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The group of LiChrospher alkyl-diol silica (ADS) phases that make part of the unique family of restricted-access materials, have been developed as special packings used in the liquid chromatographic integrated sample processing of biofluids. The advantage of these phases lies in the possibility of direct injection of untreated plasma. An on-line elimination of the protein matrix is achieved with a quantitative recovery together with an on-column enrichment. The present method describes a hand-operated on-line switching high-performance liquid chromatographic system for the determination of meloxicam. Spiked plasma samples were introduced on the ADS precolumn using a 0.05 M phosphate buffer, pH 6.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.05 M phosphate buffer-30% (v/v) acetonitrile (ACN)-25 mM t-butylamine (TBA) at a pH of 7.0, thus transferring the analyte to the analytical column LiChrocart 125-4 LiChrospher RP-8. The eluent was monitored by a UV-detector set at 364 nm. The developed column-switching method is fully applicable to plasma injections.

Direct liquid chromatographic separations of the enantiomers of metoprolol and bisoprolol have been developed, using (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid as chiral stationary phase (CSP). The separations were achieved in a normal phase system employing a mobile phase containing n-hexane, 1,2-dichloroethane and methanol. Column efficiency was strongly dependent on the composition of the mobile phase. The eluent contents of methanol and of 1,2-dichloroethane were optimized, and so was flow-rate and column temperature. Under the optimal conditions, linear responses for (R)-metoprolol and (S)-metoprolol are obtained in the range of 0.079-1.38 and 0.015-5.80 mg/ml, with detection limits of 0.008 and 0.002 mg/ml, respectively. As for bisoprolol, the linear ranges of (R)-isomer and (S)-isomer are 0.05-1.31 and 0.02-1.00 mg/ml with detection limits of 0.001 and 0.008 mg/ml, respectively. The relative standard deviation (R.S.D.) of each enantiomer did not exceed 0.90%. The method has been successfully applied to the determination of enantiomers in pharmaceuticals.

The present paper provides the principles of chemiluminescence (CL) and its powerful applications in analytical chemistry, mainly in the area of flow injection analysis, column liquid chromatographic and capillary electrophoretic separating systems, and its potential in immunoassays. CL is light produced by a chemical reaction. The most common advantages of chemiluminescent reactions are the relatively simple instrumentation required, the very low detection limits and wide dynamic ranges, which have contributed to the interest of CL detection in flow injection analysis, high performance liquid chromatography, including miniaturized systems, and, most recently, the exploding area of capillary electrophoresis. The latter powerful microanalytical separation technique offers high numbers of theoretical plates and relatively short analysis times requiring only small sample volumes, the migrating system comprising aqueous buffer solutions. In non-isotopic immunoassays, covering a great variety of applications in human and veterinary medicine, forensic medicine, agriculture and food industry, the radioisotope is replaced by a fluorescence or chemiluminescent label. The use of CL as a detection principle permits quantitative determination of various compounds at low concentrations. Disadvantages of the CL-based technique may include lack of sufficient selectivity and sensitivity to various physicochemical factors.

Capillary zone electrophoresis was applied to the determination of ascorbic and isoascorbic acid, analysing the various parameters of influence such as the separation voltage, the buffer pH and concentration, the type of separation capillary or the loading conditions. Both analytes could be adequately determined within 5 min. The proposed method uses a 20 cm x 25 microns i.d. coated column, 0.1 M phosphate buffer pH 5.0, 8 kV separation voltage and light absorption detection at 265 nm. Linear calibration curves were obtained in the 0-1 mg mi-1 range, with detection limits of 0.5 micrograms ml-1. This method proved to be very rapid, simple and practical for the qualitative and quantitative determination of ascorbic acid in lemon and orange juices, as well as in a commercially available pharmaceutical formulation.

The application of high-performance thin-layer chromatography (HPTLC) with fluorescence scanning densitometry provides a simple, rapid and reliable system for the qualitative and quantitative determination of several thiols of biological and pharmacological interest. The determination of a mixture of thiols (captopril, coenzyme A, cysteamine, cysteine and glutathione), together with their disulphides may readily be performed by pre-chromatographic derivatization with the thiol-specific fluorobenzoxadiazole reagents SBD-F and ABD-F, followed by HPTLC separation on silica gel plates using isopropyl ether-methanol-water-acetic acid (9:8:2:1, v/v/v/v) as the developing solvent, and fluorodensitometric measurement of the fluorescing derivatives. Detection limits of about 30 pg (coenzyme A) to 6 pg (cysteamine) per spot were achieved; the relative standard deviation (RSD) of the complete procedure was 1.16-3.2%.

Several thiols of biological and pharmacological interest, including glutathione, coenzyme A, acetylcysteine and captopril were derivatized with the fluorogenic reagents SBD-F and ABD-F and analysed by high-performance thin-layer chromatography (HPTLC)-fluorodensitometry on silica gel 60 plates, using isopropyl ether-methanol-water-acetic acid (9:8:2:1, v/v/v/v) as the developing solvent. The luminescence was considerably increased when several types of enhancers were applied as dipping reagents: Triton X-100, liquid paraffin and cyclodextrins; thus the detectability of the thiol fluorophores was improved. The influence of enhancer concentration, method of application, sample concentration, drying conditions and measuring time after plate dipping were investigated. The greatest enhancement was achieved using a 40% (v/v) solution of Triton X-100 in toluene as a dipping reagent for the determination of SBD-acetylcysteine; more than a 10-fold increase of the fluorescence signal was obtained, allowing low picogram detection limits.

Luminescence emission from drugs is strongly dependent on their physicochemical environment. Several biomedically and environmentally important compounds and pharmaceuticals exhibit sufficient intrinsic luminescence properties to allow their determination by high-performance liquid chromatography (HPLC) with fluorimetric, chemiluminescence or room temperature phosphorimetric detection. In the case of weakly fluorescing compounds it is possible to use the dependence of the emitted radiation on the molecular environment at the moment of measurement. The composition of the eluent, i.e. solvents, added salts and buffers, pH and ionic strength, oxygen content and temperature, are of the highest importance for the luminescence detection of drugs in solution (e.g. in liquid chromatography) or adsorbed onto solid surfaces (e.g. in thin-layer chromatography). Post-column or post-plate acid-base manipulation and the use of specific reagents may remarkably enhance the observed luminescence of several molecules. The term "enhancement" of luminescence comprises various sample treatments leading to an increase of the emitted radiation. These treatments include the addition of non-fluorescent compounds to, or the creation of organized media (surfactants, cyclodextrins, heavy atoms) in, the sample to be measured. They may also involve changes in molecular environment, pH, the application of excessive drying conditions, the removal of oxygen, the protection of adsorbed compounds against non-radiative decay mechanisms by means of specific spraying or dipping conditions, amongst others. The use of organized media in luminescence spectroscopy is growing. Many of the recent studies have involved micelles for enhancing the fluorescence, room temperature phosphorescence and chemiluminescence of several chemicals. Cyclodextrins are increasingly used for various analytical applications. Liquid paraffin, triethanolamine, dodecane, Triton X-100 and Fomblin Y-Vac are commonly used fluorescence enhancers in chromatographic assays. Examples of these systems in drug analysis are presented.