Getu Kahsay

Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics

This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis.

Simultaneous determination of lidocaine hydrochloride, hydrocortisone and nystatin in a pharmaceutical preparation by RP-LC

A liquid chromatographic (LC) method was developed to analyze a formulation (mouthwash) containing lidocaine hydrochloride, hydrocortisone and nystatin. A single LC method with UV detection was developed. A Waters Symmetry C18 HPLC column (150 mm × 4.6 mm, 5 μm) was used as stationary phase and the assay was performed with gradient elution using mobile phases containing methanol - 0.1M NaH(2)PO(4) with a pH that was previously adjusted to 4.5 with dilute phosphoric acid. The sample pretreatment was performed by treating the formulation with methanol followed by filtration. After method development, the influence of the different chromatographic parameters on the separation, the interference of other active compounds and excipients, linearity, accuracy, repeatability and intermediate precision were investigated. The method was shown to be selective, linear, accurate, precise and repeatable. Finally, the content of the compounds in the formulation was determined.

Kinetic performance comparison of fully and superficially porous particles with a particle size of 5 µm: intrinsic evaluation and application to the impurity analysis of griseofulvin.

After the great commercial success of sub-3 µm superficially porous particles, vendors are now also starting to commercialize 5 µm superficially porous particles, as an alternative to their fully porous counterparts which are routinely used in pharmaceutical analysis. In this study, the performance of 5 µm superficially porous particles was compared to that of fully porous 5 µm particles in terms of efficiency, separation performance and loadability on a conventional HPLC instrument. Van Deemter and kinetic plots were first used to evaluate the efficiency and performance of both particle types using alkylphenones as a test mixture. The van Deemter and kinetic plots showed that the superficially porous particles provide a superior kinetic performance compared to the fully porous particles over the entire relevant range of separation conditions, when both support types were evaluated at the same operating pressure. The same observations were made both for isocratic and gradient analysis. The superior performance was further demonstrated for the separation of a pharmaceutical compound (griseofulvin) and its impurities, where a gain in analysis time of around 2 could be obtained using the superficially porous particles. Finally, both particle types were evaluated in terms of loadability by plotting the resolution of the active pharmaceutical ingredient and its closest impurity as a function of the signal-to-noise ratio obtained for the smallest impurity. It was demonstrated that the superficially porous particles show better separation performance for griseofulvin and its impurities without significantly compromising sensitivity due to loadability issues in comparison with their fully porous counterparts. Moreover these columns can be used on conventional equipment without modifications to obtain a significant improvement in analysis time.

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.

Development and validation of a liquid chromatographic method for purity control of clopidogrel–acetylsalicylic acid in combined oral dosage forms

A reversed phase liquid chromatographic method with UV detection for the simultaneous determination of clopidogrel and acetylsalicylic acid and their related substances in combined oral formulations was developed and validated. Good separation was achieved on a Luna C18 column (150 mm × 4.6 mm, 3 μm) using gradient elution at a flow rate of 1 mL/min and a column temperature of 35 °C. UV detection was performed at 220 nm. The validation was performed according to the ICH guidelines. The method proved to be specific, sensitive (LOQ=0.975 μg/mL and 0.0384 μg/mL for clopidogrel and acetylsalicylic acid, respectively), linear in the concentration range from LOQ to 325 μg/mL for clopidogrel and from LOQ to 650 μg/mL for acetylsalicylic acid, precise (RSD values for intermediate precision <1%) and accurate with mean recovery values of 100.7% and 100.2% for clopidogrel and acetylsalicylic acid, respectively. Moreover, the solution stability and method robustness were examined. The method gives satisfactory separation of impurities of clopidogrel and acetylsalicylic acid and so it is suitable for quantification of the related substances as well as for the assay of the actives.

