Control of dispersion in capillary electrochromatography coupled to UV and mass spectrometric detection.

Capillary electrochromatography (CEC), along with other miniaturised chromatography techniques, such as capillary LC, offers the most benefit when efficiently coupled to mass spectrometric (MS) detectors. In conventional one-piece CEC columns, dispersion in the open connecting tube between the packed column and MS source reduces chromatographic performance to unacceptable levels. This paper examines the effect on dispersion of various column-tube arrangements and offers suggestions as to the most practical way of connecting CEC-UV-MS. Comparisons of theoretical and measured values for these different arrangements are shown.

Determination of paracetamol and its metabolites in urine by high-performance liquid chromatography using ion-pair systems.

Paracetamol (P) and its four main metabolites, the sulphate (S), glucuronide (G), cysteine (C) and mercapturic acid (M) conjugates, are separated on ODS/TMS silica using a standard eluent, water-methanol-formic acid (86:14:0.1, v/v/v), in the order S, G, P, C, M. On addition of the ion-paring cations dioctylammonium (DOA) and tetrabutylammonium (TBA), the retention of S is vastly increased while those of G and M are substantially increased and that is C is reduced. The excessive retention of S and to a lesser extent that of M and G can be controlled by the addition of a suitable concentration of a co-ion such as nitrate. The order of elution is then G, C, P, M, S, although this can be varied by adjusting the amount of nitrate present. Loading of DOA is slow as it is very strongly adsorbed and typical concentrations in the eluent are below 7 mg/l. Loading by TBA is rapid with typical concentrations being around 200 mg/l. The effects of added co-ions such as nitrate can be explained in terms of simple ion-pair equilibria. Equilibration with respect to added salts is rapid. Application of the technique to analysis of therapeutic and overdose urines shows the presence of at least three additional metabolites, one of which is identified by mass spectrometry as most probably 3-methoxyparacetamol. Another appears to be a methoxymercapturic acid derivative. There is further evidence for a group of metabolites that elute unresolved from overdose urines as a broad band after the main metabolites.

Determination of paracetamol and its metabolites in urine by high-performance liquid chromatography using reversed-phase bonded supports.

Paracetamol and its four main metabolites, the sulphate (S), glucuronide (G), cysteine (C) and mercapturic acid (M) conjugates, are readily separated in a synthetic mixture using slightly acidic aqueous alcoholic eluents (e.g. water-methanol-formic acid, 85:15:0.15, v/v/v) on either octadecyl silica (ODS silica) or octadecyl silica which has been further silanized to remove residual hydroxyl groups (ODS/TMS silica). The dependences of k' upon alcohol, acid and added salt concentrations are reported for both materials. The latter material gives the higher plate efficiencies and is much superior when applied to analysis of urines taken after therapeutic doses and overdoses of paracetamol. At least four additional metabolites are reported in overdose urines. Mass spectrometric analysis (high and low resolution) has confirmed the identity of M and identified one of the additional metabolites as methoxyparacetamol. Mass spectra of the remaining additional metabolites enable major structural features to be deduced. One of these metabolites may be associated with liver damage.

Separation of catecholamines and their metabolites by adsorption, ion-pair and soap chromatography.

The separation of catecholamines and their metabolites has been carried out by high-performance liquid chromatography using three systems: liquid-solid adsorption, ion-pair partition, and soap chromatography. In the last newly developed technique a reversed-phase packing is used in conjunction with an aqueous organic eluent containing a detergent. The detergent is chosen so that its ion can form ion-pairs with ions of the solutes. Soap chromatography proved the best technique in terms of column efficiency (giving 3000-5000 plates in 125 mm), resolution and sensitivity of detection. Noradrenaline, adrenaline and dopamine, their 3-O-methyl derivatives, L-3,4-dihydroxyphenylalanine, homovanillic acid and other related compounds could be separated in less than 10 min at the 10-50-ng level on columns whose plate heights were in the range of 20-40 mum. The dependence of retention on the concentration of organic modifier and on detergent concentration for three anionic detergents is reported. The method is applied to the direct analysis of urine and the potential of the method for such analyses, especially of pathological urines is demonstrated. Soap chromatography is likely to enlarge the scope of application of high-performance liquid chromatography to biochemical analysis. It is a powerful method for the separation of ionizable compounds which could replace conventional ion-exchange chromatography.

Separation of tetracyclines by high-speed liquid chromatography.

The separation of tetracycline and its four commoninpurities has been studied by high-speed liquid chromatography. A preliminary study of the effectiveness of ion-exchange, adsorption, liquid-liquid partition and reversed-phase ion-pair chromatography indicated that only the last method showed promise. By developing special hydrocarbon-bonded stationary phases a rapid and complete resolution of all five tetracyclines has been obtained within 10 min. Plate heights using a derivatised 18-mum partisil are in the range 0.15-0.3 mm. The method can be used to quantify the impurities in tetracycline at around the 1% level.

Separation of tricyclic psychosedative drugs by high-speed ion-pair partition and liquid-solid adsorption chromatography.

The application of high-speed ion-pair partition and liquid-solid adsorption chromatography to the separation of twenty common tricyclic tranquilizers and antidepressant drugs is described. In the ion-pair system, amine-perchlorate ion-pairs were extracted from an aqueous stationary phase supported on 10-mum silica gel by organic eluents containing a chloromethane and a higher aliphatic alcohol, and chromatographic parameters for elution by eight eluent mixtures are presented. Using 5 mm times 120 mm columns good separations, according to chemical class, were achieved. For adsorption chromatography, the components were eluted from 20-mum spherical alumina using eluents containing methylene chloride, n-hexane or n-pentane, and acetic acid. Chromatographic parameters are given for eight eluent compositions. Components differing little in structure are well separated by liquid-solid adsorption chromatography. Compared with ion-pair partition chromatography, adsorption chromatography is much more selective for compounds of the same chemical type. The two methods are therefore complementary. Both methods gave plate heights in the range of 0.1 to 0.3 mm.