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In this paper, a reliable stability-indicating generic MEEKC method for the analysis of five commonly used fluoroquinolones (FQs) has been developed and optimized by a central composite circumscribed experimental design. The separation was carried out using a fused silica capillary (60.2 cm total length) and a microemulsion (ME) composed of 81.75% (w/w) of a 125 mM NaH2 PO4 solution having a pH of 2.75, 2.65% (w/w) SDS, 1.00% (w/w) n-octanol, 6.60% (w/w) n-butanol and 8.00% (w/w) 2-propanol. A voltage of 28 kV was applied in a reverse polarity mode. A linear relationship was established from 0.04 to 0.48 mg/ml with R2 values higher than 0.98 for all five FQs. Both repeatability and intermediate precision were less than 3% and accuracy ranging from 97 to 100%. Of note, ciprofloxacin impurity A and ofloxacin impurity A could be separated from the respective drug substance in a single run which cannot be achieved using the official HPLC method from the European Pharmacopoeia. Forced degradation of all FQs under heat, in acidic and alkaline medium, in the presence of oxidizing agents and under neutral hydrolysis conditions was investigated. The highest yield of degradation products was observed using oxidative hydrogen peroxide. Hence, the proposed MEEKC method can be used for the quantitative determination of the five FQs and their potential impurities within a total runtime of 20 min.

An electrophoretically mediated microanalysis (EMMA) approach, used to perform on-line chemistry between two small molecules, has been characterized and optimized. The plug-plug type EMMA method involved electrophoretic mixing and subsequent reaction of nanoliter plugs of kanamycin-containing samples and 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid within the confines of the capillary column, which acts as a microreactor. Analyses were performed by pressure-injecting a plug of kanamycin sandwiched in two reagent plugs. A potential of 375 Vcm- 1 was then applied to electrophoretically mix the two reactants, and an incubation time of up to 5 min allowed the reaction to proceed prior to the application of a separation potential of 588 Vcm - 1 . UV detection was at 335 nm. The background electrolyte was 30 mM sodium tetraborate at pH 10.0, containing 16% of methanol. The method was validated in terms of linearity, limits of quantitation and detection, and precision. The method allows determination of kanamycin in bulk samples as a fully automated procedure.

This paper describes a system for integration of a one-step-microscale chemical derivatization and analysis by a methodology known as electrophoretically mediated microanalysis (EMMA). Differential electrophoretic mobility between an analyte, reagent, and their product offers EMMA a unique capability to selectively carry out electrophoretic mixing, control product formation, and separation. This system was successfully applied to perform derivatization and separation of the multicomponent aminoglycoside antibiotic gentamicin using 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid as labeling reagents. A multivariate approach based on central composite experimental design was used to optimize the derivative yield. Full automation of the derivatization and analytical procedure, high derivatization efficiency, high sample throughput, and precision are the excellent features of the present method. In addition, this methodology offers short analysis time, as well as selectivity and sensitivity suitable for impurities determination. Separation of gentamicin C1, C1a, C2, C2a, C2b, sisomicin, and several minor components was achieved. For the first time separation and identification of three impurities, namely garamine, 2-deoxystreptamine, and paromamine are described.

We describe the development of a capillary electrophoresis method for the determination of gentamicin C1, C1a, C2a, and C2 components in human serum. Using a weak cation-exchanger with 20 mM phosphate buffer, pH 7.4, 200 mM borate buffer, pH 9.0, and ammonia/methanol, solid-phase extraction (SPE) of gentamicin components from the human sera was performed. The extract was derivatized with 1,2-phthalic dicarboxaldehyde/mercaptoacetic acid reagent. The derivatives were separated with a background electrolyte comprising 60 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer at pH 9.5 containing 31.6% m/v methanol, and quantified with UV-light absorption detection at 230 nm. The identity of the gentamicin components was confirmed by mass spectrometry. The SPE recovery of the gentamicin ranged from 78% to 93%. The calibration curves were linear from the concentration limit of quantitation (LOQ) to 30 mg/L for the gentamicin mixture. The LOQ for gentamicin C1 was 0.33 mg/L, for C2a 0.23 mg/L, C2 0.25 mg/L, C1a 0.27 mg/L and the concentration limit of detection (LOD) for C1 was 0.15 mg/L, C2a 0.11 mg/L, C2 0.12 mg/L, C1a 0.13 mg/L. Intra-assay relative standard deviation (RSD) values were for C1 (5%), C1a (7%), C2 (6.5%) and C2a (9%); inter-assay RSD values were for C1 (11%), C1a (13.3%), C2 (15%) and C2a (14%). The Pearson's correlation between capillary electrophoresis and immunoassay revealed a linear relationship between these two techniques with r = 0.9. This method for determination of gentamicin C1, C1a, C2a, and C2 in human serum can thus be used in the entire therapeutic concentrations range of gentamicin.

Recent developments in the use of capillary electrophoretic techniques for the in-line study of enzyme reactions and derivatization protocols are reviewed. The article is divided into two parts: (i) in-line enzyme reactions and (ii) in-line derivatization. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview is provided, starting from 1996, and the investigated enzymes are classified into two tables based on the mode of engagement (i.e., continuous or transient) of the developed EMMA-based assay. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization protocols based on EMMA. Reported derivatization procedures are discussed and classified in tables, according to the functional group that is derivatized.