Search

A capillary electrophoresis method for determination of remoxipride-related impurities was developed using a dynamically tetraalkylammonium-coated C8capillary. The shape of the overloaded principal peak was regulated by substituting tetraalkylammonium ions of different size for sodium ions, thereby altering the conductivity in the background electrolyte. An adequate separation of the compounds was obtained after addition of methyl-β-cyclodextrin as structural selector in combination with a reversed electroosmotic flow. The detection level for the impurities was 0.05 % (w/w) with the Beckman Pace 2100 using UV-detection. The limit of detection was substantially improved for the fluorescent compound FLA 797 using a LIF-detector equipped with a He-Cd laser as excitation source achieving detection levels below 0.0005 %.

Summary: A chiral capillary electrophoresis system for the highresolution separation of the enantiomers of the local anaesthetics mepivacaine, ropivacaine, bupivacaine and prilocaine is described. Triethanolamine was added to the background electrolyte to obtain a negative electroosmotic flow and hence higher resolutions. The interactions of the local anaesthetics and their chemical analogues with the chiral selector, dimethyl-beta-cyclodextrin, were studied. From a model describing chiral capillary electrophoresis, the association equilibrium constants were determined by curve-fitting. The separation of mepivacaine, ropivacaine and bupivacaine was due to the different mobilities of the free analytes in solution, whereas the separation of a pair of enantiomers of a single analyte was due to differences between the association equilibrium constantsK 1 andK 2. Branching of the alkyl chain, which was situated close to the cavity in the inclusion complex, had strong effects on the chiral separation of the enantiomers.

A sensitive post-column fluoresence method based on the reaction of reducing sugars with a fluorigenic reagent (benzamidine) under alkaline conditions and elevated temperature is described. Optimum reaction conditions suitable for the detection of fluorescent reaction products have been investigated. A porous graphitized carbon column (shandon Hypercarb) was employed as the chromatographic solid phase and elution was isocratic by an aqueous acetonitrile mixture.

Summary: Retention models for ionic compounds in reversed-phase ion-pair chromatographic systems have been developed on the basis of the capacity ratios of solutes and ionic mobile phase components using indirect detection. The binding to the adsorbent, μBondapak Phenyl, has been expressed by ion-pair distribution constants and tested in the retention models by non-linear regression. All the ionic compounds followed adsorption models of the Langmuir type comprising two kinds of binding sites with widely different adsorption capacities and binding constants. The results indicate certain differences between the two sites regarding the binding of cationic and anionic components, respectively.

Summary: Since the late 1970’s several techniques involving the direct injection of biological fluids, like plasma and serum have been published. Most of the systems presented utilize a precolumn for the reception of the biological fluids, trace enrichment and preliminary clean-up before the analyte fraction is transferred to the separation column. The most common solid phase for the precolumn is silanized silica, but many other types have been used. One was especially designed for the purpose of direct injections of biological fluids: the Internal Surface Reversed Phase support. The main problem with direct injections is the development of column instabilities due to the denaturation and precipitation of plasma proteins on the support. It has, however, been possible to design systems which can handle the injection of 25ml total plasma volume without the need to exchange precolumn. The most promising techniques in this respect are the so called “pre-column” venting plug technique and the use of micellar mobile phases. Losses of analytes may be a problem when protein-binding extends 95%. However, remedies are available, such as the use of more hydrophobic solid phases, the addition of competitive binders and pH adjustments of injected solutions.

Summary: Ionic compounds without inherent detectable properties show good response in reversed phase ion-pairing systems containing a detectable ionic compound (probe) in the mobile phase. The basis for the indirect detection is the common interaction effect for solutes and probe caused by the limited binding capacity of the adsorbent. Quantitative models for solutes and probe with the same or opposite charges show a simple dependence of the response on the retention of the solute and the coverage of the binding surface by the probe. Applications of the models have shown good agreement between calculated and found response. The binding to sites of different kinds on the adsorbent and possibly ion-pair formation in the mobile phase have an influence on the indirect response. Uncharged solutes give low response in such systems owing to low binding to the competitive sites.

