used and they permitted to select a solvent system for liquid doxycycline hyclate formulations with low rate of degradation even after several months of storage. So, the inclusion of ethanol together with propylene glycol as main excipient was found to be beneficial, while no benefit was observed concerning the addition of citric acid. Once administered to drinking water, the solutions were stable for 24 h with no influence of the solvent system. ispartof: JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS vol:159 pages:60-65 ispartof: location:England status: published

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.

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.

The estimation of chronological age in cadavers, human remains and in living human beings by various methods is discussed. These methods, which are based on the age dependent non-enzymatic changes of l-form amino acids to d-form amino acids, mainly aspartic acid, are among the most reliable and accurate methods to date. Most of these methods use gas chromatography (GC). In this review, results of aspartic acid racemization in dentin at different targets are discussed. In addition, pre-considerations and guidelines are given for the selection of dentin from teeth. A pilot project was run to evaluate the efficiency of high performance liquid chromatography (HPLC) coupled with fluorescence detection. New buffer conditions were found to obtain stable derivatives of aspartic acid enantiomers for the estimation of racemization.

The application of liquid chromatography–ion trap mass spectrometry for the characterization of linear and cyclic polypeptide antibiotics was investigated. The aim was on-line identification of impurities in those antibiotic complexes without recourse to time-consuming isolation and purification procedures. Hyphenated techniques, such as liquid chromatography coupled to mass spectrometry, are ideally suited for this purpose. Characterization was performed with an ion trap mass spectrometer offering MS n capability; this enables more structural information to be obtained. Liquid chromatography in combination with ion trap mass spectrometry was successfully applied for the characterization of impurities in gramicidin, polymyxin B, polymyxin E, and bacitracin and the study of the degradation products of polymyxins B and E.

An isocratic liquid chromatographic method with UV detection at 215 nm, which is suitable for the analysis of azithromycin (AZT) in bulk samples, is described. AZT is separated from its synthesis intermediates and a degradation product as well as from six unknown impurities on an XTerra RP18 column at 70 degrees C using a mobile phase consisting of acetonitrile-pH 6.5 0.2M K2HPO4-water (35:10:55, v/v/v) at 1.0 mL/min. The XTerra stationary phase contains methyl groups that are incorporated in the bulk structure of the material. This allows for special selectivities. Robustness is evaluated by a full factorial design experiment. The method shows good selectivity, repeatability, linearity, and sensitivity.

Organic impurity determination is a critical aspect since it has a great impact on the assigned content of chemical reference standards (CRS). This is especially the case for CRS of natural products (CRSNPs) which are mostly obtained from medicinal plants or animals. Total amount of organic impurities of CRSNPs is generally determined by high performance liquid chromatography based on official or reported methods. Since the brand of column is not always prescribed or when the brand prescribed is not available in the laboratory, this can cause serious consequences, especially when the sample contains a basic or weak acid compound with p K a values ranged from 3 to 6. In this paper, columns were originally selected based on a peer-reviewed column characterization database. Firstly two columns with orthogonal selectivity (selectivity factor F ≥6) were selected from the database to determine the purity of the respective CRSNPs. The selection was performed to minimize the risk to obtain different values caused by use of various brands of columns. If there is no significant difference between their results, then the purity result is cross-validated. Otherwise, a third column has to be selected from the database and its selectivity should be similar to the above one which give better separation ( F ≤2). The approach has been illustrated by the purity determinations of N - trans - p -coumaroyltyramine and (-)-epicatechin-3- O -gallate CRSNPs, and it is most likely to be applied in more CRSNPs, especially those of basic or weak acid ones.

Preparative-scale separation of colistin sulphate bulk sample was carried out on a preparative poly(styrene-divinylbenzene) stationary phase. Isocratic elution with acetonitrile-sodium sulphate solution (0.7% m/v; pH adjusted to 2.5 with TFA) - water (16:50:34, % v/v/v) was carried out at a flow rate of 4.0 ml min(-1). Six colistin components were isolated and characterized using 1H and 13C NMR. The molecular weights were confirmed by mass spectrometry. The structures of 2 components were determined for the first time. Polymyxin E7 was identified as having the same composition as polymyxin E1, except that the fatty acid moiety was 7-methyloctanoic acid. Isoleucine polymyxin E8 was characterized as having the same composition as isoleucine polymyxin E1 with 7-methylnonanoic acid as the fatty acid moiety.

A method is described for quantitative analysis of sorbic acid in fat soluble cream formulations. A poly(styrene-divinylbenzene) column (PLRP-S 8 μm 100 A, 250×4.6 mm I.D.), maintained at 60°C was used. The mobile phase was acetonitrile–0.2 M phosphate buffer (pH 7.0)–water (1:5:94, v/v/v). The flow-rate was 1.0 ml min −1 and UV detection was performed at 254 nm. Sample preparation was carried out by treatment of the cream formulations with a mixture of hexane and mobile phase. The method showed good repeatability, linearity and recovery. It was observed that in most of the creams examined the stability of sorbic acid was poor.

