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A goal in proteomics is the analysis of proteins by LC-MS. The proteins are enzymatically digested and the resulting peptides are chromatographically separated and introduced into a tandem MS. The obtained MS data are used for a search in sequence databases, providing identification scores for the proteins. A method to improve that score is to increase the chromatographic separation and peak capacity. In this study, the chromatographic conditions were optimized for a relatively large gradient time by varying the flow rate and gradient composition. The influence of the monolithic column length (15 and 64 cm) and particle diameter (1.8 μm; 15-cm length) on the sample peak capacity, productivity and identification score was studied. For comparison of gradient systems, a scaling factor was introduced to normalize the properties/performance of columns for material, diameter and length. As model proteins/digests, a simple (myoglobin) and a larger (BSA) protein were used. The smallest peak width, highest identification scores (54 and 89% for BSA and myoglobin, respectively) and productivity (5.0 and 4.0, respectively) were obtained for the 15-cm particulate column. The study also demonstrates that a further increase in the chromatographic performance is beneficial for BSA but hardly increases the identification score for the relatively small myoglobin., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of monolithic liquid chromatography (LC) columns for proteomics, covering the scientific literature from 2004 to the beginning of 2011, is reviewed. Attention is paid to recent developments in column technology and materials, focusing on silica-based and organic (polystyrene and methacrylate) monolithic capillary columns for proteomics. The applicability of these columns is illustrated by examples of the analysis of (complex) protein digests and proteins conveniently summarized in tables. Furthermore, characteristics of column materials are compared and future trends and prospects are presented., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper describes the hyphenation of cIEF and MALDI-TOF MS via a fractionation or spotting device. After focusing in cIEF the compounds are hydrodynamically mobilized and deposited on a MALDI target plate using a sheath liquid interface, which provides the catholyte solution and the electrical ground. From previous experiments, sample conditions that resulted in a high resolution in cIEF and acceptable protein signal intensity in MS were selected [Silvertand et al., Electrophoresis, 2008, 29, 1985-1996]. Besides the mixture of test proteins, the sample solution contains 1% Pharmalyte, 0.3% hydroxyethyl cellulose and 0.1% Tween 20 and is used for both optimization as well as characterization of the cIEF-MALDI-TOF MS system. Hyphenation problems encountered are mainly due to transfer of the liquid from the needle to the MALDI target plate and are solved by choosing the proper sheath catholyte (200 mM NH4OH in 50% methanol with 0.1% Tween20). MS electropherograms were reconstructed by plotting the intensities of the m/z values corresponding to the proteins versus migration time (related to spot number). Reproducibility, peak width and signal intensity for different focusing and spotting (fractionation) times were calculated using these reconstructed MS electropherograms as well as the UV electropherograms. The best results were obtained with focusing time of 75 min (no under- or overfocusing) and a spotting time of 5 s (highest protein signal intensity in MS). The applicability of the system is demonstrated by the analysis of a biopharmaceutical (glucagon) and its deamidation product.

An automated, on-line system for protein quantification and identification, employing Surface Plasmon Resonance (SPR), enzymatic protein digestion, nanoLC and tandem-MS (MS/MS), has been developed. For the experiments recombinant human interferon-gamma (rhIFN-gamma) in buffer or diluted bovine plasma was used as a model protein. Upon injecting 90muL of a 1mugmL(-1) solution of rhIFN-gamma in diluted plasma at a flow rate of 10muLmin(-1), 320fmol of protein was reproducibly bound to the sensor surface. After desorption of the isolated protein from the SPR surface using 10mM glycine pH 1.3, on-line digestion, nanoLC and MS/MS analysis, rhIFN-gamma could be identified on basis of peptide masses and MS/MS fragmentation data. A sequence recovery of 66% was found when a pepsin micro reactor was used. For a trypsin micro reactor the sequence recovery was 50%. In the latter case, the desorbed protein solution was pH-tuned with a TRIS buffer for optimal enzyme activity. With the identified trypsin- and pepsin-produced peptides and because parts of their amino acid sequences overlap, the protein sequence can be largely elucidated showing the potential for the analysis of unknown proteins. The SPR-digestion-nanoLC-MS/MS platform provides unattended analysis of a sample within 60min.

