Hugo Bohets

In situ dissolution testing using potentiometric sensors

Potentiometric sensors can be used to determine the amount of API dissolved in the dissolution medium in function of time by measuring directly in the dissolution vessel of a Paddle (USP type 2) and Basket (USP type 1) apparatus. The prototype potentiometric sensor instrumentation showed very promising results for a selection of APIs with different physico-chemical properties. The applicability, benefits and limitations of the prototype were explored. The applicability of the measurement technique strongly depends on the log(P) of the API. Here, it is shown that measurements can easily be performed for APIs with a log(P)>4. Electrode performance however decreases with decreasing logP of the APIs due to decreased drug selectivity in comparison to the excipients and ionic strength of the applied dissolution medium. The potentiometric sensors are shown to be insensitive towards undissolved particles and air bubbles as opposed to UV spectrometric measurement where these can lead to severe light scattering. For the tested APIs, the obtained dissolution profiles are very reproducible and show a low variation compared to the measurements using manual sampling and UV or HPLC analysis. The measurements demonstrate that potentiometric sensors are a very promising technology that can become a standard for in situ dissolution measurements.

A label-free potentiometric sensor principle for the detection of antibody-antigen interactions.

We report here on a new potentiometric biosensing principle for the detection of antibody-antigen interactions at the sensing membrane surface without the need to add a label or a reporter ion to the sample solution. This is accomplished by establishing a steady-state outward flux of a marker ion from the membrane into the contacting solution. The immunobinding event at the sensing surface retards the marker ion, which results in its accumulation at the membrane surface and hence in a potential response. The ion-selective membranes were surface-modified with an antibody against respiratory syncytial virus using click chemistry between biotin molecules functionalized with a triple bond and an azide group on the modified poly (vinyl chloride) group of the membrane. The bioassay sensor was then built up with streptavidin and subsequent biotinylated antibody. A quaternary ammonium ion served as the marker ion. The observed potential was found to be modulated by the presence of respiratory syncytial virus bound on the membrane surface. The sensing architecture was confirmed with quartz crystal microbalance studies, and stir effects confirmed the kinetic nature of the marker release from the membrane. The sensitivity of the model sensor was compared to that of a commercially available point-of-care test, with promising results.

Online monitoring of dissolution tests using dedicated potentiometric sensors in biorelevant media.

The performance of the Ion-Selective Electrode (ISE) for in vitro dissolution testing using biorelevant media was evaluated in this study. In vitro dissolution was carried out using USP apparatus 2 (paddle method) with classical and with updated biorelevant media to simulate the pre- and postprandial states. The ISE was used as an analytical stand-alone system and in combination with a single-point HPLC-UV measurement. A modified method enabling the use of the ISE for very poorly soluble substances is also proposed. In terms of f(2)-factor, the results acquired using the ISE for the drug diphenhydramine-HCl were found to be very similar to the results obtained by manual sampling followed by HPLC-UV analysis. In Fed State Simulated Gastric Fluid (FeSSGF), a medium containing 50% milk, the ISE is more practical since the need to separate proteins from the analyte prior to HPLC-UV analysis is eliminated. Further work will be needed to establish ISE methodology for Fed State Simulated Intestinal Fluid (FeSSIF) media. In summary, the ISE has promise as an analytical tool for research and development applications.

Unique potentiometric detection systems for HPLC determination of some steroids in human urine

Isocratic HPLC with potentiometric detection is used for the determination of some 17-ketosteroids (17-KS), e.g., androsterone, dehydroepiandrosterone and estrone, and their respective sulfated conjugates (17-KSS). Glassy carbon or composite electrodes containing a mixture of graphite and poly(vinyl chloride), PVC, were used as substrate electrodes. These substrates were covered either by montmorillonite or potassium tetrakis(p-chlorophenyl) borate containing PVC-based rubber phase membranes. The neutral 17-KS compounds were derivatized with Girards reagent P (GP) to obtain cationic pyridinium acetohydrazones prior to the HPLC/potentiometric detection assay. No side reactions were observed, and the GP itself was not interfering. The method yielded accurate and reproducible results and was applicable to samples containing down to micromolar concentrations. Next, the 17-KSS compounds, acting as anionic charged molecules, were determined directly in human urine samples with the HPLC/potentiometry combination without preliminary derivatization. For this purpose, a new anion-sensitive potentiometric electrode was developed using a macrocyclic polyamine containing, PVC-based, rubber phase membrane. The three 17-KSS compounds were also determined accurately down to micromolar concentrations. Especially, the main androgen metabolites as dehydroepiandrosterone sulfate and androsterone sulfate could be selectively determined with a developed potentiometric sensor in human urine samples without time-consuming cleanup and preconcentration step.

Response of DNA Fragments to Potentiometric Sensors Studied Using HPLC

Potentiometric sensors are studied as viable candidates for the construction of high throughput DNA arrays. For preliminary investigations, such sensors were used in an HPLC setup in the present work. This avoided errors due to ionic contaminants or additives in the commercial samples. The oligonucleotides dT(10), dT(20) and dT(30) were used as test substances. The potentiometric sensors were of the coated wire type, containing PVC, DOP, MTDDACl and a synthetic podand urea receptor. The HPLC system consisted of a reversed phase column eluted with a phosphate buffer, triethylammoniumacetate (TEAA), and an acetonitrile gradient. Molar responses and sensitivities increased with increasing chain length of oligonucleotides, yielding detection limits as low as 10(-6)M (dT(30), injected concentration). The slopes of the calibration graphs were at least 23 mV/decade (dT(10)), which was much higher than expected. The results are discussed in view of the potential use of this sensor type in high throughput microarrays.