Liquid Chromatographic Analysis of Various Formulations Containing Emtricitabine

A gradient liquid chromatographic (LC) method for control of emtricitabine (FTC) was validated for the analysis of FTC formulations (capsules and oral solution) and fixed-dose-combination tablets containing FTC [FTC combined with tenofovir disoproxil fumarate (TDF) and FTC combined with TDF and efavirenz (EFV)]. The method is based on the purity test recently prescribed in the International Pharmacopoeia and uses a Hypersil BDS C18 column (25 cm × 4.6 mm i.d.), 5 μm kept at a temperature of 35 C. Other reversed-phase columns were also investigated. The mobile phases for gradient elution consist of acetonitrile, phosphate buffer and water. The flow rate is 1.0 mL min -1 and UV detection is performed at 280 nm. The method is capable of separating the main components from one another, from the inactive ingredients and from the main degradation products. The method was validated with respect to accuracy, precision, sensitivity and linearity for each component and the solution media were optimized. Finally, commercial FTC capsules, FTC oral solution, FTC/TDF tablets and FTC/TDF/EFV tablets were examined. © 2013 Springer-Verlag Berlin Heidelberg.status: publishe

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 (250mmÃ4.6mm i.d., 5μm), isocratic elution was carried out with a mobile phase, containing sodium sulfate (35g/L), sodium octanesulphonic acid (1g/L), tetrahydrofuran (14mL/L) and 0.2M phosphate buffer pH 3.0 (50mL/L). Using these experimental conditions, the limit of quantification (LOQ, S/N=10) was 5ng. The linearity was examined in the range LOQ-60μg/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. Keywords: Liquid chromatography-Pulsed electrochemical detection, Tobramycin, Aminoglycoside antibiotic

LC with electrochemical and UV detection for analysis of a formulation containing gentamicin and parabens

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.status: publishe

Analysis of Temocillin and Impurities by Reversed Phase Liquid Chromatography: Development and Validation of the Method

© 2014, Springer-Verlag Berlin Heidelberg. A robust, specific, precise and sensitive high-performance liquid chromatographic method has been described for purity control of temocillin. Chromatographic separation was achieved using a Symmetry C18 (150 × 4.6 mm, 5 µm) column kept at 30 °C. The mobile phase consisted of a gradient mixture of mobile phases A (5 g/L solution of Na2HPO4·2H2O, pH 7) and B (ACN-MeOH-H2O, 50:10:40 v/v/v) pumped at a flow rate of 1.0 mL/min. UV detection was performed at 235 nm. The developed method was validated according to the ICH guidelines for its robustness, selectivity, sensitivity, precision and linearity. An experimental design was applied for the robustness study. Linearity was assessed both at impurity level in the range from LOQ to 10 % and assay level from 25 % to 150 % (0.6 mg/mL = 100 %). It is the first liquid chromatographic method described for the separation of temocillin and its potential impurities. It was possible to identify four degradation products from the forced degradation studies. The degradants do not interfere with the main peak and other known impurities showing that the method is specific and stability-indicating.status: publishe

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.status: publishe

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 5 µm, 250 × 4.6 mm I.D. maintained at 45 °C. The mobile phase is composed of acetonitrile – phosphate buffer (pH 3, 0.2 mol.l-¹) – tetrabutylammonium hydrogen sulphate 0.2 mol.l-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 65 min. UV detection is performed at 232 nm and the flow rate is 1 ml/min. The method shows good selectivity, precision, linearity and sensitivity. Five commercial bulk samples were analyzed.status: publishe

Analysis of Dideoxyadenosine Triphosphate by Capillary Electrophoresis with Fluorescence Detection. Derivatization Through the Adenine Group

The adenine base of 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) was chosen as the target for precapillary derivatization for its analysis by CE with fluorescence detection. Several fluorophores, such as chloroacetaldehyde, 9-fluorenylmethyloxycarbonylchloride (FMOC), fluorescamine (Fluram), o-phthaldialdehyde (OPA) and complexation with terbium (Tb), gadolinium (Gd), and phenanthroline (phen) were tried. Precapillary derivatization of ddATP with chloroacetaldehyde showed poor peak shape for the epsilon-ddATP adduct and degradation peaks of epsilon-adenine, possibly due to the low sample pH of 4.0. Precapillary derivatization of ddATP with OPA/-mercaptoethanol, Fluram, and FMOC showed no fluorescent derivatization products. Complexation of ddATP with Tb-Gd-phen produced a sharp fluorescent peak. The sensitivity of the Tb-Gd-ddATP-phen complex was found to be 20 times higher than UV detection of ddATP.status: publishe

Liquid chromatographic separation of hexopyranosylated cytosine nucleosides from their degradation products

Development of a liquid chromatographic method which can separate each of a series of hexopyranosylated cytosine nucleosides from their degradation products formed at acid, neutral and basic pH is described. Both silica-based reverse-phase and polymer columns were examined. Influence of the mobile phase pH, ion-pairing agent, concentration of the buffer and type and concentration of organic modifier were systematically investigated. The concentration of the ion-pairing agent and the buffer were found to have a major effect on selectivity. Samples were finally analyzed on a poly(styrene-divinylbenzene), PLRP-S 100 Angstrom (8 mu m) 250 x 4.6 mm I.D. column at 60 degrees C and with a mobile phase consisting of acetonitrile-sodium octanesulphonate (pH 2.5; 0.02 M)-potassium phosphate buffer (pH 2.5; 0.2 M)-water (X:25:50:25 -X, v/v, where X is variable). (C) 1997 Elsevier Science B.V