This work describes two separate liquid chromatographic (LC) methods which were developed to control gentamicin sulphate and its preservatives methylparaben and propylparaben in an injectable formulation for veterinary purposes. Owing to the absence of a UV absorbing chromophore in the gentamicin molecule, LC combined with pulsed electrochemical detection (LC-PED) was found to be suitable for the determination of this drug in the formulation. The LC-PED methods from edition 7.0 (valid till 06/2012) and 7.5 (supplement valid from 07/2012) of the European Pharmacopoeia (Ph. Eur.) were compared. The currently recommended LC-PED method allowed good separation between the main gentamicin components and their impurities without interference from additives. Because of its better performance the actual method was used to investigate the degradation profile of the gentamicin sulphate injection. Results obtained from the forced stability study showed that the formulation was stable to oxidative and thermal stress. For the control of the preservatives, a LC-UV method was applied. Both methods were found to be specific, linear and precise. Hence, these two LC methods can be used for routine analysis of the gentamicin sulphate injection. © 2013 The Royal Society of Chemistry. ispartof: Analytical Methods vol:5 issue:10 pages:2491-2496 status: published

A liquid chromatography method has been developed for quantitative analysis of a mixture of methyl and propyl p-hydroxybenzoates in water and fat soluble ointment formulations. The method employs a PLRP-S 8 μm 100 A, 250 × 4.6 mm i.d. column maintained at 60°C and a mobile phase acetonitrile-potassium phosphate buffer (pH 7.0; 0.2 M)-water (35:5:60, v/v/v), at a flow rate of 1.0 ml min−1. The analytes were detected by UV at 254 nm. Water soluble formulations were dissolved in acetonitrile -water (40:60, v/v). Fat soluble formulations were dissolved in hexane and extracted with acetonitrile-water (60:40, v/v). The method showed good selectivity, repeatability, reproducibility, linearity and recovery.

An isocratic liquid chromatographic method for the assay and purity control of josamycin using wide-pore poly(styrene-divinylbenzene) as the stationary phase is presented. Josamycin has been well separated from all the related leucomycins, present as impurities in commercial samples. Wide-pore PLRP-S 8 μm 1000 A has shown the best selectivity. The separation has been carried out at 65°C with a mobile phase of 2-methyl-2-propanol/0.2 mol/l potassium phosphate buffer pH 10.5/water (24.5: 5: 70.5, V/V) and a flow rate of 1.0 ml/min. UV detection has been performed at 232 nm. The total analysis time has been about 40 min. The method has been used to analyse commercial bulk samples and tablets containing josamycin base.

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. ispartof: Journal of Pharmaceutical Analysis vol:3 issue:3 pages:161-167 ispartof: location:China status: published

Cefpiramide is susceptible to many factors, including high temperature. Two solid states were observed in cefpiramide: a crystalline form and an amorphous one, and both the accelerated (40°C ± 2°C /75% ± 5% relative humidity (RH)) and stress (60°C ± 2°C / 75% ± 5% RH) stability study demonstrated that the crystalline form of cefpiramide was much more stable than its amorphous form, with related substances, high molecular mass polymers and assay as critical stability indicating parameters. Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and water content results indicated that each molecule of crystalline cefpiramide may contain two molecules of crystal water, which can explain its high thermal stability. This study provides valuable information for cefpiramide and its drug products with respect to the improvement of its thermal stability through crystallization.

A commercial erythromycin formulation containing erythromycin A (EA) as the major compound showed the presence of an unknown degradation compound that was co-eluted with erythromycin E (EE) in the European Pharmacopoeia (Ph. Eur.) liquid chromatographic (LC) method. The amount of the degradation compound increased with respect to time. To separate this unknown (UNK1), investigation was performed with different LC methods coupled to ultraviolet detection (LC-UV). With the present Ph. Eur. method, the degradation compound could not be well separated. However, with the most selective LC-UV method (XTerra method), two more degradation products (UNK2 and UNK3) were found in the formulation which could not be observed using other methods because of their poor separation. By combining the results obtained with LC-UV, LC/MS and LC/NMR, the degradation products were identified as pseudoerythromycin A hemiketal (PsEAHK), erythromycin A enol ether carboxylic acid and erythromycin C enol ether carboxylic acid. PsEAHK is known to be a base-catalysed degradation product of EA, whereas the other two degradation products were newly identified.

