Potentiometric detection in UPLC as an easy alternative to determine cocaine in biological samples.

The analytical methods which are often used for the determination of cocaine in complex biological matrices are a prescreening immunoassay and confirmation by chromatography combined with mass spectrometry. We suggest an ultra-high-pressure liquid chromatography combined with a potentiometric detector, as a fast and practical method to detect and quantify cocaine in biological samples. An adsorption/desorption model was used to investigate the usefulness of the potentiometric detector to determine cocaine in complex matrices. Detection limits of 6.3 ng mL(-1) were obtained in plasma and urine, which is below the maximum residue limit (MRL) of 25 ng mL(-1). A set of seven plasma samples and 10 urine samples were classified identically by both methods as exceeding the MRL or being inferior to it. The results obtained with the UPLC/potentiometric detection method were compared with the results obtained with the UPLC/MS method for samples spiked with varying cocaine concentrations. The intraclass correlation coefficient was 0.997 for serum (n =7) and 0.977 for urine (n =8). As liquid chromatography is an established technique, and as potentiometry is very simple and cost-effective in terms of equipment, we believe that this method is potentially easy, inexpensive, fast and reliable.

Concentration-related response potentiometric titrations to study the interaction of small molecules with large biomolecules.

In the present paper, the utility of a special potentiometric titration approach for recognition and calculation of biomolecule/small-molecule interactions is reported. This approach is fast, sensitive, reproducible, and inexpensive in comparison to the other methods for the determination of the association constant values (Ka) and the interaction energies (ΔG). The potentiometric titration measurement is based on the use of a classical polymeric membrane indicator electrode in a solution of the small-molecule ligand. The biomolecule is used as a titrant. The potential is measured versus a reference electrode and transformed into a concentration-related signal over the entire concentration interval, also at low concentrations, where the millivolt (y-axis) versus log canalyte (x-axis) potentiometric calibration curve is not linear. In the procedure, Ka is calculated for the interaction of cocaine with a cocaine binding aptamer and with an anticocaine antibody. To study the selectivity and cross-reactivity, other oligonucleotides and aptamers are tested, as well as other small ligand molecules such as tetrakis(4-chlorophenyl)borate, metergoline, lidocaine, and bromhexine. The calculated Ka compared favorably to the value reported in the literature using surface plasmon resonance. The potentiometric titration approach called "concentration-related response potentiometry" is used to study molecular interaction for seven macromolecular target molecules and four small-molecule ligands.

Undetected components in natural mixtures: how many? What concentrations? Do they account for chemical noise? What is needed to detect them?

By definition, information about the set of components in a complex mixture below the detection limit is not directly available. However, if the composition of natural mixtures follows a natural law, the application of this law would enable the prediction of analytically important characteristics of that "hidden" fraction of the mixture. We have found that the analytical responses of compounds in three disparate natural mixtures (extracellular metabolites, light crude oil, and plant extracts) follow a log-normal (LN) distribution to a very high degree of correlation. Through the application of the LN model, the total number of components potentially detectable and the LN parameters of their analytical response distribution have been determined. From this distribution, one can predict the degree of analytical selectivity and dynamic range that would be required to detect any additional fraction of the components present. The data analyses of the studied mixtures reveal that the LN distribution parameters differ from one mixture type to another and that important information regarding the sample and the method employed is obtained. Further, the background level or "chemical noise" in the determinations studied agrees with the predicted cumulative responses of the undetected components. If generally applicable, the LN model will provide characterization parameters for mixture types, a means to assess completeness of analytical methods, and a model for theorists in mixture composition.

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 Girard's 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.

Development of in situ ion selective sensors for dissolution.

