Martin Steppe

Validation of HPLC and UV spectrophotometric methods for the determination of meropenem in pharmaceutical dosage form

A high-performance liquid chromatographic method and a UV spectrophotometric method for the quantitative determination of meropenem, a highly active carbapenem antibiotic, in powder for injection were developed in present work. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by reversed-phase technique on an RP-18 column with a mobile phase composed of 30 mM monobasic phosphate buffer and acetonitrile (90:10; v/v), adjusted to pH 3.0 with orthophosphoric acid. The UV spectrophotometric method was performed at 298 nm. The samples were prepared in water and the stability of meropenem in aqueous solution at 4 and 25 degrees C was studied. The results were satisfactory with good stability after 24 h at 4 degrees C. Statistical analysis by Students t-test showed no significant difference between the results obtained by the two methods. The proposed methods are highly sensitive, precise and accurate and can be used for the reliable quantitation of meropenem in pharmaceutical dosage form.

Determination of dexamethasone acetate in cream by HPLC

The aim of this research was to validate a high performance liquid chromatographic method for the quantitative determination of dexamethasone acetate contained in cream preparation. A MetaSil octadecyl silane (250 x 4.6 mm, 5 microm) column, a methanol: water (65:35; v/v) mobile phase (1.0 ml min(-1)) and an UV detector (set at 254 nm) were used to evaluate the parameters: linearity, precision, accuracy, specificity, as well as, quantitation and detection limits. The calibration curve showed a correlation coefficient of 0.9999. The precision was demonstrated by the relative standard deviation (RSD) of 0.53. The recovery test resulted in an average of 97.85%, what confirmed the accuracy of the method. The quantitation and detection limits determined were 1.41 and 0.47 microg ml(-1), respectively. The specificity test showed there was no interference in the drug peak.

Microbiological assay for azithromycin in pharmaceutical formulations.

The validation of a microbiological assay, applying the cylinder-plate method, for the determination of the antibiotic azithromycin is described. Using a strain of Micrococcus luteus ATCC 9341 as the test organism, azithromycin at concentrations ranging from 0.1 to 0.4 microgml(-1) could be measured in capsules and suspensions. A prospective validation of the method showed that it was linear (r=0.998), precise (RSD=1.40-capsules; RSD=1.19-powder for suspension and RSD=1.73-oral suspension) and accurate (it measured the added quantities). We conclude that the microbiological assay is satisfactory for quantitation of in vitro antibacterial activity of azithromycin.

Simultaneous determination of aliskiren and hydrochlorothiazide from their pharmaceutical preparations using a validated stability‐indicating MEKC method

A stability-indicating MEKC method was developed and validated for the simultaneous determination of aliskiren (ALI) and hydrochlorothiazide (HCTZ) in pharmaceutical formulations using ranitidine as an internal standard (IS). Optimal conditions for the separation of ALI, HCTZ and its major impurity chlorothiazide (CTZ), IS and degradation products were investigated. The method employed 47 mM Tris buffer and 47 mM anionic detergent SDS solution at pH 10.2 as the background electrolyte. MEKC method was performed on a fused-silica capillary (40 cm) at 28°C. Applied voltage was 26 kV (positive polarity) and photodiode array (PDA) detector was set at 217 nm. The method was validated in accordance with the ICH requirements. The method was linear over the concentration range of 5-100 and 60-1200 μg/mL for HCTZ and ALI, respectively (r(2) >0.9997). The stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Precision and accuracy evaluated by RSD were lower than 2%. The method proved to be robust by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of ALI and HCTZ both individually and in a combined dosage tablet formulation to support the quality control.

Stability-Indicating RP-LC Method for the Determination of Vildagliptin and Mass Spectrometry Detection for a Main Degradation Product

A simple, precise and stability-indicating reversed-phase liquid chromatography method was developed and validated for the determination of vildagliptin (VLG) in pharmaceutical dosage form. The chromatographic separation was obtained within 6 min and was linear in the range of 20-80 µg/mL (r(2) = 0.9999). Limit of detection and limit of quantitation were 0.63 and 2.82 µg/mL, respectively. The method was validated in accordance with International Conference on Harmonization acceptance criteria for specificity, linearity, precision, accuracy, robustness and system suitability. Stress studies were carried out and no interference of the degradation products was observed. The excipients did not interfere in the determination of VLG. Furthermore, the main degradation product obtained from the stress studies (thermal, oxidative and alkaline hydrolysis) was evaluated for mass spectrometry and its molecular structure was predicted. The proposed method was successfully applied for the quantitative analysis of VLG in tablet dosage form, which will help to improve quality control and contribute to stability studies of pharmaceutical tablets containing this drug.

Development and Validation of Derivative Spectrophotometric Method for Determination of Rabeprazole Sodium in Pharmaceutical Formulation

Abstract The aim of this work is to develop and validate the derivative spectrophotometric method for determination of the proton pump inhibitor rabeprazole sodium in pharmaceutical formulations. The technique was applied using water (pH 10.0) as diluent. The first‐order derivative spectra were obtained at N=5, Δλ=4.0 nm, and determinations were made at 304 nm. The method showed high specificity in the presence of formulation excipients and good linearity in the concentration range of 6.0 to 18.0 µg/mL−1. The intra‐ and interday precision data demonstrated the method has good reproducibility [Relative Standard Deviation ((RSD)=1.0 interdays)]. Accuracy was also evaluated and results were satisfactory (mean recovery of 99.15%). The detection and quantitation limits were 0.055 and 0.17 µg/mL−1, respectively. The method was demonstrated to be adequate for routine analysis in quality control.