The estrogenic and anti-inflammatory activities of 3-methoxy-16, 17-seco-16-norestra-1,3,5-trien-15-(2'-indoxyliden)-17-oic acid is reported. After intraperitoneal administration, the dose of this compound required to reduce swelling of the rat paw by 50% (ED 50 ) was 14.1 mg/kg using the carrageenan-induced rat paw oedema anti-inflammatory assay method. Indomethacin had an ED 50 of 3.2 mg/kg in this assay while dexamethasone had an ED 50 of 1.7 mg/kg. The estrogenic activity of the compound after intramuscular administration in rats was 0.72 relative to diethylstilbestrol, when the two compounds were assayed at three dose levels of 1.0, 0.3 and 0.1 mg/kg. Key Words: Steroidal indoxyl, synthesis, estrogenic, anti-inflammatory East and Central African Journal of Pharmaceutical Sciences Vol.5(3) 2002: 44-48

A gradient liquid chromatographic method which can separate isoniazid, pyrazinamide and rifampicin is described. A Hypersil C18, 5 mm, 250 mm x 4.6 mm internal diameter column was maintained at 40°C. The method was developed by systematic evaluation of the influence of the buffer concentration, column temperature and the mobile phase pH. The method proposed uses isocratic elution with potassium phosphate buffer (pH 6.0; 0.05 M) for 10 min, followed by linear gradient to potassium phosphate buffer (pH 6.0; 0.05 M)-methanol (40:60, v/v) in 5 min, isocratic elution at the same composition for a further 15 min and then linear gradient back to potassium phosphate buffer (pH 6.0; 0.05 M) in 5 min. The flow-rate was 1 ml/min and UV detection was at 254 nm. The method was validated and it has been used for routine analysis of tablets containing isoniazid, pyrazinamide and rifampicin. Analysis time is 35 minutes. (E & C Afr Jnl Pharm Sci: 2002 5(1): 8-14)

A new liquid chromatography method for the analysis of mangiferin (I) from Mangifera indica L. stem bark extract (Vimang®), and pharmaceuticals is described. Screening experiments were performed by an experimental design to find the influence of some important chromatographic variables (methanol, column temperature and acetic acid) on the retention times and resolution between critical pairs in the separation. This design also permitted to estimate method robustness and optimal conditions to achieve the best resolution. The best separation was found using a LiChrospher RP 18, 5 µm, 250 x 4.6 mm I.D. column maintained at 25°C, a mobile phase comprising methanol-2.5% v/v aqueous solution of acetic acid (280:720, v/v) at a flow rate of 1.0 ml/min, and detection by UV at 254 nm. The method resolves I, the major component, from other components of the extract. The method showed good selectivity, repeatability (RSD < 2%) and linearity (r = 0.9998). The limits of detection and quantitation were 0.008% (0.9 ng) and 0.05% (6.2 ng), respectively, relative to a 0.6 mg/ml standard solution, injection volume 20 µl. This method was used to quantify I in some aqueous extracts from the natural product and pharmaceuticals.

Five isocratic reversed-phase liquid chromatography (LC) methods have been examined for the separation of phenoxymethylpenicillin and its related substances. A method previously selected for the analysis of benzylpenicillin gave the best selectivity. The mobile phase of this method is composed of 0.5 M phosphate buffer (pH 3.5)—water—methanol (10:50:40), the amount of organic modifier was slightly adapted when it was used with different columns. Similar selectivity is obtained not only on different C18 materials but also on C8 packings. The selectivity on poly(styrenedivinylbenzene) is less good. As method performance test a resolution test with benzylpenicillin was used. The robustness of this method was examined by applying a full factorial design to test the influence of the content of organic modifier in the mobile phase, the pH of the mobile phase, the concentration of buffer in the mobile phase and the temperature of the column. The results show that the method is robust.

The identification of five novel compounds, pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-N-demethylerythromycin A-6,9-hemiketal, 5-O-beta-D-desosaminylerythronolide A and 15-nor-erythromycin C, in mother liquor concentrates of Streptomyces erythraeus is described. The pseudo-erythromycin derivatives are characterized by a 12-membered macrocyclic ring as a result of C13----C11 trans-lactonization. The five compounds have very little antimicrobial activity.

The ultraviolet, infrared, and proton magnetic resonance spectra of 16 commercially available cephalosporins were recorded. The spectra are interpreted, and their value for identification is discussed.

The MICs of erythromycins A, B, C, and D and some of their derivatives were determined against 21 gram-positive and 15 gram-negative microorganisms. Antibacterial activity was confined to gram-positive and very few gram-negative bacteria. Erythromycin B was somewhat less active than erythromycin A, and erythromycin C and D showed about half that activity or even less. Most other derivatives had negligible activity. Determination of potency by diffusion and turbidimetric assays were in line with MICs. The examination of the results of these assays, however, revealed that there are differences between the data of different laboratories, depending on the microorganisms and conditions used.