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A method for the detection of oxidized, reduced and protein-bound glutathione in eye lenses has been developed. Homogenized lens samples are deproteinated with acetonitrile and perchloric acid. Protein-bound glutathione is reduced by 1,4-dithiothreitol. Separation of the different forms of glutathione and dithiothreitol is performed by ion-pair reversed-phase high-performance liquid chromatography with sodium octylsulphate as the ion-pairing agent. The compounds are detected amperometrically using on-line-generated bromine, which oxidizes thiols and disulphides. In this way two samples can be analysed in triplicate in a single day. The lower detection limits are 80 and 48 nmol per gram wet lens for reduced and oxidized glutathione, respectively. The amounts of free reduced and protein-bound glutathione in calf lenses, determined with this method, are 6.8 +/- 0.4 and 0.96 +/- 0.03 mumol per gram wet lens, respectively. That of oxidized glutathione is less than 0.048 mumol per gram wet lens.

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Dansylation of pentachlorophenol was carried out after solid-phase extraction of acidified human serum and desorption with dichloromethane, in a dichloromethane-water two-phase system using tetrabutyl-ammonium bromide as the phase-transfer catalyst. Derivatisation was complete within 2 min at room temperature. After evaporation of an aliquot of the organic phase, the residue was dissolved in methanol and injected into a reversed-phase chromatographic system, equipped with a post-column photochemical reactor. In the reactor, dansylated pentachlorophenol is converted into highly fluorescent products. The recovery of the analyte from serum was 85 +/- 4%. Calibration graphs for horse and human serum were linear over two decades, with correlation coefficients ranging from 0.996 to 0.999. The detection limit of pentachlorophenol in horse and human serum was 400 pg ml-1. The reproducibility of the total procedure for a human serum sample containing 4 ng ml-1 was 4.5%.

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Dynamic quenching of Eu(III) and Tb(III) luminescence by inorganic anions as a detection method in ion chromatography was investigated. To obtain a high luminescence intensity, lanthanide(III) complexes are formed with ligands which make indirect excitation of the ions possible. Only a few anions (e.g., nitrite, chromate) induce efficient dynamic luminescence quenching. Chromate is an efficient quencher of Tb-acac luminescence. Samples of tap water and surface water, spiked with chromate, were injected into a high-performance liquid chromatographic system with post-column addition of the luminescent complex. In this way, a detection limit of 1.1 . 10(-7) M (13 ppb) of chromate could be obtained.

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The increasing interest in the rapid trace analysis of large series of biomedical samples using column liquid chromatographic techniques requires the use of well balanced combinations of sample pretreatment, separation and detection techniques. In such work, selectivity, sensitivity and reproducibility are the key parameters. The application of automated or semi-automated on-line pre-column technology and/or post-column reaction detection are excellent ways to meet these requirements. A critical review is presented of the theoretical background of on-line post-column reactors with emphasis on their categorization, viz., open-tubular, packed-bed, segmented-stream and hollow-fibre membrane reactors. The evaluation of these reactor systems is performed by discussing selected applications of, for instance, systems based on electrochemical and redox, hydrolytic, photochemical, ion-pairing, true chemical derivatization, peroxyoxalate chemiluminescence and solid-phase reactions. As automation is becoming even more important, a number of labelling procedures, which can be performed in an on-line pre-column mode, are briefly discussed and a comparison is made between the potential of on-line pre- and post-column procedures.

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A method was developed for the determination of 3'-azido-3'-deoxythymidine (AZT) in plasma. The method is based on the trace enrichment of AZT on a pre-column packed with a silver-loaded thiol stationary phase at pH 11.6. On-line desorption to the reversed-phase liquid chromatographic system is performed by injecting a plug of 50 microliters of 1 M perchloric acid on the silver (I)-thiol pre-column. Two different sample pretreatment methods - protein precipitation with perchloric acid and on-line clean-up via a polymeric PRP-1 pre-column - were applied for the determination of AZT in human plasma. The latter method allows the direct injection of plasma samples into the analytical system and can therefore easily be automated. With both methods detection limits in the order of 10(-8) M AZT were obtained after preconcentration of 1.0 ml of plasma, using UV detection at 267 nm.

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Several analytes such as the inorganic anions bromide, iodide, sulphite and nitrite and organic compounds as substituted anilines and sulphur compounds cause quenching of peroxyoxalate chemiluminescence. A detection method for liquid chromatography based on the quenching phenomenon has been developed. It makes use of an immobilized luminophore, i.e. 3-aminofluoranthene covalently bound via an alkyl-spacer on controlled pore glass, packed in the detector cell. The mechanism behind the quenching has been elucidated by investigating the roles of luminophores (both in the liquid and in solid state) and oxalates in peroxylate CL with respect to quenchers. Most probably the quencher destroys the radical ion pair produced after electron transfer in the last stage of the CIEEL reaction scheme, thus preventing the formation of electronically excited luminophore.