The dissolution of formulations of the drugs dapoxetine, paliperidone, cinnarizine, tetrazepam, mebeverine, loperamide, galantamine and ibuprofen was studied by an in-line potentiometric measurement system. The transpose of a Nikolskii-Eisenman type function performed the conversion of potential to percentage of dissolution. A novel gradient membrane electrode was developed especially for dissolution, varying continuously in composition from an ionically conducting rubber phase to an electronically conducting solid state PVC/graphite composite. The gradient part had a thickness of 200 microm. The electrodes life span exceeded 6 months. An ion exchange procedure was used to prepare them for one specific drug. This enabled us to use one universal electrode built to measure a wide array of drugs. The system parameters such as accuracy, reproducibility and linearity were presented with the data obtained for the drug dapoxetine. In dissolution, accurate measurements were possible from 10(-9) to 10(-3) M concentrations, for high log P drugs. The effect of t(90) response times on the measurement error was estimated. The t(90) response times of the electrodes were concentration dependent, and varied between 50 and 10 s for, respectively, 10(-6) and 10(-3) M concentrations. Potential drift was studied in detail. The measurements performed with these electrodes showed an accuracy of 1%, and inter- and intra electrode variabilities of 0.6 and 1.7%, respectively. The electrodes were successfully applied in colloidal media containing suspended matter, typically formed during dissolution of tablets. The advantages and pitfalls of potentiometry over the presently used techniques for dissolution testing are discussed.

Coated wire potentiometric detection for capillary electrophoresis studied using organic amines, drugs, and biogenic amines.

Capillary electrophoresis was coupled successfully and reliably to potentiometric sensors, which are based on an ionically conductive rubber phase coating, applied on a 250 microm diameter metal substrate. The membrane components included potassium tetrakis(p-chlorophenyl)borate (TCPB), bis(2-ethylhexyl)sebacate (DOS), and high molecular mass poly(vinyl chloride) (PVC). Potentiometry reveals a very sensitive CE detection mode, with sub-micromolar detection limits for amines and the randomly chosen drugs quinine, clozapine, cocaine, heroine, noscapine, papaverine, and ritodrine. The lowest detection limit, 1 x 10(-8) M injected concentration, was obtained for the quaternary ammonium compound tetrahexylammonium chloride. The more polar lower aliphatic amines and the biogenic amines dopamine, adrenaline, and cadaverine have much higher detection limits. The detection limits are log P dependent. Addition of a commercially available calixarene molecule or a synthetic macrocyclic amphiphilic receptor molecule to the electrode coatings enhanced the sensitivity respectively for the lower aliphatic amines and for the biogenic amines. A transpose of the Nikolskii-Eisenman-type function was suggested and used to convert the signal of the detector to a concentration-dependent signal.

[Potentiometric detection of beta-adrenolytic and beta-adrenergic drugs in HPLC systems]. / Potencjometryczna detekcja leków beta-adrenolitycznych oraz beta-adrenergicznych w ukladach HPLC.

This paper presents a comprehensive review of reference literature and the principal results of our own studies on the use of inclusion effects of macrocyclic compounds in the potentiometric detection of hypotensive and antiarrhythmic drugs as well as bronchodilators following their separation in HPLC systems. It was found that the properties of the liquid membrane electrodes embodying some macrocyclic compounds (i.e. trioctylated alpha-cyclodextrin, dibenzo-18-crown-6 or calix[6]arene hexaethylester) enabled reasonable prediction of their sensitivity in terms of the computed hydrophobicity and polarizability parameters of the analyzed drugs. The statistically significant equations enabling the optimalization of the composition of liquid membrane electrodes and the prediction of their detection limits in relation to the molecular structures of cationic drugs were established by applying multiparameter regression procedures. In particular it was indicated that the use of a liquid membrane electrode with trioctylated alpha-cyclodextrin enables sensitive detection (<10(-8) M) of highly hydrophobic drugs (logP >2.5 on the Parham-Hall-Kier scale) such as clenbuterol, bufuralol, and propanolol under HPLC conditions. These results indicate that the developed potentiometric detection method of hypotensive drugs can be considered as a promising alternative to the immunoassay procedures (ELISA) currently recommended for the toxicological determination of trace amounts of the drugs in biological samples. It was also shown that the set of experimentally determined detection limits characterizing the sensitivity of the four different liquid membrane electrodes used in the cation-exchange HPLC system enabled reliable screening and proper pharmacological classification of the beta-adrenergic and beta-adrenolytic drugs analyzed. Using principal component analysis (PCA) of the potentiometric data, it was stated that a differentiation of cardioselective from non-cardioselective beta1-blockers commonly administered as the hypotensive drugs was possible. This result indicates that the new method of potentiometric detection in HPLC systems can be applied as an effective high-throughput screening (HTS) procedure of large combinatorial libraries of newly synthesized drug-like candidates. This may increase the possibilities of identifying the lead compounds to be directed to further specialized biological tests, thus enabling the successful design and formulation of a new generation of more selective and pharmacogenome-oriented hypotensive drugs with reduced side-effects.

