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Even though it is known that the enantiomers of a drug can exhibit different pharmacokinetic and dynamic properties, fencamfamine has been used in clinical practice as a mixture of four isomers, arising from the two centres of chirality in the molecule. The purpose of this investigation was to establish a capillary electrophoretic method for the discrimination of the isomers of fencamfamine and to determine its isomeric composition. The chiral selectors studied were uncharged α-cyclodextrin (α-CD), β-CD, heptakis (2,3-di- O -acetyl)β-cyclodextrin, γ-CD and octakis (2,3-di- O -acetyl)γ-cyclodextrin, and the negatively charged sulfobutylether-β-CD. In order to optimize the separation conditions, the selector concentration, pH values and buffer strength were varied. With Diac-β-CD and γ-CD, a very high resolution of the four isomers was found. Interestingly, the migration order of the diastereomers was different for these two selectors. In each case NMR spectroscopic studies revealed multiple isomer–cyclodextrin complexes to be responsible for the good discrimination of the isomers. In contrast to the classical inclusion complexes mostly described for CDs, fencamfamine was found to interact mainly with the outside region of the CD cavity.

The effect of acetylation of β-cyclodextrin (CD) on the chiral discrimination of a series of phenethylamines has been investigated using capillary electrophoresis and nuclear magnetic resonance (NMR) spectroscopy. Therefore, pure, fully derivatized CDs were synthesized. Capillary electrophoresis measurements revealed that the 2,3-diacetylated CD was a better discriminator than the 6-acetylated and the native CD. NMR investigations of the complexes suggest that the phenyl ring of the phenethylamines is inserted in the cavity and the side chain interacts with the hydroxyl rim of the macrocycle. The structures of the complexes indicated by the NMR results were supported by molecular models of the derivatised CDs.

The enantio-resolution of propranolol and four derivatives was examined by free solution capillary electrophoresis (FSCE) using various cyclodextrin molecules. Of the three modified cyclodextrins investigated, hydroxyethyl-β-cyclodextrin provided the largest chiral resolution values for all five analytes. This may be linked to its extended hydrogen bonding chains on the cyclodextrin rim. Methyl-β-cyclodextrin and heptakis (2,3-di-O-acetyl) β-cyclodextrin gave lower maximum analyte resolutions, probably due to differences in their macrocyclic structure and hydrogen bonding ability. The presence of a bulky, non-polar alkyl grouo on the analytes was found to enhance chiral recognition. Methanol was found to have a varied effect on chiral resolutions, dependent on the type of cyclodextrin and structure of the analyte.

The extraction and isolation of polychlorinated biphenyls (PCBs) from sample matrices such as freeze-dried milk is a lengthy and expensive process that conventionally requires the isolation of microgram amounts of PCBs from large volumes of flammable organic solvents used in the extraction and chromatographic stages. Supercritical-fluid extraction (SFE) and chromatography (SFC) using carbon dioxide has been investigated as a replacement procedure using a Simplex optimisation approach to optimise the working conditions. The SFE conditions required to extract PCBs and milk fat from freeze-dried milk were investigated, and it was found that although extraction was rapid (10 min), the conditions required were identical so that there was no separation of PCBs and fat. However, SFC conditions have been developed that permit the resolution of PCBs from milk fat in 15 min. Thus, a combination of SFE and SFC have the potential to replace the existing methods of extraction and isolation with procedures that are quicker, cheaper, and avoid the use of flammable organic solvents.

The semi-preparative reversed-phase HPLC separation of brompheniramine enantiomers using β-cyclodextrin as a chiral mobile phase additive is described. Enantiomers were recovered free from mobile phase components on-line by a novel column switching procedure. On a 250 mm × 10 mm I.D. cyano column, a throughput of 8 mg per hour (0.65 mg per gram column packing per hour) was achieved at product optical purities of 88% and above. This was achieved using two passes through the system. Comparison is made with other reported semi-preparative chiral separations using β-cyclodextrin, and the potential utility of the described method discussed.

