Jan Saevels

Study of the competitive inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine using capillary zone electrophoresis

Abstract This article describes how capillary zone electrophoresis was used to evaluate the inhibitory behavior of EHNA or erythro-9-(2-hydroxy-3-nonyl)adenine on the enzymatic deamination of adenosine by adenosine deaminase. The technique used for carrying out the assay was electrophoretically mediated micro analysis (EMMA). A fused-silica capillary was filled with phosphate buffer containing the inhibitor at a concentration that was adequate to inhibit the enzyme. After subsequent injection of the enzyme adenosine deaminase and the substrate adenosine, enzymatic reaction, electrophoretic separation and quantification of substrate and reaction product all took place inside the capillary. Reaction velocities were estimated from the peak area of the reaction product inosine. Michaelis-Menten and Lineweaver-Burk plots were constructed and the inhibitory constant Ki of EHNA was determined by comparison of the Km of adenosine deaminase in the presence of inhibitor with the Km in a solution without inhibitor.

Interlaboratory studies on two high-performance liquid chromatographic assays for tylosin (tartrate)

An interlaboratory study was performed on two high-performance liquid chromatographic methods to determine tylosin. The first method is a reversed-phase HPLC on a C18 column, while the second is a method using a polymeric stationary phase. The first method is described in several pharmacopoeia monographs on tylosin, to determine the composition of a tylosin mixture, while the second method is recently proposed to determine both the composition and the contents in such a mixture. The interlaboratory studies were set-up and interpreted according to ISO guidelines. This paper is written as a tutorial type of article explaining the principles and methods of these guidelines. The results of both methods were compared. Both were found to have disadvantages but in general the old method is still preferred, both for composition determination and to assay the components.

In-house packing and testing of capillaries for capillary electrochromatography using simple equipment

The feasibility of producing packed capillaries for capillary electrochromatography (CEC) is evaluated. Emphasis is put on the fact that only material was used that is already available in any LC/CE orientated laboratory. An experimental set-up is developed for filling the capillaries by use of an ordinary LC pump, and frits are sintered with a glowing resistance wire, fed by a d.c. power supply. Electrochromatography was carried out in an in-house built capillary electrophoresis apparatus, without pressurizing the vials. Under these conditions, the capillaries performed well, producing up to 190,000 plates per meter. Bubble formation did not appear, on condition that the mobile phase was thoroughly helium degassed. Even at ambient temperature, electrophoresis obeyed Ohms law up to a voltage of 30 kV, proving that Joule heating was not a major concern.

Interlaboratory study comparing the microbiological potency of spiramycins I, II and III

An interlaboratory study has been performed to determine the relative potencies of spiramycins (SPMs) I, II and III by diffusion or/and turbidimetric assays with Bacillus subtilis or Staphylococcus aureus as the test organisms. Six laboratories from three countries participated. Experimental procedures were according to the European Pharmacopoeia, 3rd ed. The activity of SPM I is markedly higher than that of SPM II and III. By diffusion, the activities of SPM II and III relative to SPM I were found to be 57 and 72%, respectively. The interlaboratory relative standard deviations (RSD) varied from 3.6 to 16.3%. By turbidimetry, the activities of SPM II and III relative to SPM I were found to be 45 and 52%, respectively. The interlaboratory RSD values varied from 2.6 to 7.7%. The results of the study were analyzed according to the ISO 5725-2 guidelines to determine the repeatability, the between-laboratory and the reproducibility variances of both methods.

Investigation of unexpected migration behavior of adenosine in capillary zone electrophoresis

SummaryThe capillary zone electrophoresis of two common nucleosides, adenosine and inosine, was investigated. Both compounds were resolved in a 0.1 M sodium phosphate buffer, pH 7.5. Contrary to expectations, adenosine behaved at this pH— 5 pH units lower than the literature pKa— as a negative ion, migrating behind mesityloxide (neutral marker) when working in normal polarity mode. To confirm the migration order, peaks were identified from absorption maxima, by high-speed scanning detector. The change in electrophoretic mobility with pH was investigated for the nucleosides, and 10 other background electrolytes were tried to match the separation capabilities of the sodium phosphate buffer. Most inorganic buffers showed comparable separation, while organic, Good-type buffers lacked selectivity.