The developments in capillary isoelectric focusing (cIEF) over the period 2003-2007 are reviewed. With the focus on technological aspects, cIEF papers published in the fields of methodology, new techniques, detection, multidimensional systems, miniaturization and applications are summarized. The methodology section covers recent research in ampholytes composition, detergents and other additives, carrier ampholyte free cIEF, coatings and other capillary modifications. In the section on new systems adjustments to the technique (e.g. dynamic IEF), different applications of cIEF (e.g. as injection system) and new devices are reported. Systems focusing on whole column imaging, fluorescence and chemiluminescence detection and coupling to mass spectrometers are discussed in the section on detection. Interfacing cIEF with MS via RPLC systems and hyphenation of cIEF with capillary electrochromatography and other capillary electrophoresis modes are also summarized. Papers focusing on miniaturization are reviewed in the section on microfluidic devices. The section on applications will show analysis of biopharmaceutical compounds and isolated proteins for metabolomic studies. For the analysis of complex biological matrices, generally multidimensional systems are needed, which are mentioned throughout this review.

On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin beta- and alpha-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.

Low repeatability of migration time, peak area, and linearity (pI vs. mobilization time) is a problem often encountered in capillary IEF (cIEF) and is mainly caused by protein precipitation and protein-wall interactions. In order to study the influence of these phenomena, the effect of different classes of additives on repeatability of migration time, peak area and linearity of a mixture of seven model proteins has been investigated. Moreover, the influence of these additives on protein signal suppression in MALDI-TOF MS has been studied. The optimal ampholyte blend (stabilizes pH gradient) to be used depends on the selected UV detection wavelength. All tested ampholyte blends show a significant and comparable signal suppression in MS. The best detergent (to prevent precipitation and wall interaction) should be determined for each sample individually, but generally polyethylene oxide and zwitterionic detergents show good repeatability for migration time (RSD <4.5%) and peak area (majority <10%). The RSD of R(2) is <1.3% for the hydrophilic protein mixture. However, these components cause severe signal suppression in MS. Therefore glucoside detergents should preferably be used for MS coupling. Viscosity-increasing agents (for hydrodynamic wall coating and to minimize diffusion) in particular cellulose derivatives, give good repeatability for migration times (RSD <4.5% at lower concentrations), peak area (except for high concentration methylcellulose and hydroxyethylcellulose all within 7.5%), and correlation (pI vs. migration time), but severe signal suppression is observed in MALDI-TOF MS. Overall, cIEF repeatability and linearity can significantly be improved by adding the appropriate components. However, when the system is coupled to a MALDI-TOF MS, compromises have to be made between high repeatability and linearity on one hand and MS signal intensity on the other.

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm(-2) trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 degrees C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol microL(-1) horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm x 50 microm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.

This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP-CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE-CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.

A method was developed for the fast separation of a myoglobin digest using a monolithic RP 18 silica capillary column of 100 microm I.D. The results were compared with those obtained with a particulate RP 18 silica capillary column of 100 microm I.D. at a flow-rate between 0.6 and 1.2 microl/min. The digest was analyzed at the monolithic column at a flow-rate up to 2.8 microl/min. This high flow-rate could not be applied to the particulate column due to the high back-pressure. When the starting composition of the gradient was changed from 0 to 20% and a gradient steepness of 16%/min was used, the analysis time was less than 4 min. A positive Mascot identification score of 115 was achieved for the MS-MS data. When a lower gradient steepness was employed, the chromatographic resolution and the peak capacity did not increase for most compounds. The intraday repeatability for the retention time of the monolithic column was better than 1.5% at 2.8 microl/min and even less than 0.5% using a flow-rate of 0.6 or 1.0 microl/min. For the particulate column, it was between 0.5 and 1.4% for a flow-rate of 0.6 microl/min, probably due to the high column back-pressure. The interday reproducibility for the retention time of the monolithic column was less than 0.9% using a flow-rate of 1.0 microl/min.

