Improved liquid chromatography combined with pulsed electrochemical detection for the analysis of etimicin sulfate.

This paper describes an improved liquid chromatography method combined with pulsed electrochemical detection for the analysis of etimicin sulfate. In total, 22 impurities could be separated. A TSK-GEL C18 column (250 mm × 4.6 mm i.d., 5 µm) is used, and the mobile phase is composed of 40 mL of acetonitrile and 960 mL of an aqueous solution containing trifluoroacetic acid (15 mL/L), pentafluoropropionic acid (500 µL/L), 50% sodium hydroxide (8 mL/L) and sodium sulfate (1.5 g/L). The pH of the aqueous solution is adjusted to 3.5 with 0.8 M sodium hydroxide. The influence of the different chromatographic parameters on the separation was investigated. A quadruple potential-time waveform was applied to the electrodes of the detection cell. 0.8 M sodium hydroxide was added post column to raise the pH to at least 12 before detection. A central composite experimental design was used to describe the relationship between factors and response values and to establish factorial analysis. Compared to previously published investigations, this improved method shows higher sensitivity, better separation ability and robustness and has been incorporated by the Chinese Pharmacopoeia 2015 for analysis of etimicin sulfate. A number of commercial samples of etimicin sulfate were also analyzed using this method.

Analysis of impurity profiling of arbekacin sulfate by ion-pair liquid chromatography coupled with pulsed electrochemical detection and online ion suppressor-ion trap-time off light mass spectrometry.

Ion-pair liquid chromatography with pulsed electrochemical detection (LC-PED) was established for the analysis of impurities in arbekacin (ABK) sulfate. APursuit pentafluorophenylpropyl (PFP) column was used as stationary phase. This novel method showed greater separation and sensitivity ability. In a representative ABK sample, 24 impurity peaks were detected in LC-PED, where of only 9 were monitored by a post-column derivatization method prescribed by the Japanese Pharmacopoeia (JP). For identification of the chemical structures of the impurities detected by LC-PED, LC-Mass Spectrometry (MS) was used. Two challenges had to be overcome in this work. The first was the transfer of the MS incompatible mobile phase to an MS compatibleone while maintaining the elution order of the peaks in the chromatograms. Previously reported approaches such as two-dimensional (2D)LC were hardly applicable in this case due to the lack of ultraviolet (UV) absorbing chromophores in ABK and its impurities. The sodium hydroxide solution was replaced by aqueous ammonia to adjust the pH of the mobile phase used in LC-PED. The other challenge encountered was the ion suppression effect caused by trifluoroacetic acid (TFA) and pentafluoroproponic acid (PFPA) in the mobile phase. Some strategies such as "TFA-fixed" and its modifications were tried, but they were inconvenient and severe contamination of the MS was observed. A cationself-regenerating suppressor (CSRS), which was originally designed for increasing analyte conductivityof ammonia and amines analysis in ion chromatography (IC), was coupled between the LC and Ion Trap-Time of Flight (IT-TOF)-MS and almost all TFA and PFPA in the mobile phase were removed. The limit of detection (LOD) of ABK in this integrated system improved significantly to 20 ng/mL. The chemical structures of the 28 impurities were elucidated and 15 impurities were reported for the first time.

Indirect pulsed electrochemical detection following high-performance reversed-phase liquid chromatography.

For detection using pulsed electrochemical detection (PED), analytes must preadsorb to the working gold electrode. Indirect pulsed electrochemical detection (InPED) exploits this requirement by including a PED-active reagent in the mobile phase. The background signal provided by oxidation of this reagent is attenuated by the adsorption of analyte molecules to the electrode. In this paper, a method has been developed to allow the use of InPED in combination with high-performance reversed-phase liquid chromatography (RPLC). Biotin was used as a probe molecule to determine that the use of acetonitrile as the organic modifier in the mobile phase provided superior results to the use of methanol. In addition, a silica-based, high surface area C18 column (i.e., Varian Pursuit XRs) was found to give better results than a polymer-based reversed-phase column (i.e., Dionex IonPac NS1). Optimized experimental conditions were used todetermine lipoic acid, tiopronin, and penicillamine, obtaining detection limits of ≤1 µM (30 pmol injected). The analytical utility of RPLC-InPED was demonstrated by an assay of an over-the-counter-formulation containing lipoic acid.

Indirect pulsed electrochemical detection of aliphatic carboxylate-containing analytes following high performance anion-exchange chromatography.

The mechanism of detection in pulsed electrochemical detection (PED) requires preadsorption of the analyte to the working electrode prior to its subsequent oxidation. Indirect detection is accomplished by the addition of a PED-active reagent to the mobile phase, whose signal is attenuated by an analyte that more strongly adsorbs to the electrode surface. Here, indirect PED (InPED) is applied to the determination of aliphatic carboxylate-containing compounds separated using high performance anion-exchange chromatography (HPAEC). Limits of detections of 0.05-2 ppm (10-400 pmol) are found for most analytes tested using an optimized potential-time waveform at a gold working electrode. The analytical utility of InPED is demonstrated for assays of gabapentin, biotin, proline and several over-the-counter formulations.

A review of pulsed electrochemical detection following liquid chromatography and capillary electrophoresis.

Pulsed electrochemical detection (PED) has progressed as a highly sensitive and selective detection technique following aqueous-based separation systems over the past three decades. The application of on-line pulsed potential cleaning to electrocatalytic noble metal electrodes has significantly increased the number of applications formerly achieved with conventional electrochemical (EC) detection. Electrochemical cells are easily miniaturized, providing the ability to apply detection by PED at microelectrodes and onto microchips utilizing electrophoretic separations. In addition, recent advances in PED waveforms and instrumentation have enabled the detection technique to be easily coupled with high pressure separation systems which require rapid detection to maintain separation integrity. As a result, advanced applications for the determination of carbohydrates as well as the expansion of PED for the detection of other organic aliphatic compounds have been recently accomplished. This review will focus on developments and methods utilizing PED following liquid chromatography (LC) and capillary electrophoresis (CE). Publications are reviewed in chronological order to emphasize the advancement of the detection method and the sustained relevance of its applications.

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 (250 mm×4.6 mm i.d., 5 µm), isocratic elution was carried out with a mobile phase, containing sodium sulfate (35 g/L), sodium octanesulphonic acid (1 g/L), tetrahydrofuran (14 mL/L) and 0.2 M phosphate buffer pH 3.0 (50 mL/L). Using these experimental conditions, the limit of quantification (LOQ, S/N=10) was 5 ng. The linearity was examined in the range LOQ-60 µg/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.