The production of cyanobacterial toxins as anatoxin-a in the UK by blue—green algae such as Oscillatoria and Anabaena flos-aquae is a potential health hazard, especially to animals, birds and fish. A sensitive reversed-phase ion-pair high-performance liquid chromatographic (RP-HPLC) method is described for the measurement of the neurotoxins anatoxin-a (AnTx) and homoanatoxin (HomoAnTx) in the presence of lyophilized algal extract. A base-deactivated C18 column material was used with acetonitrile—phosphate buffer (pH 3) and sodium dodecyl sulfate as ion-pair reagent with ultraviolet detection at 227 nm. Linear calibrations were obtained between 2 and 93 ng on-column for AnTx·HCI and 2–112 ng on-column for HomoAnTx·HCI with limits of detection of 1 and 2 ng on-column, respectively. A sample of Oscillatoria bloom material collected from Loch Insh, Scotland, in 1991 was found by this method to contain approximately 0.8 mg of AnTx·HCl per gram of lyophilized extract. Homoanatoxin was not present. The identity of AnTx was confirmed by the isolation of the AnTx peak by RP-HPLC, its derivatization to N-butylAnTx and its analysis by RP-HPLC and gas chromatography—mass spectrometry.

A procedure is described which allows the on-line recovery of enantiomers following semi-preparative chiral separation by RP—HPLC using β-cyclodextrin in the mobile phase. By this method, the phenothiazine antihistamine trimeprazine (I) was resolved into its antipodes at greater than 95% optical purity at a throughput of more than 1 mg of each enantiomer per hour, using 4 mm i.d. columns. The recovered trimeprazine was found to be free from cyclodextrin.

Ampicillin trihydrate, sodium salt, in aqueous solution has a pH of about 8. No complete degradation pathway has been proposed to explain the degradation of ampicillin under alkaline conditions and the information available explains the formation of only certain products. The present work was carried out with the aim of providing this information. The formation of degradation products of ampicillin trihydrate, sodium salt, produced in aqueous solutions (pH 12 and 37°C) have been studied as an accelerated form of the possible degradation that may occur in aqueous solutions at pH 8. Some of the degradation products formed under these conditions were then obtained either by synthesis or by degradation of ampicillin sodium followed by isolation using semi-preparative HPLC. These compounds were characterized by 1H NMR spectroscopy. The information obtained from the experiments by HPLC and NMR spectroscopy made it possible to propose a degradation pathway for ampicillin under the conditions described above. 5R-penicilloic acid is the first degradation product of ampicillin and subsequently undergoes epimerization at C-5 to form the 55 isomer via the imine tautomer. Mechanisms for the formation of compounds previously believed to form only under acidic conditions are proposed, i.e. ampicillin penilloic acid and 2-hydroxy-3-phenylpyrazine. The formation of ampicillin polymers was detected in dilute solutions (1% w/v) within a few hours of dissolution. The presence of ampicillin penicillenic acid and ampicillin penamaldic acid was detected by 1H NMR and their main spectroscopic features determined.

The resolution of nine sympathomimetic phenethylamine racemates by beta-cyclodextrin and heptakis(2,3-di-O-acetyl)beta-cyclodextrin has been investigated by capillary electrophoresis and 1H NMR spectroscopy. The NMR and capillary electrophoresis results showed that beta-cyclodextrin probably formed stronger complexes with the amines than did heptakis(2,3-di-O-acetyl)beta-cyclodextrin but was a poorer chiral discrimination agent in both techniques. The addition of heptakis(2,3-di-O-acetyl)beta-cyclodextrin to the capillary electrophoresis buffer gave baseline resolution of enantiomer peaks for seven of the compounds studied while beta-cyclodextrin resolved only three of the racemates.

The measurement of cocaine and its major metabolites has been achieved by an HPLC method that compensates for their different solubilities and detection properties. Although ecgonine methyl ester is a major metabolite it is generally not measured by HPLC because it is poorly detectable by UV, and its water solubility makes recovery from urine difficult. Using modified solid-phase extraction procedures recoveries of 85% for ecgonine methyl ester, 97% for cocaine, 106% for benzoylecgonine and 80% for ethylcocaine have been obtained from urine. Increased chromatographic retention and detection sensitivity has been obtained by formation of the t-butyldimethylsilyl derivative of ecgonine methyl ester which was found to be stable in the HPLC mobile phase for at least 1 week. Alkylation of norcocaine and benzoylecgonine has improved their detection sensitivity and also chromatographic resolution. All calibrations were linear over the range 200-1000 ng ml-1 in urine with correlation coefficients > 0.99.

