Stefan Tyski

CE versus LC for simultaneous determination of amoxicillin/clavulanic acid and ampicillin/sulbactam in pharmaceutical formulations for injections.

A rapid, capillary electrophoresis method was evaluated for determination of amoxicillin and clavulanic acid in Augmentin as well as ampicillin and sulbactam in Unasyn preparations for injections. Phosphate-borate buffer at pH 8.66 containing 14.4% sodium dodecyl sulfate was used as a mobile phase. The method was validated. Reproducibility, precision, accuracy and assay linearity in concentration of amoxicillin 0.05-3.03 mg/ml and ampicillin 0.05-3.08 mg/ml, as well as clavulanic acid 0.02-2.02 mg/ml and sulbactam 0.05-2.08 mg/ml were established. This new method is fast, inexpensive and limits consumption of organic solvents when compared with alternative high performance liquid chromatography (HPLC) method, used for drug analysis. Statistical analysis by Students t-test showed no significant differences between the results obtained by the two methods t(calculated) 0.32 and 1.69 for amoxicillin and clavulanic acid and 0.67 and 1.93 for ampicillin and sulbactam were smaller than t(tabulated).

Recent development of potent analogues of oxazolidinone antibacterial agents

The oxazolidinones are a new and potent class of antimicrobial agents with activity mainly against Gram-positive strains. The commercial success of linezolid, the only FDA-approved oxazolidinone, has prompted many pharmaceutical companies to devote resources to this area of investigation. Until now, four types of chemical modifications of linezolid and oxazolidinone-type antibacterial agents, including modification on each of the A-(oxazolidinone), B-(phenyl), and C-(morpholine) rings as well as the C-5 side chain of the A-ring substructure, have been described. Division into sections according to side chain modification or the type of ring will be used throughout this review, although the process of synthesis usually involves the simultaneous modification of several elements of the linezolid substructure; therefore, assignment into the appropriate section depends on the structure-activity relationships (SAR) studies. This review makes an attempt to summarise the work carried out in the period from 2006 until mid-2012.

Adaptation of capillary electrophoresis to the determination of selected cephalosporins for injection

The migration behaviour of cephazolin, cefuroxime sodium, ceftriaxone sodium, cefoperazone sodium and ceftazidime in a mixture was studied. Phosphate-borate buffer pH 5-8 alone and with addition of sodium dodecylsulfate (SDS) was used. In capillary zone electrophoresis of all research compounds separation was not achieved. It was observed that supplementation buffer pH 6.5 with SDS (10 g/l) improved resolution of cephalosporins, but addition of pentanesulfonic acid (17.4 g/l) to the running buffer at pH 6.5 results in separation of each cephalosporin. In this condition good repeatability of migration times as well as repeatability of peak area were confirmed.

Determination of linezolid and its achiral impurities using sweeping preconcentration by micellar capillary electrophoresis.

Linezolid is the first compound of a truly new class of antibiotics--the oxazolidinones. The elaborated method of capillary electrophoresis (CE) of linezolid separation from its achiral impurities was successfully performed using sweeping preconcentration, followed by UV absorption detection at 254nm. The best results were obtained with 125mM Tris buffer, pH 2.0, with addition of 20% (v/v) methanol as background electrolyte. Sodium dodecyl sulfate (150mM) was added to the electrolyte in the inlet vial as the sweeping agent. The separation was carried out at negative polarity. Then, the optimized method was validated in terms of linearity, accuracy and precision. Sweeping preconcentration of linezolid provides detection limit at 0.05microg/ml level. The evaluated CE method was applied in the analysis of medicinal product containing linezolid-linezolid solution for infusion.

Determination of enantiomeric impurity of linezolid by capillary electrophoresis using heptakis-(2,3-diacetyl-6-sulfato)-β-cyclodextrin

A method for the enantioseparation of linezolid, the first compound of a truly new class of antibiotics-the oxazolidinones, was developed. The elaborated method of linezolid enantiomers separation was successfully performed using an anionic single-isomer cyclodextrin-heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) as a resolving agent with the help of the charged resolving agent migration model (CHARM model). The best results were obtained with 27.5mM HDAS-beta-CD dissolved in 50mM borate buffer, pH 9.0, 15 degrees C, normal polarity. The facile strategies for the reversal of the enantiomers elution order are also described. Afterwards, the optimized method was validated in terms of sensitivity, linearity, accuracy and precision.

Different sample stacking strategies for the determination of ertapenem and its impurities by micellar electrokinetic chromatography in pharmaceutical formulation.

