Luc Nagels

Potentiometric detection of organic acids in liquid chromatography using polymeric liquid membrane electrodes incorporating macrocyclic hexaamines.

Potentiometric detection employing coated-wire electrodes was applied to the determination of organic acids in liquid chromatography (LC). Poly(vinyl chloride)-based liquid membranes, incorporating lipophilic macrocyclic hexaamines as neutral ionophores were used as electrode coatings. The selectivity and sensitivity of the macrocycle-based electrodes were found to be superior to an electrode based on a lipophilic anion exchanger (a quaternary ammonium salt). Sensitive detection was obtained for the di- and tricarboxylic acids tartaric, malonic, malic, citric, fumaric, succinic, pyruvic, 2-oxoglutaric and maleic acids after separation in reversed-phase LC. Detection limits (signal/4sigmanoise=3) of 6 pmol for malonic acid and 2 pmol for maleic acid were attained. The detection was explained using a molecular recognition model. The hexaamine-based potentiometric electrodes had a 1-s response time at 1 ml min(-1) flow-rates. They were stable for at least 4 months, with an intra-electrode variation of 3.2% (n=5).

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.

Convection/diffusion- and diffusion-controlled rapid-scan voltammetry in liquid chromatographic systems with a large-volume wall-jet detector

Abstract On-line voltammetric measurements under liquid chromatographic (LC) conditions yield voltammograms which are either purely diffusion- or purely convection/diffusion-controlled, or a combination of both. The dependence of this behaviour on flow-rate, scan-rate and electrode diameter is investigated for a large-volume wall-jet detector. It is shown that for 4 V s−1 scan-rates at 1 ml min−1 flow-rates (conventional LC conditions), S-shaped (convection/diffusion-controlled) voltammograms can be obtained with macro-electrodes (⩾ 1 mm diameter). Distortion of voltammogram shape by the cell time constant is discussed for macro-electrodes. The behaviour of the cell in microbore and micro-LC applications is demonstrated. The advantage of being able to change from convection/diffusion-controlled to diffusion-controlled behaviour is discussed.

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.

Potentiometric detection of exogenic beta-adrenergic substances in liquid chromatography

Potentiometric detection with poly(vinyl chloride) (PVC) based liquid membrane electrode coatings is presented for a series of 18 exogenic beta-adrenergic substances in cation-exchange-HPLC and RP-HPLC systems. In both types of HPLC modes employing hybrid polymer-silica packings we observed that use of tetrakis(p-chlorophenyl)borate (TCPB) containing electrodes yielded limits of detection (DL) down to 10(-7)-10(-8) M (injected concentrations). The use of eluents with high concentrations of acetonitrile (up to 55%) yielded detection limits down to 10(-9) M (injected concentrations). A quantitative structure-potentiometric response activity relationship (QSAR) was developed for the set of beta-adrenergic substances and for a set of PVC-based electrodes using TCPB alone or in admixture with trioctylated alpha-cyclodextrin, dibenzo-18-crown-6, or calix[6]arene hexaethylester. A multiple linear regression model based on a computationally derived set of 14 molecular descriptors allowed prediction of the detection limits of beta-adrenergic substances and other amine substances from their molecular structure.

Characterization of an immobilized hexose oxidase reactor for mono- and oligosaccharide determination by liquid chromatography

Abstract The enzyme oxidase was extracted from the red sea-weed Chondrus crispus and immobilized in an enzyme reactor. The characteristics of this reactor were studied for the post-column liquid chromatographic detection of carbohydrates (separation on a cation exchange column). The reactor converted the eluting carbohydrates to hydrogen peroxide, which was detected amperometrically. Several saccharides could be determined at low μg levels. Conversion efficiencies for 12 mono- and oligosaccharides were measured at different flow-rates. The flow-rate versus conversion efficiency behaviour of the reactor and its selectivity is compared to the behaviour of a glucose oxidase reactor with very high enzyme loading in this respect. The differences between both reactors are explained theoretically. The practical use of both reactors in chromatographic analysis of carbohydrates is discussed and demonstrated.

Integrated Acquisition of Analytical and Biopharmaceutical Screening Data for beta-Adrenergic-Drugs Employing Diversified Macrocycle Supported Potentiometric Detection in HPLC Systems