The interaction of beta-cyclodextrin with a series of structurally related chiral thromboxane antagonists was investigated using NMR and RP-HPLC. HPLC studies used both a cyclodextrin bonded phase (Cyclobond I), and beta-cyclodextrin as a mobile phase additive with an achiral C8 column. Many of the compounds exhibited chiral recognition with beta-cyclodextrin in each technique, but only partial correlations between the three data sets were observed. HPLC and ROESY NMR data suggested the possibility of bimodal inclusion.

A phase separator is described that is suitable for post-column HPLC applications. It operates with commonly used HPLC eluents and immiscible organic solvents as long as the two phases remain immiscible. It is compatible with gradient elution systems. Separation efficiency is routinely better than 0.8, which ensures that analyte peak heights are about 95% of the maximum height under these conditions. An application for the detection of pethidine, cocaine, methadone, piritramide and dipipanone at 0.8–1.8 ng on-column loadings is described.

A rapid, sensitive assay for tryptophan and some of its metabolites in urine, plasma and saliva has been developed using sodium dodecylsulphate as a pairing ion in a surfactant ion-pair high-performance liquid chromatography technique. The method is highly selective for tryptophan which is separated from its main indoleamine metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, and from kynurenine. The usefulness of the assay has been demonstrated in plasma level and urinary excretion studies of orally administered tryptophan.

The behaviour of functional groups having widely differing physico-chemical character has been examined in ion-pair reversed-phase high-performance liquid chromatography using surface active pairing ions. The effects of temperature, mobile phase, organic modifier type and percent composition, stationary phase carbon loading and type, ionic strength, and pairing ion structure, charge and concentration on extra-thermodynamic functional group contribution values, have all been determined. Analysis of group behaviour within the framework provided by solvophobic theory is often found to be possible using linear free-energy relationship appraoches, and it is shown that retention behaviour can be described as ion-pair formation in the mobile phase followed by distribution to the stationary phase. In addition, substituent behaviour is found to exhibit linear enthalpy-entropy compensation behaviour, suggesting further that a common retention mechanism can be described for all ionized solutes using these pairing ions.

The selection of columns containing cyanopropyl-bonded silica (3.5% carbon loading) and their use under reversed-phase conditions has permitted short retention times with good resolution to be obtained for 16 basic drugs of abuse and three metabolites. Predominantly aqueous mobile phases enable the determination of these weakly UV-absorbing compounds over the concentration range 0.7–18 μg ml −1 with detection at 205 nm. Trimethylsilyl-bonded silica (2.6% carbon loading) also provided good resolution but required longer analysis times.

The qualitative and quantitative determination of large anionic solutes, including the drug sodium cromoglycate, by an ion-pair high-performance liquid chromatographic technique using alkylbenzyldimethylammonium chlorides as pairing ions is described. The method provides a sensitive and selective assay for the solutes examined in both simple aqueous solutions and in complex biological fluids, and requires no pre-extraction step. Retention mechanisms are discussed.

Reversed-phase ion-pair conditions are used for the determination of d -penicillamine and penicillamine disulphide. Two chromatographic systems were employed, one for penicillamine and the other for penicillamine disulphide. The procedures permit the determination of total penicillamine (protein-bound, free and as disulphides) in whole plasma, and total penicillamine (free and as disulphides) in plasma ultrafiltrate, using an incubation step in the presence of dithiothreitol. Free penicillamine and penicillamine disulphide may be determined independently by direct injection of plasma ultrafiltrate. Both solutes may be measured at an on-column sensitivity of 10 ng, utilizing an electrochemical detector based on a glassy carbon electrode.

The analysis of ionized solutes by high-performance liquid chromatography (HPLC) may be facilitated by the use of ion-pairing agents. This contribution discusses the development of ion-pair HPLC during the past decade, and points to those aspects of the technique which have greatest significance. Mathematical descriptions of various probable ion-pair retention mechanisms are given and related to the practical usage and efficiency of the methods. A comprehensive listing of reported chromatographic systems, pairing ions and solutes studied is given.