Summary: The distribution equilibria of cationic compounds in reversed-phase chromatographic systems (ion-pair chromatography) have been studied on the basis of their effect on a detectable mobile phase component. The solid phase was a polystyrene-divinylbenzene copolymer and the detectable component, a quaternary ammonium ion, 1-methylpyridine. The solutes were mono- and divalent amines and quaternary ammonium ions. The cations can be retained by ion-pair adsorption and ion exchange. Expressions for the ion-pair retention of the solutes and the mobile phase cation (system peak) have been developed assuming Langmuir distribution of ion pairs to a solid phase with one kind of binding site. The validity of the expressions has been tested by evaluation of ion-pair distribution constants using non-linear curve fitting techniques. Good agreement for the constants of common ion pairs was obtained from different kinds of capacity ratio expressions. Ion exchange retention can appear beside ion-pair retention, and it has been observed in the pH range 1.6–6.1. The effect depends not only on cations in the mobile phase, but also on the nature of the buffering systems.

Retention models for anionic analytes and mobile phase compounds in reversed-phase ion-pair chromatographic systems have been studied. The solid phase was a polystyrene-divinylbenzene copolymer. The analytes were mono- and divalent anions monitored by indirect detection technique. The mobile phase was highly alkaline and contained a detectable anion, sulfanilic acid.

A new technique for the separation of carbohydrates as ion-pairs in strongly alkaline solution is presented. Carbohydrates are weakly acidic and partly present as anions at pH≥12 [1]. They are retained as ion-pairs on polymeric solid phases (PRP-1 and PLRP-S) with a hydrophobic quaternary ammonium counter ion present in the mobile phase. The effects of nature and concentration of mobile phase components on the retention of carbohydrates have been investigated and an ion-pair distribution model is proposed. The influence of temperature indicated no changes in retention mechanism with high counter ion concentration, but the resolution decreased with increasing temperature. Saccharides added to the mobile phase were shown to increase the retention and the selectivity.

At alkaline pH, carbohydrates are highly retained on a sulphonated polystyrene resin loaded with rare earth or uranyl metal ions. The complex formation is governed by the ionization of the carbohydrate moiety. pH and the type of metal ion were found to have a decisive influence on the complex formation. Binding of the metal ion to the cation exchanger and to different carbohydrates is described. The competitive complexation between solute and hydroxide to the metal ions is described as a ligand exchange reaction and a retention model is proposed. The ligand exchange column was used as precolumn in a coupled column separation system and the high selectivity is demonstrated by the separation of paracetamol glucuronide from a urine sample after filtration and direct injection.

The relative bioavailabilities of single oral doses of ampicillin, amoxycillin, and bacampicillin were compared with and without concomitant administration of a six-times higher molar dose of cyclacillin. As the absorption of cyclacillin has been shown to involve a capacity-limited transport system in animals, it was selected as the reference compound for the study. The treatments were given to 14 fasting volunteers using a randomized, complete crossover design. The drugs in plasma and urine were determined by liquid chromatography. Renal clearance was 17%, 10% and 19% lower when ampicillin, amoxycillin, and bacampicillin were given together with cyclacillin. Consequently, differences in the relative bioavailability were based on urinary recoveries assuming constant non-renal clearance. When amoxycillin was given with cyclacillin there was a 67% delay in the time of the plasma peak concentration, and an 8% lower urinary recovery than when it was given alone. There was a 50% and 33% delay in the tmax of ampicillin and bacampicillin when combined with cyclacillin; the urinary recovery of ampicillin in the combination was 10% lower but that of bacampicillin was similar. There was also a 20% delay in the tmax of cyclacillin when combined with amoxycillin. The differences in renal clearance indicate an interaction in the renal elimination of the drugs, but the effect was probably not the explanation for the marked shift in time of the absorption of these rapidly absorbed drugs. The results support the existence of a capacity-limited transport system for aminopenicillins in the human gut.

