A Standard Protocol for the Calibration of Capillary Electrophoresis (CE) Equipment

Calibration of complex analytical systems is always a difficult task. Nevertheless, a suitable approach has to be designed before the systems can be introduced into routine analysis. In literature, many methods have been described for the purpose of calibrating such systems, but only a few of them deal with capillary elctrophoresis. Here, we want to demonstrate a general approach to how the calibration of this type of analytical instrument becomes feasible

Rapid Determination of Diclofenac in Pharmaceutical Formulations by Capillary Zone Electrophoresis

Capillary electrophoresis is competitive to HPLC and other chromatographic methods, predominantly when charged analytes have to be separated. The time of analysis can be reduced by the use of very short capillaries applying a high voltage. In most instruments which are commercially available the so-called ‘short end’ of the capillary can be used for separation, leading to very rapid separations. In this contribution we want to demonstrate this approach by using Diclofenac Sodium as an analyte

Quality Control and Stability Studies with the Monoclonal Antibody, Trastuzumab: Application of 1D- vs. 2D-Gel Electrophoresis

Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well

Transport Rankings of Non-Steroidal Antiinflammatory Drugs across Blood-Brain Barrier In Vitro Models

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison

Formaldehyde—A Key Monad of the Biomolecular System

Experiments will be presented and reviewed to support the hypothesis that the intrinsic reactivity of formaldehyde may lead to the formation of a rather comprehensive set of defined biomolecules, including D-glucose, thus fostering concepts of evolution considering the existence of a premetabolic system as a primordial step in the generation of life

Group transport study of NSAIDs across RBMEC.

*<p>Ratio to Diazepam is calculated by average PE_cell data of the investigated NSAID and the corresponding diazepam value.</p><p>Summary of permeability data of the group study with NSAIDs piroxicam, ibuprofen, meloxicam, tenoxicam and diclofenac across the RBMEC cell layers (n = 3, data are presented as means ± SD).</p

Characterization of the BBB model based on primary rat brain microvascular endothelial cells (RBMEC) and astrocytes.

<p>RBMECs grow in endothelial cell typical spindle-like morphology proven by light and scanning electron microscopy (SEM). Transmission electron microscopic (TEM) images confirmed that RBMEC grow as a monolayer. The enlarged part of the image shows two RBMECs connected to each other directly over a pore of the Transwell insert membrane (A). mRNA expressions of tight junction proteins ZO-1, occludin, claudin-3, claudin-5 and claudin-12, and of adhesion molecules PECAM-1, VCAM, ICAM-1 and CD44. All data were related to endogenous control GAPDH which was set to 1000 (B). Immunofluorescence images of PECAM-1, ZO-1, occludin, claudin-3 and claudin-5 confirmed the protein’s presence and localization in RBMEC layers (C). Transport studies with paracellular marker APTS-dextran ladder confirmed functionality of the barrier. RBMEC layers were able to differentiate between the different dextran fractions in a molecular size-dependent manner. Comparison of the permeability coefficients for APTS-dextran across PBMEC/C1-2, ECV304 and RBMEC layers is presented in the table on the right side (D).</p