Influence of the solvent system on the stability of doxycycline solutions.

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.

Combined use of liquid chromatography with mass spectrometry and nuclear magnetic resonance for the identification of degradation compounds in an erythromycin formulation.

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.

Mass spectrometric characterization of gentamicin components separated by the new European Pharmacopoeia method.

Liquid chromatography combined with pulsed electrochemical detection (LC-PED) is the method of choice in the European Pharmacopoeia for the determination of gentamicin and its related substances. A recently approved improved LC-PED method, with a reversed-phase C(18) column and a mobile phase consisting of trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), sodium hydroxide and acetonitrile, showed better separation and more sensitive detection of the gentamicin components than the previous method using a polymer column. More unknown peaks can be separated from the main components and from each other. As the LC-PED method cannot be directly coupled to a mass spectrometer (MS), the unknown substances were collected after the LC column, desalted and analyzed by MS. The structures of the unknown compounds were deduced based on comparison of their fragmentation patterns with those of reference substances investigated by MS(n) experiments using an electrospray ion trap mass spectrometer. A comparison was also made with an already previously published LC-MS method using a volatile mobile phase.

An interlaboratory study on the suitability of a gradient LC-UV method as a compendial method for the determination of erythromycin and its related substances.

A liquid chromatographic (LC) method for the analysis of erythromycin and related substances has been adapted from an isocratic method developed by Chepkwony et al. (2001). The suitability of the method for general application as a compendial (pharmacopoeia) method has been assessed by means of an interlaboratory (collaborative) study. The method involves LC separation on a XTerra C18 column kept at 65 degrees C and UV detection at 210 nm. Five laboratories, located in Europe and the United States (US), participated in the study. Four erythromycin samples were tested. The main components (erythromycin A (EA), erythromycin B (EB), erythromycin C (EC)) and the impurities were determined. The analysis of variance was carried out on the results of the five laboratories to evaluate the between-laboratory consistencies and the laboratory-sample interaction. The estimates for the repeatability and reproducibility of the method, expressed as relative standard deviation (RSD) of the result of the determination of EA, were calculated to be 0.8% and 1.4% respectively. It is concluded that the method examined is a good replacement for the methods currently described in the European Pharmacopoeia (Ph. Eur.) and the United States Pharmacopoeia (USP), especially for its enhanced selectivity.

Controlled release of chlorhexidine from amorphous microporous silica.

A new system for the controlled release of the antiseptic chlorhexidine is presented. Amorphous microporous silica (AMS) excipient material was synthesized via an acid catalyzed sol-gel method and shaped as powder or coating. Chlorhexidine diacetate was introduced into the pores of the AMS silica via the incipient wetness impregnation method. This silica reservoir maintained a slow release of chlorhexidine over more than 7days. Chlorhexidine release was controlled by configurational diffusion in the AMS pores having free diameters of less than 1nm. The release of chlorhexidine was fine tuned by adapting particle size and pore diameter. Controlled release of chlorhexidine from an AMS coating on silicon wafer was demonstrated.

Characterization of impurities in tobramycin by liquid chromatography-mass spectrometry.

The characterization of unknown impurities present in tobramycin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed-phase (RP)-LC method using a volatile mobile phase containing a perfluorinated ion-pair reagent was developed and coupled with an ion trap mass spectrometer. The structures of the unknown impurities were deduced by comparison of their fragmentation patterns with those of the available reference substances obtained by LC-MS(n) experiments.

Development of a liquid chromatographic assay for an anti-HIV tablet containing lamivudine, zidovudine and TMC278.HCL.

A liquid chromatographic method was developed to analyse a tablet containing three anti-human immunodeficiency virus (HIV) compounds: lamivudine, zidovudine and a compound with the code name TMC278.HCl. Due to the presence of UV absorbing chromophores in the three active components, a single LC method with UV detection was developed. A Hypersil BDS C(18) column was used as stationary phase and the assay was performed with gradient elution using mobile phases containing acetonitrile, 0.2M potassium dihydrogen phosphate and water. The sample pretreatment is performed by treating the formulation with dimethyl sulfoxide-water (1:1) followed by filtration. After method development, the influence of the different chromatographic parameters on the separation, the interference of other active compounds and excipients, the repeatability and the linearity were investigated. The method was shown to be robust, selective, linear and repeatable. Finally, the content of the compounds in the tablet was determined.