The systemic availability of a new antidepressant, zimelidine, and of its pharmacologically active metabolite, norzimelidine, was studied in six healthy male volunteers. Three single doses of zimelidine (25 mg and 100 mg orally and 25 mg i.v.) and two single doses of norzimelidine (25 mg orally and i. v.) were given to each volunteer allowing at least seven days between administrations. Plasma concentrations of zimelidine and norzimelidine were determined in serial blood samples by HPLC. Following oral zimelidine peak plasma concentrations of the metabolite were attained about 3 h after dosing. Oral administration of norzimelidine itself resulted in a plasma concentration profile for this compound that was similar to that observed after oral zimelidine. Utilising the plasma concentration data following intravenous infusion of each compound, the elimination half-lives for zimelidine and norzimelidine were calculated to be 5.1 h (range 4.3–6.0) and 15.5 h (range 10.6–22.9) respectively. The total body clearances of the 2 compounds were similar at 0.52 l · min-1 (range 0.26–0.70) for zimelidine and 0.56 l · min-1 (range 0.28–0.83) for norzimelidine. The substantially longer elimination half-life of norzimelidine was apparently the result of a larger volume of distribution (9.4 l · kg-1; range 7.8–11.4) for this metabolite, as compared to zimelidine (3.21 · kg-1; range 1.6–4.9). The calculated bioavailability of zimelidine was 26% (range 9.1–39) after the 25 mg oral dose, and 29% (range 14–46) after the 100 mg dose. The bioavailability of norzimelidine was 66% (range 36–91). However, oral administration of zimelidine resulted in as much or more norzimelidine reaching the systemic circulation, as the oral administration of norzimelidine itself. This is important as a large part of the activity of the drug may be due to the metabolite.

The pharmacokinetics of bacmecillinam and pivmecillinam were studied in healthy fasting volunteers given tablets in a cross-over, randomized order. The mean (±SD) peak levels of plasma mecillinam were 1.43±0.34, 2.73±0.43, and 4.62±1.41 mg/l after bacmecillinam 100, 200, and 400 mg and 2.38±0.65 mg/l after pivmecillinam 400 mg. The corresponding areas under plasma Vs time curves (AUC) were 2.21±0.19, 3.99±0.63, and 7.74±1.38 mg·h·l-1 for bacmecillinam and 5.35±0.93 mg·h·l-1 for pivmecillinam. The elimination half-lives were 0.8–1.1h for bacmecillinam and 0.7h for pivmecillinam. The 12 h urinary recovery of unchanged mecillinam after the 400 mg doses was 41% for bacmecillinam and 30% for pivmecillinam. The 400 mg dose of bacmecillinam gave a significantly higher plasma peak (p<0.001), AUC (p<0.001) and urinary recovery (p<0.001) than did pivmecillinam 400 mg. The plasma peaks appeared earlier and the rate of absorption was higher after bacmecillinam than after pivmecillinam (p<0.05). In conclusion, bacmecillinam had a better bioavailability than pivmecillinam in the tablet formulations studied. The AUC increased linearly with increasing doses of bacmecillinam.

An indirect UV-detection method in the separation of saccharides by reversed-phase LC was developed. Porous graphitic carbon was used as the solid phase and sorbic acid, muconic acid and furanacrylic acid were tested as UV-absorbing markers included in the alkaline sodium hydroxide-methanol mobile phase. The indirect response of the saccharides was in general found to follow earlier developed theories. The most important parameters for optimizing the response were the retention of the solutes relative to the marker, which should be close to 1, and a high loading of the marker on the solid phase. The detection sensitivity can be improved by optimizing the marker and organic solvent concentrations in the mobile phase, the pH as well as the temperature. The stability of the saccharides was found to be adequate for the alkaline conditions used in the separation systems.

Peak compression and deformation effects were studied in a reversed phase ion-pair adsorption chromatographic system, comprising silanized (C18) silica as solid phase and an acidic mobile phase. An indirect detection technique was used, protriptyline (a secondary hydrophobic amine) being the probe, and substituted benzamides the analytes.

Adsorption of a secondary amine, protriptyline, as phosphate ion-pair, to Nucleosil C18, was studied. Two different techniques were compared; the traditional break-through and the system peak technique. Both techniques indicated that the adsorption of the ion-pair was best fitted to a 2-site adsorption model of the Langmuir type, indicating a heterogenous solid phase surface.The reliability of the system peak technique was tested by comparing the retention volumes of the system peaks for different kind of equilibrium disturbances introduced into the system. When injecting finite amounts of analytes the system peak retention was affected by the analyte concentration, charge and hydrophobicity, keeping the eluent composition constant. The effects could qualitatively be explained in light of the developed model for the retention.