Ertapenem, a Group 1 carbapenem, is most recently introduced into the market. It is a beta-lactam antibiotic that possesses a broad antibacterial spectrum including common community-acquired Gram-positive and Gram-negative aerobic and anaerobic pathogens, but low activity against some nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter spp., enterococci and methicillin-resistant staphylococci. The elaborated method of micellar electrokinetic chromatography (MEKC) of ertapenem separation from its impurities was successfully performed using normal stacking mode (NSM) and stacking with reverse migrating micelles (SRMM), followed by UV absorption detection at 214 nm. The best results were obtained with 60mM sodium dihydrogen phosphate and 20mM boric acid buffer pH 6.0, as background electrolyte. Uncoated fused-silica capillary and neutral-coated capillary with normal and reverse polarity, and voltage values of +18 and -12 kV, respectively, were used throughout the investigation. Sodium dodecyl sulfate was employed as the pseudostationary phase. A comparison of applied techniques, including sensitivity enhancement factors and limits of detection (LOD), is presented. The optimized method was validated in terms of linearity, accuracy and precision. Comparable LOD was obtained using both stacking methods (0.3 microg/mL) but better efficiency of ertapenem peak was obtained using NSM. Under the optimum stacking conditions, about 183-4.75-fold and 1289-4.07-fold improvements in peak areas were obtained for NSM and SRMM, respectively, compared to the usual hydrodynamic sample injection (10s). The reproducibility, expressed by relative standard deviations (RSD) of the migration times, for NSM was about 0.96-1.25 and for SRMM was 0.32-0.45. The RSD of corrected peak areas, for NSM was about 1.07-8.14 and for SRMM was 0.74-8.12. The difference in separation time between the two techniques was not obvious. Satisfactory separation was possible after less than 11min of electrophoresis. The evaluated MEKC method was applied in the analysis of medicinal product containing ertapenem: Invanz-ertapenem for injection.

Adaptation of capillary electrophoresis to piperacillin drug analysis

Abstract A capillary electrophoresis method was developed for the pharmaceutical analysis of piperacillin, a β-lactam antibiotic, in preparations for intramuscular and intravenous injections. Disodium hydrogenphosphate and sodium tetraborate buffer pH 6.2 or 8.7 supplemented with sodium dodecyl sulfate 7 g/l and electrophoresis voltage −18 kV, seem to provide optimal conditions for piperacillin CE assay. The method was validated and good reproducibility, precision, accuracy and assay linearity in antibiotic concentrations 0.08–2.00 mg/ml for both electrolytes were observed. The detection limit for piperacillin was 0.01 mg/ml. Preliminary experiments showed the usefulness of CE for separation and determination of piperacillin and β-lactamase inhibitor co-existing in the drug Tazocin. The results obtained by CE were also compared with those obtained by liquid chromatography. Statistical analysis by Student’s t-test showed no significant differences between the results obtained by the two methods.

Activity of ozonated water and ozone against Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

Summary Background The known bactericidal properties of ozone have not been checked in relation to its action on bacterial biofilms. This is especially true of ozonated fluids. The aim of this study was to investigate the bactericidal activity of ozonated water and that of a mixture of ozone and oxygen against biofilms. Material/Methods Eighteen clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa exhibiting various levels of antibiotic sensitivity were investigated. Bacteria were cultured in biofilm form on polystyrene titration plates for periods of 2 to 72 hours. The biofilms formed in this way were exposed to in statu nascendi ozonated water produced in a prototype device that had been tested in clinical conditions, or to a mixture of oxygen and ozone generated in the same device. Live cells in the biofilm were stained with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide solution. The degree of reduction of viable bacteria following ozone exposure was determined. Results Ozonated water was found to be an effective bactericidal agent against biofilms after as little as 30 seconds of exposure, while the bactericidal activity of the ozone-oxygen solution was much lower. Prolongation of the duration of biofilm exposure to the gaseous disinfectant to 40 minutes led to a reduction in the viable cell count, which nevertheless remained high. Conclusions Unlike the ozone-oxygen mixture, ozonated water effectively destroys bacterial biofilms in vitro.

Determination of doripenem and related substances in medicinal product using capillary electrophoresis

Doripenem, the latest carbapenem antibiotic licensed in the United States (15 October 2007) and the European Union (25 July 2008), has been implemented into therapeutic use along with imipenem, meropenem and ertapenem. The described method of zone electrophoresis in a low pH buffer for the separation of doripenem from its impurities has been successfully performed using field-amplified sample stacking (FASS), followed by UV absorption detection at 214 nm. The best results were obtained with phosphate buffer (100 mM) pH 2.9 containing 10% (v/v) of methanol, as the background electrolyte. Uncoated fused-silica capillary (60/52 cm; 75 μm id) with normal polarity, and voltage values of 25 kV, was used throughout the investigation. The optimised method of doripenem determination was validated in terms of linearity, accuracy and precision, and provides a detection limit of 3.0 μg/mL of doripenem. The repeatability, expressed by relative standard deviation (RSD) of the migration time, for doripenem and its degradation products varied from 1.37 to 2.51%, whereas the corrected peak areas were about 0.91-9.87%. Satisfactory separation was achieved within 20 min of electrophoresis; moreover, all carbapenems (imipenem, meropenem, ertapenem and doripenem) were well separated from each other during this time. The evaluated CZE method was applied in the analysis of a medicinal product containing doripenem Doribax(®) powder for solution for infusion.

Application of micellar electrokinetic chromatography to the determination of sultamicillin in oral pharmaceutical preparations

A micellar electrokinetic capillary electrophoretic method for determination of sultamicillin in Unasyn oral preparations--tablets and suspension--was evaluated. Phosphate-borate buffer at pH 7.0 containing 1.0% sodium dodecylsulfate was used as a mobile phase. The elaborated method ensures separation of sultamicillin from p-toluenesulfonic acid and the impurities, ampicillin, sulbactam and penicillamine. The method was validated for specificity, reproducibility, precision, accuracy and assay linearity (in a concentration range of sultamicillin of 0.05-1.5 mg/ml). Statistical analysis by Students t-test showed no significant differences between the results obtained by micellar electrokinetic chromatography and HPLC, t(calculated) 0.519 for suspension assays and 0.284 for tablets assays were smaller then t(tabulated).