A study was initiated to investigate the use of surface plasmon resonance (SPR) for the detection in plasma of a high pI model protein, recombinant human interferon-gamma (IFN-gamma). Initially a number of self-assembled monolayers (SAMs) and hydrogel-derivatised SAM-coatings were characterised for the adsorptive and desorptive properties of plasma components. Next a monoclonal anti-IFN-gamma antibody, MD-2, was covalently attached to dextran-modified mercaptoundecanoic acid surfaces that performed best. On coatings consisting of carboxyl-modified dextran (CMD) a difference in interaction behaviour was observed when IFN-gamma was injected in either buffer or diluted plasma. During the injection of IFN-gamma in buffer, an acceleration of the interaction process was observed and the signal continued to increase after the injection plug had passed. Upon injection of diluted plasma spiked with IFN-gamma, the response increased without acceleration of the binding process. After the injection was finished, some of the bound material desorbed as expected, resulting in a signal decrease. On non-charged dextrans, the interaction between the antibody-modified surface and IFN-gamma in either plasma or buffer was similar. During sample injection the response increased with a binding rate depending on the concentration of IFN-gamma present in solution. When the injection was finished, some of the bound material was washed away from the surface and only a minor contribution of non-specific adsorbed plasma components was noticeable. From the coatings tested, the non-modified dextran-coated SPR sensor disks prove to be best suited for the detection of IFN-gamma in complex matrices like plasma. The interaction of IFN-gamma in both diluted plasma and buffer is comparable and concentrations of IFN-gamma of 250 ng ml-1 and higher can be detected in both buffer and 100x-diluted plasma. The non-specific adsorption of plasma components is low, whereas the specific IFN-gamma response is relatively high.

The research on our flow-injection, label-free, non-faradaic impedimetric immunosensor for interferon-gamma (IFN-gamma) has been extended. The sensor is prepared by immobilization of anti-IFN-gamma antibodies on a self-assembled monolayer (SAM) of acetylcysteine, deposited on polycrystalline gold. A multi-frequency impedance method is described, which allows time-resolved measurement of Nyquist plots. To these plots, an equivalent circuit was fitted, which is discussed in terms of a two-layer structure (inner and outer layer) of the interfacial region. Because binding of IFN-gamma mainly causes a decrease of Q (a constant-phase element), this element is considered as the outer layer. Several aspects of the impedimetric sensor response are studied, including the dependence on detection frequency, target concentration and applied dc potential. For quantitative detection of IFN-gamma, an optimum of the signal-to-noise (S/N) ratio of the out-of-phase impedance component (Z'') was found at about 100 Hz. At a dc-potential of +0.2 V versus a saturated calomel reference electrode, the sensor response is higher than at 0.0 V. Logarithmic dose-response curves of IFN-gamma in the concentration range of 10(-18) to 10(-9) M were obtained using two procedures: by successive injections over a single electrode, and by using freshly prepared electrodes for each measurement. Using the latter method, the repeatability is impaired. The need for in situ complementary techniques for a correct interpretation of the studied parameters is discussed.

An electrochemical immunosensor for direct detection of the 15.5-kDa protein interferon-gamma (IFN-gamma) at attomolar level has been developed. Self-assembled monolayers (SAMs) of cysteine or acetylcysteine are formed on electropolished polycrystalline Au electrodes. IFN-gamma adsorbs physically to each of these SAMs. With injections of 100 mM KCl, IFN-gamma can be removed in the flow without damaging the acetylcysteine SAM. However, the cysteine SAM is affected by these KCl injections. In an on-line procedure in the flow, a specific antibody (MD-2) against IFN-gamma is covalently attached following carbodiimide/succinimide activation of the SAM. The activation of the carboxylic groups, attachment of MD-2, and deactivation of the remaining succinimide groups with ethanolamine are monitored impedimetrically at a frequency of 113 Hz, a potential of +0.2 V versus SCE, and an ac modulation amplitude of 10 mV. Plots of the real (Z') and imaginary (Z") component of the impedance versus time provide the information to control these processes. In the thermostated setup (23.0 degrees C), samples of unlabeled IFN-gamma (in phosphate buffer pH 7.4) are injected and the binding with immobilized MD-2 is monitored with ac impedance or potential-step methods. While the chronoamperometric results are rather poor, the ac impedance approach provides unsurpassed detection limits, as low as 0.02 fg mL-1 (approximately 1 aM) IFN-gamma. From a calibration curve (i.e. Z" versus the amount injected), recorded by multiple 50-microL injections of 2 pg mL(-1) of IFN-gamma, a dynamic range of 0-12 pg mL(-1) could be derived. However, when nonspecific adsorption is taken into account, which has been found to be largely reduced through injections of 100 mM KCl, a much smaller dynamic range of 0-0.14 fg mL(-1) remains. The immunosensor can be regenerated by using a sequence of potential pulses in the flow by which the SAM with attached MD-2 and bound IFN-gamma is completely removed. When the developed procedures described above are repeated, the response of the immunosensor is reproducible within 10%.