Three pre-column derivatization reagents, namely o -phthalaldehyde, 4-chloro-7-nitrobenz-2,1,3-oxadiazole, sodium naphthaquinone-4-sulphonate and two ion-pair reagents, namely, naphthalene-2-sulphonate and sodium dodecylsulphate have been investigated for their suitability for the qualitative and quantitative analysis of urine and plasma samples containing amphetamines. The derivatization method employing sodium naphthaquinone-4-sulphonate was found to be selective and sufficiently sensitive for the routine determination of amphetamine and methylamphetamine in urine and plasma samples at the ng/ml level.

The separation of organochlorine pesticides and polychlorinated biphenyls from a high concentration of lipid has been achieved using a polymeric column designed for high-performance liquid chromatography. The method, which can be used as a clean-up step in residue analysis procedures, has a high lipid capacity but low solvent consumption. Recoveries of both lipid and selected organochlorine compounds were routinely over 90% at p.p.m. levels.

The chromatographic separation of tetracycline, oxytetracycline, 6-demethyl-chlortetracycline, methacycline and minocycline, and the common impurities of tetracycline, oxytetracycline and chlortetracycline has been optimised on a Hamilton PRP-1 column using citrate-phosphate buffer with propan-2-ol and tetrahydrofuran as organic modifiers. Column efficiency was approximately doubled by the inclusion of 1% v/v dichloromethane in the mobile phase.

Codeine and norcodeine have been extracted together from a 7-l batch of microbial transformation liquor using an XAD-4 resin column. The extract was divided equally and used to compare normal-phase (NP) and reversed-phase (RP) preparative high-performance liquid chromatography for the recovery of pure codeine and norcodeine. The conditions for each column were optimised for maximum sample throughput with complete resolution of both compounds. The aqueous fractions of codeine and norcodeine from the RP column were also passed through an XAD-4 resin column to extract the compounds finally, whereas the organic solvent fractions from the NP column did not require this step. The RP procedure gave recoveries of 71.25% of codeine (98.6% purity) and 80.69% of norcodeine (97.5% purity) and the NP procedure gave recoveries of 79.20% of codeine (96.7% purity) and 83.97% of norcodeine (97.2% purity). Owing to its superior sample throughput and lower operating costs, the RP procedure is recommmended for routine use.

A previously described routine procedure involving the use of a silica gel column in determining diosgenin and yamogenin has been improved by using water-containing solvents. The advantages are that there is less variation between duplicate results, that each column can be used at least five times and that component bands are eluted in predictable volumes of solvents. An apparatus and solvent sequence is described that allows twelve columns to be developed simultaneously.The method has been successfully applied to crude extracts from Dioscorea deltoidea tuber and to oily crude extracts from the seeds of Trigonella foenumgraecum(fenugreek) and Balanites aegyptiaca. The over-all error of the procedure, including sampling, extraction and infrared spectrophotometric determination for duplicate analyses of 2·5-g samples of the fenugreek seed used, expressed as a 95 per cent. confidence interval of the mean sapogenin value, was 1·04 ± 0·025 per cent. for diosgenin plus yamogenin, 0·64 ± 0·019 per cent. for diosgenin and 0·40 ± 0·023 per cent. for yamogenin.As the method does not permit the separation of tigogenin from diosgenin nor that of neotigogenin from yamogenin, the results indicate maximum yields for diosgenin and yamogenin in fenugreek seed. The results exclude sterols, steryl esters, dihydroxysapogenins and spirostadienes.

A routine procedure is described for the determination of diosgenin and yamogenin in fenugreek seed. A crude extract is obtained by acidic hydrolysis of the seed, neutralisation and extraction of the insoluble matter with light petroleum. By means of column chromatography this extract is freed from fixed oil and other substances, thus yielding a mixture of diosgenin and yamogenin suitable for infrared spectrophotometric analysis. The results calculated for duplicate analyses of 2·5-g samples of commercial Moroccan seed, expressed as a 95 per cent. confidence interval of the mean sapogenin value, are 0·96 ± 0·017 per cent. for diosgenin plus yamogenin, 0·58 ± 0·008 per cent. for diosgenin and 0·38 ± 0·016 per cent. for yamogenin. The procedure has been found to be satisfactory for column loadings of up to 75 mg of diosgenin plus yamogenin in the presence of up to 600 mg of fixed oil, which is approximately three times the weight of extractive from 2·5 g of Moroccan seed.