Potentiometric quasi-array employing calixarene derivatives for the high-throughput similarity/diversity screening of beta-adrenergic and beta-blocking chiral drugs by HPLC.

Performance of potentiometric quasi-array detection system consisted with the seven poly (vinyl chloride) (PVC) based liquid membrane electrodes in the cation-exchange HPLC using acetonitrile--40 mM phosphoric acid (15 : 85, v/v, pH* 2.35) for assessing of molecular similarity/diversity in a mini-library of beta-adrenergic and beta-blocking chiral drugs was presented. Macrocyclic compounds differing in stability of their conformers as well as in a size, steric hindrance and polarity of its internal cavities, comprising a series of five calix[6]arene derivatives completed with one modified calix[4]resorcinarene, were used as neutral ionophores to compose mentioned set of PVC-based electrodes. The output potentiometric responses were registered in the linear supernerstian range of calibration graph of each electrode, i.e. for a constant injected concentration 2.0 x 10(-4) M of investigated drugs, which is related to the amount frequently used at in vitro studies on pharmacological effects of these drugs. The impact of symmetry oriented supramolecular interactions on the responses of developed electrodes were characterised with proposed series of the highly significant quantitative structure-potentiometric response relationships (QSPRRs) combining both three-dimensional (3D) molecular descriptors of analysed drugs as well as lipophilicity and volume polarizability of calixarene-type ionophores. The principal components analysis (PCA) and unweighted hierarchical clustering analysis (HCA) were used as the pattern recognition techniques into collected potentiometric database for extraction of the useful information on the molecular and pharmacological similarity/diversity of analysed drugs, thus a high-throughput and consistent identification of therapeutically relevant agonists of beta2- and beta3-adrenoceptors and antagonists of beta1-adrenoceptor was especially achieved. This evidence supports also a hypothesis formulated by results of homology modelling on the subtle significance of an unrecognised supramolecular insertion processes of the chiral drug molecule into the flexible hydrophobic pocket(s) formed by seven helical transmembrane moving domains of beta-adrenoceptors on their final activation, sequestration, down-regulation or blockade.

Simultaneous high-throughput determination of clenbuterol, ambroxol and bromhexine in pharmaceutical formulations by HPLC with potentiometric detection.

Potentiometric detection of clenbuterol, ambroxol and bromhexine in marketed pharmaceuticals was described in six isocratic HPLC systems. The podant- and macrocyclic-type neutral ionophores, N,N,N',N'-tetracyclohexyl-oxybis(o-phenyleneoxy)diacetamide (TOPA) and hexakis(2,3,6-tri-O-octyl)-alpha-cyclodextrin (OCD), were applied in poly(vinyl)chloride (PVC)-based liquid membrane electrodes. Both types of neutral ionophores improve the sensitivity for all mentioned drugs when compared with a tetrakis(p-chlorophenyl)borate (BOR)-based electrode as well as with single wavelength UV detection. Detection limits (S/N=3) of 2.6 x 10(-10) mol l(-1) (injected concentration) for the highly hydrophobic bromhexine were achieved with the TOPA-based electrode and a cyano reversed-phase (RP)-HPLC with Uptisphere UP5CN-25QS silica column (250 x 4.6 mm i.d.) eluted with acetonitrile (AcN)-ethanol-perchloric acid (1.66 mM) (60:2:38, v/v/v) (pH* 2.45). Comparable result was obtained with OCD-based electrodes and an XTerra RP18 hybrid silica-polymer column eluted with AcN-phosphoric acid (20 mM) (25:75, v/v) (pH* 2.60). In the mobile phases containing 60-75% v/v AcN or methanol, stable and reproducible response of both types of neutral ionophore-based electrodes was observed for at least 3 days. The results of the validated procedure for reliable simultaneous determination of the drugs in fortified representative samples of pharmaceuticals were also presented.

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.

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.

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.