FLA 908 was identified as a metabolite of remoxipride in human urine after enzymatic hydrolysis. The identity was proven by comparison of its retention time in LC and its UV and mass spectra to authentic FLA 908.

The selective inhibitors of neuronal 5-hydroxytryptamine (5-HT) and noradrenaline (NA) uptake, zimelidine and desipramine, were compared in a double blind crossover study of 40 inpatients with endogenous depression. The clinical effects of these two drugs and some biochemical variables related to the monoamine systems were studied during 4 weeks' treatment. Patients with a low pretreatment level of 5-hydroxyindoleacetic acid (5-HIAA) in the cerebrospinal fluid (CSF) (<20 ng/ml) responded significantly better to zimelidine treatment than those with a high pretreatment level (>20 ng/ml). In the group treated with desipramine no difference in therapeutic outcome was obtained between patients with low and high pretreatments levels of 5-HIAA in the CSF. Attempts to correlate the steady state plasma concentrations of zimelidine, norzimelidine and desipramine with the therapeutic effect were unsuccessful. The plasma concentration of norzimelidine demonstrated a singificant (P<0.05) positive correlation with age. The mean value of the uptake of 14C-5-HT in the patients' platelets, when measured before the treatment, was significantly (P<0.05) lower than found in a control group. Zimelidine, mainly via its metabolite norzimelidine, caused a pronounced inhibition of uptake of 14C-5-HT in platelets, decrease in whole blood 5-HT and inhibition of accumulation of 14C-5-HT in rat hypothalamic synaptosomes incubated in the patients plasma. Desipramine produced a slight inhibition of accumulation of 14C-5-HT in rat synaptosomes, but a marked inhibition of uptake of 14C-5-HT in the patient's platelets and a decrease in whole blood 5-HT. The accumulation of 3H-NA in rat synaptosomes incubated in the patients' plasma was strongly inhibited by desipramine treatment but only slighty affected by zimelidine.

Summary: Dynamic coating of bonded C8 fused silica capillaries with cationic amphiphiles is shown to minimize adsorption of basic drug molecules but also to facilitate control/reversal of electroosmotic flow. Optimization of the peak shapes was achieved by addition of cationic amphiphiles with a suitable mobility and adsorption to the capillary surface. The surface modification described, combined with the addition of cyclodextrins as selectors into the background electrolyte, resulted in unique CE systems showing excellent resolution for structural analogues. The separation of rotamers and enantiomers is demonstrated. Applications to the antipsychotic compound remoxipride and its related substances and to the chiral separation of asthma- and local anaesthetic drug enantiomers are given.

An indirect UV-detection method in the separation of saccharides by reversed-phase LC was developed. Porous graphitic carbon was used as the solid phase and sorbic acid, muconic acid and furanacrylic acid were tested as UV-absorbing markers included in the alkaline sodium hydroxide-methanol mobile phase. The indirect response of the saccharides was in general found to follow earlier developed theories. The most important parameters for optimizing the response were the retention of the solutes relative to the marker, which should be close to 1, and a high loading of the marker on the solid phase. The detection sensitivity can be improved by optimizing the marker and organic solvent concentrations in the mobile phase, the pH as well as the temperature. The stability of the saccharides was found to be adequate for the alkaline conditions used in the separation systems.

Die untersuchten Amine finden Verwendung als Arzneimittel und können in Mengen von ng/ml bestimmt werden. Anthracen-2-sulfonat dient als Gegenion. Die Eignung zur Ionenpaarextraktion wird durch Bestimmung der Extraktionskonstanten geprüft. Die Fluorescenzeigenschaften werden durch Messung der Quantenausbeuten der Fluorescenz bestimmt. Die erforderlichen Eigenschaften eines Lösungsmittels werden diskutiert. Methylenchlorid erweist sich dabei als gut geeignet. Die Bedingungen für quantitative Extraktion und Bestimmung werden angegeben. Die Methode wurde für die Bestimmung von Amitriptyline im Bereich 10–100 ng/ml ausgearbeitet, wobei sich ein Extraktionsgrad von 100% und eine Präzision von 5% ergaben.