Development of a liquid chromatography method for the analysis of josamycin.

Out of three methods for the analysis of josamycin, the best one was selected and used as starting point for further development. A central composite design was applied to find the most influencing parameters and to optimize the chromatographic conditions and a full factorial design was used to perform a robustness study. The final method uses a Hypersil ODS column 5microm, 250mmx4.6mm i.d. maintained at 45 degrees C. The mobile phase is composed of acetonitrile-phosphate buffer (pH 3, 0.2moll(-1))-tetrabutylammonium hydrogen sulphate 0.2moll(-1)-water (21:5:3:71, v/v/v/v). Strongly retained impurities after the main peak require gradient elution, which is obtained by increasing linearly the acetonitrile concentration (from 21% to 50%, v/v) and decreasing the TBA concentration (from 3% to 0%, v/v) in the mobile phase. The total run time was 65min. UV detection is performed at 232nm and the flow rate is 1ml/min. The method shows good selectivity, precision, linearity and sensitivity. Five commercial bulk samples were analyzed.

Improved reversed-phase liquid chromatographic method combined with pulsed electrochemical detection for the analysis of amikacin.

A two-step gradient liquid chromatographic method combined with pulsed electrochemical detection is described for the determination of amikacin and its impurities. The mobile phase is composed of an aqueous solution containing 1.8 g/l sodium 1-octanesulphonate, 14 ml/l tetrahydrofuran, 50 ml/l of phosphate buffer pH 3.0 and sodium sulphate, which was 20 g/l in mobile phase A and 28 g/l in mobile phase B. 0.5 M sodium hydroxide was added post-column to enhance the detection. An investigation of different reversed-phase columns indicated that the Discovery (C18, 5 microm, 250 mm x 4.6 mm i.d.) column was the most suitable. Compared to previously published investigations, the proposed method showed higher sensitivity and efficiency, allowing the separation of the main component amikacin from 16 impurities, 7 of which were of unknown identity. A central composite experimental design was used to assess the robustness. The method showed good repeatability and linearity in the assay range. The method was further applied to analyze some commercial samples.

Characterization of impurities in dirithromycin by liquid chromatography/ion trap mass spectrometry.

With a recently developed liquid chromatographic (LC) method, using a phosphate buffer, several unknown impurities present in dirithromycin samples were separated. In this paper, a reversed-phase liquid chromatography-tandem mass spectrometry method is described for the investigation of dirithromycin and related substances. The method employed uses a Zorbax Extend C18 column (250 mm x 4.6 mm I.D.), 5 microm, and a mobile phase consisting of acetonitrile, 2-propanol, water and ammonium acetate solution pH 8.5. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ion (ESI) source operated in the positive ion mode. The LCQ is ideally suited for the identification of related substances because it provides on-line LC/MS(n) capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation behavior of dirithromycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total the structures of nine impurities were elucidated, among which three were different analogues with a modification in the side chain on the oxazine ring. Two impurities showed a different alkyl group in position C13. In two impurities the desosamine sugar was involved with changes in the degrees of methylation of the amino group. One unknown impurity was identified as dirithromycin F and another unknown was characterized as dirithromycin N-oxide.

Evaluation of an International Pharmacopoeia method for the analysis of indinavir sulfate by liquid chromatography.

A gradient LC method for the determination of indinavir sulfate (IDV) and its impurities has been recently published in a consultation document of the International Pharmacopoeia, WHO Drug Information. The method uses a base-deactivated reversed-phase C18 column (25 cm x 4.6 mm i.d.), 5 microm kept at a temperature of 40 degrees C. The mobile phases consist of acetonitrile, phosphate buffer pH 7.5 and water. The flow rate is 1.0 ml/min. UV detection is performed at 220 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards IDV components was investigated on 16 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm i.d.), 5 microm. A central composite design was applied to examine the robustness of the method. The method shows good precision, linearity, sensitivity and robustness. Six commercial samples were examined using this method.