Potentiometric detection with poly(vinyl chloride) (PVC) based liquid membrane electrode coatings is presented for a series of eighteen beta-adrenoceptor binding drugs (five agonists and thirteen antagonists) in cation exchange-HPLC and RP-HPLC systems. Incorporation of lipophilic cation-exchanger tetrakis(p-chlorophenyl)borate (TCPB) alone or in combination with trioctylated alpha-cyclodextrin into the polymeric liquid membrane gives very sensitive responses for racemic forms of bufuralol, propranolol, carazolol, clenbuterol, mabuterol, cimaterol, bisoprolol, oxprenolol, alprenolol, tertatolol, and bevantolol, especially in the cation-exchange HPLC system applying acetonitrile -- 40 mM phosphoric acid (15: 85, v/v, pH* = 2.35) as the mobile phase. In both applied orthogonal HPLC modes we observed that use of TCPB containing electrodes (no addition of neutral macrocyclic ionophores) gives more than five fold improvement in limit of detection down to 10(-7) M for mabuterol, bufuralol, alprenolol and tertatolol in comparison with UV detection. These results suggest that potentiometric detection, especially in RP-HPLC employing hybrid polymer-silica packings, can be considered as the promising alternative in the high-throughput drug abuse or doping control procedures of investigated beta-adrenergic agonists and beta-adrenolytics in humans and animals. The quantitative structure - potentiometric response relationships were developed for a set of eighteen beta-adrenenergic drugs and a set of PVC based electrodes using TCPB alone or in admixture with trioctylated alpha-cyclodextrin, dibenzo-18-crown-6 or calix[6]arene hexaethylacetate ester. A multiple linear regression model based on computationally derived set of molecular descriptors was used to predict detection limits of beta-blocking agents and beta-adrenergic agonists from their molecular structure in the developed potentiometric detectors. Principal components analysis (PCA) of data considering determined potentiometric detection limits revealed that it can be used to establish a reliable pharmacological classification of compounds with beta-adrenoceptor activity, especially for the differentiation of cardioselective and non-cardioselective beta1-antagonists.

Characterization of cellulase-based enzyme reactors for the high-performance liquid chromatographic determination of β-d-glucan oligosaccharides

Abstract Post-column enzyme reactors were used in series with an electrochemical detector for the selective high-performance liquid chromatographic (HPLC) determination of β- d -glucan oligo- and polysaccharides (degree of polymerization up to 30). Immobilized cellulase converted the eluting oligomers to β- d -glucose, which was oxidized by immobilized glucose oxidase. The production of hydrogen peroxide was measured with an electrochemical detector. The functioning of this system was verified for a whole range of glucosaccharides varying in both structure (positional isomers) and degree of polymerization. Fractional conversions and molar response factors were determined for all the compounds under study. Rate constants are discussed for the reactor system used applying a first-order kinetics model. Efficient HPLC separations were obtained for these oligo- and polysaccharides on a reversed-phase column using gradient elution. Detection limits were of the order of a few nanograms. The reactors were stable for several months.

A novel potentiometric approach for detection of beta-adrenergics and beta-adrenolytics in high-performance liquid chromatography

Potentiometric approach enabling sensitive and reliable detection for a series of 20 autonomic beta-adrenergic ligands with the use of poly(vinyl chloride) (PVC) based liquid membrane electrode coatings in the normal-bore cation exchange HPLC and narrow-bore reversed phase HPLC system is presented. It was found that in both kinds of HPLC modes with a contemporary hybrid polymer-silica packings an application of electrodes containing a tetrakis(p-chlorophenyl)borate (TCPB) gives limits of detection below to 8.0x10(-7) mol l(-1) (injected concentrations). In case of highly hydrophobic beta-adrenergic drugs the use of binary aqueous mobile phases with high concentrations of acetonitrile (up to 25% v/v) shifting an observable detection limits (DL) down to 2.0x10(-8) mol l(-1), especially for electrodes with addition of trioctylated alpha-cyclodextrin. The characteristics of developed potentiometric detectors was established by proposed a quantitative structure-potentiometric response relationships (QSPRRs) for a series of diversified beta-adrenergic compounds and for a set of the PVC based electrodes using TCPB alone as well as in combination with trioctylated alpha-cyclodextrin, dibenzo-18-crown-6, or calix[6]arene hexaethylester as the neutral macrocycle ionophore. A highly significant QSPRRs equations were obtained leading to reasonable prediction of the DL of specified electrodes in terms of the computationally derived set of molecular descriptors of beta-adrenergics and beta-blocking agents and similar amino alcohol type xenobiotics.

Carbon-polymer chips as sensitive electrochemical detectors for micro-lqiuid chromatography

Abstract The construction and characterization of an on-line electrochemical detector especially suited for flow-rates in the order of a few microlitres per minute is described. The working electrode is tubular, of length 15 mm and I.D. 90 μm (effective volume 100 nl). It is made of a small carbon-polymer chip, giving an inexpensive, practical and disposable construction. When used with packed fused-silica liquid chromatographic columns of 0.3 mm I.D., sub-picogram detection limits are obtained. The detector is linear for amounts up to 1 ng. An exceptional feature of this detector is that it stabilizes in a few seconds after potential onset (no bseline drift). The noise level is very constant and predictable. Some examples are given of the oxidative determination of phenolic compounds and catecholamines. Coulometric efficiencies, responses versus flow-rate behaviour and peak dispersion characteristics are given.