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An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17 beta-19-nortestosterone (beta-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), beta-19-NT and its metabolite 17 alpha-19-nortestosterone (alpha-19-NT) can be determined in biological samples with a detection limit of 0.05 microgram/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17 beta-laurate, the maximum concentrations of beta-19-NT (1.3 micrograms/l) and alpha-19-NT (3.1 micrograms/l) were found seven days after intramuscular administration. In a bile sample from this calf only alpha-19-NT (55 microgram/l) was detected. In meat samples from three treated calves, the concentration of beta-19-NT varied from 0.1 to 1.6 micrograms/kg and no alpha-19-NT could be detected. In liver samples from these calves, the concentrations of beta-19-NT and alpha-19-NT were less than 0.05-0.1 and 0.5-0.9 micrograms/kg, respectively. In the corresponding kidney samples, the concentrations of beta-19-NT and alpha-19-NT were 0.4-0.5 and 0.5-1.6 micrograms/kg, respectively. The application of the same immuno precolumn to the determination of 17 beta- and 17 alpha-trenbolone, two structurally related steroids, is also demonstrated.

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The automation of zone-electrophoretic sample treatment for liquid chromatography is described. The procedure is completely controlled from a liquid chromatograph. The carry-over of proteins from human serum under different experimental conditions was studied. The influence of the presence of proteins in the sample is illustrated with the anionic compound salicylic acid and the increase in selectivity for cationic compounds is demonstrated with the determination of ephedrine, norephedrine and amphetamine in urine.

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In order to obtain more information on the (de)sorption processes on a metal-loaded thiol phase, various compounds containing heterocyclic nitrogen atoms have been studied. Adenine and adenosine, which contain no carbonyl groups in a position ortho to the ring nitrogen, were chosen to study the role of the binding donor atoms and, therefore, of the 'minimum' structure required for efficient pH-dependent (de)sorption. The results indicate that a single heterocyclic nitrogen atom, preferably with a high pKa value, is sufficient to provide pH-dependent (de)sorption of chelating phases loaded with soft metal ions, such as Ag(I) or Hg(II), which can profitably be used in an on-line combination with column liquid chromatography. Barbiturates were chosen on the basis of their structural similarity with the earlier studied (halogenated) uracil derivatives. The (de)sorption behaviour of the barbiturates was studied as a function of the pH, the metal ion [Cu(II), Ag(I), Hg(II), Pd(II)] bound to the stationary phase and the type of stationary phase. The combined data show that the pKa value of an analyte and the stability constant of the complex formed with the metal ion sorbed on the stationary phase give a reliable indication of the optimum (de)sorption pH. Application of this principle is demonstrated by the determination of four barbiturates in plasma.

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A liquid chromatographic column-switching system for automated sample pretreatment and determination of the anabolic hormone beta-19-nortestosterone (beta-19-NT) and its metabolite alpha-19-nortestosterone (19-norepitestosterone) in calf urine is described. The system consists of an immunoaffinity pre-column (immuno pre-column) packed with Sepharose-immobilized polyclonal antibodies against beta-19-NT, a second pre-column packed with C18 bonded silica and an analytical C18 column. Urine (25 ml) is directly loaded on the immuno pre-column, where the analytes of interest are trapped by the immobilized antibodies. Next the analytes are desorbed selectively with a solution containing an excess of the cross-reacting steroid hormone norgestrel and transferred, via the second pre-column, to the analytical column. The recovery of beta-19-NT in spiked urine samples was over 95%. The detection limit was 50 ng/l for a 25-ml urine injection. The system showed no loss of analytical performance over a 6-month period, during which about 100 samples were analysed with the same immuno pre-column. The general applicability of this sample pretreatment method is discussed.

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A stereoselective post-column reaction detection system for eight L-amino acids that makes use of a reactor packed with immobilized L-amino acid oxidase is described. The combination of the selectivity of the enzyme and the selectivity of the peroxyoxalate chemiluminescence detection used provides an extremely selective detection system. The detection system gives linear responses over two orders of magnitude and detection limits at the 0.35.10(-6)-3.0.10(-6) M level. The method was used for the determination of selected L-amino acids in urine and beer.