Analysis of neomycin using an improved liquid chromatographic method combined with pulsed electrochemical detection.

An isocratic liquid chromatographic method with pulsed electrochemical detection is described for the determination of neomycin in the presence of its impurities. The mobile phase is composed of an aqueous solution containing 35 g/l of sodium sulphate, 1 g/l of sodium 1-octanesulfonate, 14 ml/l of tetrahydrofuran (THF) and 50 ml/l of 0.2 M phosphate buffer pH 3.0. Sodium hydroxide was added post column to enhance the detection. An investigation of different reversed-phase columns indicated that the Discovery (C18 5 microm, 250 mm x 4.6 mm I.D.) column was the most suitable. The proposed method shows high efficiency, allowing the separation of the main component neomycin B from neomycin C and 15 other impurities. A central composite design was used to assess the robustness of the method. The method showed good selectivity, repeatability, linearity and sensitivity. This method was applied to analyse commercial samples.

Determination of stigmasterol, beta-sitosterol and stigmastanol in oral dosage forms using high performance liquid chromatography with evaporative light scattering detection.

A validated and repeatable high performance liquid chromatography (HPLC) method with online evaporative light scattering (ELSD) was developed for the analysis of two sterols, stigmasterol, beta-sitosterol and a stanol, stigmastanol, found to be common in many herbal formulations and health care supplements. The method is based on the separation of the three marker compounds on a C8 column (Phenomenex Luna, 5 microm, 150 mmx4.6 mm i.d.) using methanol:water (95:5 v/v) as the mobile phase, and a flow rate of 1 ml/min to separate all the marker compounds within 12 min. Cholesterol (50 microg/ml) was used as internal standard and methanol as the extraction solvent. The ELSD response parameters were optimised and the limits of detection (LOD) and quantification (LOQ) were calculated to be 2 and 5 microg/ml, respectively, which is more sensitive than obtained by photo diode array detection (5 and 7 microg/ml). Using ELSD, the percentage relative standard deviation (%R.S.D.) of intra-day and inter-day (3 days) precision for each marker was better than 3%, the accuracy data were within 97-103% and the recovery data were found to be within 95-107% for the five commercially available products examined. This method was used to assay commercially available products formulated as oral dosage forms purported to contain African Potato and associated sterols and stanol and proved to be suitable for the routine analysis and quality control of such products.

Investigation of vancomycin and related substances by liquid chromatography/ion trap mass spectrometry.

Liquid chromatography (LC) methods compatible with mass spectrometry (MS) that are suitable for impurity profiling of vancomycin mixtures have not been described in the literature. The mobile phases of the existing methods contain non-volatile additives and/or solvents that give problems in combination with MS. In this paper, a reversed-phase LC/tandem mass spectrometry method is described for the investigation of vancomycin and related substances. The LC method uses a Zorbax Extend C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of methanol, water and ammonium acetate solution (pH 9.0). This method allows us to separate vancomycin and its impurities. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and negative ion modes. The LCQ is ideally suited for identification of impurities and related substances because it provides on-line LC/MSn capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation of vancomycin and known derivatives was studied and the structures of six substances occurring in commercial samples were elucidated.

Improved LC of minocycline drug substance.

An isocratic liquid chromatographic method is described for the separation of minocycline and its impurities. This method uses XTerra RP-18, 5 microm (25 cm x 4.6 mm I.D.), a silica-based stationary phase with reduced silanol activity. A mobile phase composed of acetonitrile-0.2 M tetrabutylammonium hydrogen sulphate pH 6.5-0.2 M ethylenediaminetetraacetic acid pH 6.5-water (20:20:20:40; v/v/v/v) was used at a flow rate of 1 ml/min. The column temperature was set at 35 degrees C. UV detection was performed at 280 nm. Optimisation of the separation method and a robustness study were performed by means of a central composite experimental design. The method allows to separate minocycline from known impurities. Some unidentified impurities are also separated. The total time of analysis is less than 20 min.