Development and validation of a reversed phase liquid chromatographic method for analysis of griseofulvin and impurities

A simple and robust reversed phase liquid chromatographic method was developed and validated for the quantitative determination of griseofulvin (GF) and its impurities in drug substances and drug products (tablets). Chromatographic separation was achieved on a Discovery C18 (250mm×4.6mm, 5μm) column kept at 30°C. The mobile phase consisted of a gradient mixture of mobile phase A (water-0.1% formic acid pH 4.5, 80:20, v/v) and B (ACN-water-0.1% formic acid pH 4.5, 65:15:20, v/v/v) pumped at a flow rate of 1.0mL/min. UV detection was performed at 290nm. The method was validated for its robustness, sensitivity, precision, accuracy and linearity based on ICH guidelines. The robustness study was performed by means of an experimental design and multivariate analysis. Satisfactory results were obtained from the validation studies. The use of volatile mobile phases allowed for the identification of three main impurities present above the identification threshold using mass spectrometry (MS). The developed LC method has been applied for the assay and impurity determination of GF drug substances and tablets. The method could be very useful for the quality control of GF and its impurities in bulk and formulated dosage forms.

Characterization of mesoporous silica used for drug delivery by sorptive interaction – multiple headspace extraction–gas chromatography

A multiple headspace extraction experiment coupled to gas chromatography (MHE-GC) is used for the classification and qualification of different mesoporous silica (MPSi) materials used for drug delivery. In this MHE experiment, a pure liquid solvent probe is fully evaporated in a sealed headspace vial in the presence of the MPSi sample, leading to a gas-solid partitioning that is theoretically described. The obtained results matched with the known characteristics of the studied samples, such as adsorption capacity due to differences in porosity and passivation treatments. Moreover, it proves the effectiveness of a poly dimethyl siloxane (PDMS) coating treatment over a thermal one in reducing the specific interactions of the MPSi. In addition, it evidences the important role of confinement effects when the pore diameter is close to the microporosity range. Finally, a simple experiment for fast screening is proposed by comparison of the results obtained for four different probes used as a mixture.

Improved liquid chromatographic method for quality control of spiramycin using superficially porous particles

Graphical abstract Figure. No caption available. HighlightsEasy‐to‐use and fast LC method for quality control of spiramycin was developed.Improved separation compared to previously published methods.A strategy using MS to profile impurities from non‐volatile LC system was applied.Structures of unknown impurities were proposed based on fragmentation data. ABSTRACT This article describes the development and validation of a liquid chromatographic method for spiramycin using a column with superficially porous particles. Gradient elution was applied and the mobile phase consisted of phosphate buffer (0.2 M; pH 8.3) – H2O – acetonitrile in a ratio 10:60:30 (v/v/v) for mobile phase A and 10:30:60 (v/v/v) for mobile phase B. UV detection was performed at 232 nm. Compared to previous methods, the analysis time was about two times faster and impurities were better separated. Furthermore, impurities which were present above 0.25% were characterized using liquid chromatography coupled with mass spectrometry (LC/MS).

Development and validation of LC methods for the separation of misoprostol related substances and diastereoisomers

Misoprostol is a synthetic prostaglandin E1 analogue which is mainly used for prevention and treatment of gastric ulcers, but also for abortion due to its labour inducing effect. Misoprostol exists as a mixture of diastereoisomers (1:1) and has several related impurities owing to its instability at higher temperatures and moisture. A simple and robust reversed phase liquid chromatographic (RPLC) method is described for the separation of the related substances and a normal phase (NP) LC method for the separation of misoprostol diastereoisomers. The RPLC method was performed using an Ascentis Express C18 (150 mm × 4.6 mm, 5 μm) column kept at 35 °C. The mobile phase was a gradient mixture of mobile phase A (ACN-H2O-MeOH, 28:69:3 v/v/v) and mobile phase B (ACN-H2O-MeOH, 47:50:3 v/v/v) eluted at a flow rate of 1.5 mL/min. UV detection was performed at 200 nm. The NPLC method was undertaken by using an XBridge bare silica (150 mm × 2.1 mm, 3.5 μm) column at 35 °C. The mobile phase contained 1-propanol-heptane-TFA (4:96:0.1%, v/v/v), pumped at a flow rate of 0.5 mL/min. UV detection was performed at 205 nm. This LC method can properly separate the two diastereoisomers (Rs > 2) within an analysis time of less than 20 min. Both methods were validated according to the ICH guidelines. Furthermore, these new LC methods have been successfully applied for purity control and diastereoisomers ratio determination of misoprostol bulk drug, tablets and dispersion.

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.