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Dansylation of phenolic steroids was carried out in chloroform-water and hexane-water two-phase systems with a tetrabutylammonium salt as phase transfer catalyst. Derivatization was complete within a few minutes on shaking at room temperature. Direct injection of part of the organic phase into a normal-phase liquid chromatography system was possible. The calibration graph of ethinyl estradiol, dansylated in a chloroform-water two-phase system, was linear over three orders of magnitude with a correlation coefficient of 0.993 (n = 8). The detection limit of dansylated ethinyl estradiol was 100 pg (signal-to-noise ratio = 2). The reproducibility of the derivatization at an analyte concentration of 200 ng/ml in chloroform was 4.1% (relative standard deviation; n = 5). A mechanism is proposed for the phase transfer catalysed dansylation of phenolic compounds.

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A selective method was developed for the determination of disulfiram and two of its metabolites, diethyldithiocarbamate (DTC) and copper (II) diethyldithiocarbamate [Cu(DTC)2], in complex (biological) samples by reversed-phase liquid chromatography (RP-LC) with post-column derivatization. In the first step, DTC is converted into lead (II) diethyldithiocarbamate [Pb (DTC)2] by adding lead (II) acetate. Disulfiram, Pb (DTC)2 and Cu (DTC)2 can be easily pre-concentrated on C18-bonded silica. After separation by isocratic RP-LC, derivation takes place in two solid state post-column reactors packed with metallic copper and copper (II) phosphate. Disulfiram reacts with metallic copper to form Cu (DTC)2. The same product is obtained by the ligand-exchange reaction between Pb (DTC)2 and copper (II) phosphate. Cu (DTC)2 can be detected selectively at 435 nm with good sensitivity (molar absorptivity, epsilon = 13,000). The derivatization reactions proceed rapidly and quantitatively, which was confirmed by comparison of absorption spectra. The applicability of this method is demonstrated for undiluted urine samples which, apart from the addition of lead (II) acetate and pre-concentration of C18-bonded silica, require no clean-up procedure.

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Post-column ion-pair extraction has been applied to the on-line radiometric determination of [(14)C]-urapidil and its main metabolises in reversed-phase liquid chromatography (LC). [(14)C]-remoxipride was used as internal standard. Chromatography was performed isocratically, using acetonitrile-water (pH = 2.2) as eluent on a cyanobonded column. Various ion-pair reagents were tested for their extraction efficiency of the analytes mentioned from the eluent into chloroform. Quantitative extraction was possible using sodium dodecylbenzenesulphonate as ion-pair reagent. The influence of the extraction solvent composition, the percentage organic modifier in the eluent and the flow-rates of eluent and scintillator on the extraction percentage and the (14)C-counting efficiency has been studied. In addition, the enhancement of sensitivity is possible using an on-line radiometric procedure. Finally, the method has been applied to the analysis of rat plasma for [(14)C] -labelled urapidil and its metabolises.

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Positive and negative chemical ionization (PCI and NCI, respectively) have been used for the characterization of ten organophosphorus pesticides in on-line liquid chromatography-mass spectrometry (LC-MS). LC analyses were performed on a 20 cm X 0.7 mm I.D. C8-bonded phase using acetonitrile-water (70:30) or acetonitrile-water-chloroacetonitrile (69:30:1) as eluent. With PCI, molecular weight information was obtained with both eluents. For NCI considerable differences in the spectra were found using the two eluents. Without chloroacetonitrile the spectra were dominated by the functional group fragment and with chloroacetonitrile the base peak was [M - R]- with R being methyl or ethyl, while the spectra further contained the functional group ions. Special emphasis was devoted to the occurrence of chloride attachment at different source temperatures. With several compounds the [M + Cl]- ion was formed and its relative intensity strongly increased when the source temperature decreased. With NCI the sensitivity was about one order of magnitude better than with PCI. This advantage was partly lost when 1% of chloroacetonitrile was used in the eluent; on the other hand, complementary structural information was obtained. As an application, the determination of three organophosphorus pesticides in sediment is reported.

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Amphetamine-type drugs with a wide polarity range have been screened in both human and horse urine using on-line pre-concentration on pre-columns packed with hydrophobic and cation-exchange sorbents in series and gradient microbore high-performance liquid chromatography. The underivatized amphetamines were identified by UV detection at 210 nm. The method has potential for the automated liquid chromatographic screening of amphetamines in urine, e.g., for doping control.

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The design of an automated cartridge exchange module for on-line sample handling in liquid chromatography is described. When combined with a low-cost purge pump, a solvent selection valve and an auto-sampler, a fully automated sample handling system is obtained. Samples are sorbed on a disposable cartridge packed with 40 micron octyl-bonded silica, purged for clean-up and eluted on-line to the analytical column. Unattended operation of the system is demonstrated for various examples, i.e., the determination of anti-epileptic drugs in serum, an anti-cancer drug in plasma, barbiturates in urine, phenylurea herbicides in river water and caffeine in a soft drink.

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