Evaluation of the stability of chlortetracycline in granular premixes by monitoring its conversion into degradation products.

A methodology for the evaluation of the stability of chlortetracycline (CTC) in granular premixes is described. This methodology is based on the monitoring of the conversion of CTC into its degradation products by an improved gradient liquid chromatography (LC) method, based on one previously described by our laboratory. Sample preparation involves the extraction of CTC and its degradation products prior to LC analysis, using acidified methanol as extraction solvent. The gradient elution LC method proved to be very sensitive, especially towards the late eluting anhydro derivatives. The use of a Hypersil C8 BDS, 5 microm, 250 mm x 4.6 mm i.d. column is recommended since this column allowed a complete separation of the different impurities from each other and from the main component CTC. The applicability of this approach was demonstrated by the analysis of stability samples.

Development of a liquid chromatographic method for ear drops containing neomycin sulphate, polymyxin B sulphate and dexamethasone sodium phosphate.

Two liquid chromatographic methods were developed to analyse ear drops containing neomycin sulphate, polymyxin B sulphate and dexamethasone sodium phosphate. This formulation will be described in the Belgian National Formulary. Since neomycin, an aminoglycoside antibiotic, has no UV chromophore and pre or post column derivatization is complicated, pulsed electrochemical detection on a gold electrode was chosen to determine neomycin. Polymyxin B sulphate and dexamethasone sodium phosphate do have a UV chromophore. So, a single LC method with UV detection was developed for the determination of polymyxin B sulphate and dexamethasone sodium phosphate. The sample pretreatment is simply done by diluting the formulation with water. For each method, the influence of the different chromatographic parameters on the separation, the interference of other active compounds and excipients, the repeatability and the linearity were investigated. Finally, the content of the actives in the formulation was studied at 0, 2, 4, 6, and 8 weeks.

Analysis of purity in 19 drug product tablets containing clopidogrel: 18 copies versus the original brand.

In this study, 18 copies of PLAVIX tablets containing clopidogrel hydrogensulfate were compared to the innovator drug product for uniformity of mass, impurity profile, content, dissolution properties and stability. In order to be able to separate the R-enantiomer of clopidogrel, an enantiospecific liquid chromatographic method was used to determine the impurities and to perform the assay. The paddle method was used for dissolution testing. Most of the copies were not similar compared to the original drug product: their amount of impurities was higher, the content of clopidogrel lower, the dissolution profiles different and after 3 months under stress conditions in the original packaging, the results for the samples and the reference were significantly different in most of the cases.

Hyphenation of liquid chromatography to ion trap mass spectrometry to identify minor components in polypeptide antibiotics.

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.

Analysis of erythromycin and benzoylperoxide in topical gels by liquid chromatography.

Gels containing a combination of erythromycin and benzoylperoxide are frequently used in the treatment of acne vulgaris. A method was developed to determine the content of both erythromycin and benzoylperoxide in these gels. Erythromycin was extracted from the gel in conditions where the oxidative power of benzoylperoxide was neutralised by addition of ascorbic acid and this extract was analysed on an Xterra RP(18) column, with a mobile phase containing acetonitrile-0.2 M K2HPO4-water (35:5:60, v/v/v). The detection wavelength was 215 nm. A second extraction procedure was developed for the analysis of benzoylperoxide. The extraction solution was analysed on a Hypersil C(18) BDS column and a mobile phase containing acetonitrile-water (58:42, v/v). Detection was performed at 254 nm. The flow rate was 1.0 ml/min in both methods. The selectivity, repeatability, linearity and recovery of both methods were examined. Special attention was given to determination of the recovery and the uncertainty on the recovery. This allowed evaluation of the bias of the extraction method. The method developed was used to examine the stability of a gel for topical use.