Gemifloxacin mesylate (GFM) stability evaluation applying a validated bioassay method and in vitro cytotoxic study.

The validation of a microbiological assay applying the cylinder-plate method to determine the quinolone gemifloxacin mesylate (GFM) content is described. Using a strain of Staphylococcus epidermidis ATCC 12228 as the test organism, the GFM content in tablets at concentrations ranging from 0.5 to 4.5 μg mL(-1) could be determined. A standard curve was obtained by plotting three values derived from the diameters of the growth inhibition zone. A prospective validation showed that the method developed is linear (r=0.9966), precise (repeatability and intermediate precision), accurate (100.63%), specific and robust. GFM solutions (from the drug product) exposed to direct UVA radiation (352 nm), alkaline hydrolysis, acid hydrolysis, thermal stress, hydrogen peroxide causing oxidation, and a synthetic impurity were used to evaluate the specificity of the bioassay. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Students t test, which indicated that there was no statistically significant difference between these two validated methods. These studies demonstrate the validity of the proposed bioassay, which allows reliable quantification of GFM in tablets and can be used as a useful alternative methodology for GFM analysis in stability studies and routine quality control. The GFM reference standard (RS), photodegraded GFM RS, and synthetic impurity samples were also studied in order to determine the preliminary in vitro cytotoxicity to peripheral blood mononuclear cells. The results indicated that the GFM RS and photodegraded GFM RS were potentially more cytotoxic than the synthetic impurity under the conditions of analysis applied.

Fesoterodine stress degradation behavior by liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry

In the present study, a rapid validated stability-indicating LC method was established and comprehensive stress testing of fesoterodine was carried out according to ICH guidelines. Fesoterodine was subjected to stress conditions of acid and basic hydrolysis, oxidation, photolysis and thermal decomposition. The degradation products formed under stress conditions were investigated by LC-UV and LC-ESI-MS. Successful separation of the drug from its degradation products was achieved on a monolithic C(18) column (100 mm × 4.6mm i.d.) maintained at 45°C using acetonitrile-methanol-0.03 mol L(-1) ammonium acetate (pH 3.8) (30:15:55, v/v/v) as the mobile phase. The flow rate was 2.4 mL min(-1) and the detection wavelength was 208 nm. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated. Chromatographic separation was obtained within 2.5 min and it was suitable for high-throughput analysis. Fragmentation patterns of degradation products formed under different stress conditions were studied and characterized through LC-ESI-MS fragmentation. Based on the results, a drug degradation pathway was proposed, and the validated LC method was successfully applied to the quantitative analysis of fesoterodine in tablet dosage forms, helping to improve quality control and to assure therapeutic efficacy.

Validation of UV Spectrophotometric Method for Quantitative Determination of Entacapone in Tablets Using Experimental Design of Plackett‐Burman for Robustness Evaluation and Comparison with HPLC

Abstract Validation of UV spectrophotometric method for quantitative determination of entacapone in tablets using acetonitrile as solvent. The validation of analytic method was realized through the study of the following analytic parameters: specify linearity, precision, accuracy, and robustness. The excipients of the formulation did not interfere at 305 nm, demonstrating the specificity of the method. The method was linear (r=0.99996) at concentrations ranging from 3.0 to 20.0 µg ml−1, precise (repeatability and intermediated precision), exact (method of standard addition), and robust. The results confirmed that the method is valid and useful to the routine quality control of entacapone in coated tablets. The method was compared to a high‐performance liquid chromatography (HPLC) method, which was previously developed and validated to the same drug. There was not a significant difference between the methods for entacapone quantitation.

A simultaneous assay method using capillary zone electrophoresis for a fixed dose combination of vildagliptin and metformin hydrochloride in coated tablets

In the present study, a quick and reliable validated capillary zone electrophoresis (CZE) method was established for the simultaneous determination of vildagliptin (VLG) and metformin hydrochloride (MET) in pharmaceutical dosage form. Successful separation of the drugs by the CZE method was achieved in a fused silica capillary by applying a potential of 25 kV (positive polarity) and hydrodynamic injection by 50 mbar for 5 s. The selected running buffer consisted of 25 mM sodium tetraborate decahydrate solution (array pH 9.0) with UV/photodiode (PDA) detection at 207 nm and 250 nm for VLG and MET respectively. Specificity, linearity, precision, accuracy, limit of detection and limit of quantitation and robustness were established for VLG and MET in accordance with International Conference on Harmonization (ICH) guidelines. The specificity and stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using PDA detection. Electrophoretic separation was obtained within 10 min and the method was linear in the range of 30–60 μg mL−1 and 300–600 μg mL−1 for VLG and MET, respectively (R2 > 0.9980). Precision of the method was reflected by RSD lower than 2.95% and accuracy results around 100% for both VLG and MET measurement. Moreover, the Plackett–Burman experimental design was used to evaluate robustness, producing results within the acceptable range.