It has been emphasized in several studies that the state of the surface, including the surface roughness, is very important for the reproducible formation of high-quality self-assembled monolayers on gold. The pulsed-potential pretreatment procedure described in this paper can, in a reproducible way, reduce the surface roughness of mechanically polished polycrystalline gold electrodes by a factor 2. The developed procedure, in which the gold is alternately oxidized and reduced, has been optimized for use in a flow system (100 mM phosphate buffer pH 7.4). The influence of the pretreatment procedure on the surface roughness of the electrodes has been studied by in-situ oxygen adsorption measurements using cyclic voltammetry. The most effective pulse regime in producing a gold surface with a reproducible and relatively low surface roughness is a triple-potential pulse waveform, with potentials of +1.6, 0.0, and -0.8 Vvs SCE and pulse widths of 100 ms for each potential. Prolonged pulsing for 2000-5000 s with the gold working electrode in a flow-through cell showed an electropolishing effect, i.e., a decrease of the roughness in time. Flow conditions are very important: the roughness decreased faster at higher flow rates, while an increase was observed without flow. A process of reconstruction and dissolution of gold during application of the potential pulses under flow conditions is assumed to account for the observed phenomena. A self-assembled monolayer of thioctic acid with reproducible characteristics, determined with impedance measurements, could be formed on a pretreated gold surface.

The Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant methionyl human granulocyte colony-stimulating factor were reduced to sulfhydryls with dithiothreitol or mercury. Both reduction reactions are dependent on the pH. The reduction reaction with dithiothreitol increased in rate with increasing pH; between pH 7-9 and above pH 10.5 this increase was less than in other regions. These observations are explained by repulsive forces between dithiothreitol and regions in granulocyte colony-stimulating factor which intensify in these pH-regions. The hydroxyl catalysis causes the overall increase in k(obs) in the pH-region studied. The reduction of the disulfides with mercury is, as could be expected from the Nernst equation for disulfide reduction, also pH dependent: the half-wave potential decreases with increasing pH as predicted by theory.

A recently developed liposome sandwich immunoassay for interferon-gamma (IFN-gamma), to be applied in microtiter plates, is tailored for surface plasmon resonance (SPR) spectrometry. The assay is performed on a thin (approximately 20 nm) polystyrene layer that covers a gold surface. This way, analytical data obtained from microtiter plate technology can directly be extrapolated toward SPR. For assaying the antigen IFN-gamma, a 16-kDa cytokine, a capture monoclonal antibody is physically adsorbed onto the polystyrene surface. After addition of the sample containing IFN-gamma, a biotinylated detecting antibody is added. Avidin is used as a bridging molecule between the biotinylated antibody and the biotinylated liposomes. All solutions are prepared with PBS buffer (10 mM, pH 7.4). This avoids additional changes in index of refraction caused by the use of various buffer solutions in immunoassays on microtiter plates for coating, binding, and washing procedures. It is shown that, when liposomes are used, a substantial enhancement of the detection limit is achieved. The "liposome" strategy improves the sensitivity for the IFN-gamma assay approximately 4 x 10(4) times and the detection limit to low picomolar. The method is generally applicable to other sandwich immunoassays.

Various aspects of the application of liposomes as a label in immunoassays are reviewed. Methods for the preparation of liposomes, from the basic film method to the more advanced dehydration-rehydration method, are discussed. Furthermore, the markers used in liposome labels, as well as the methods to conjugate liposomes to antigens or antibodies, are summarized. Liposome immunoassays are applied as homogeneous or heterogeneous assays. Homogeneous assays often rely on the lytic activity of complement on antibody-associated liposomes. Another group of homogeneous assays utilizes the inhibitory action of antibodies on the activity of conjugates of mellitin (a bee venom protein) with a hapten. Free mellitin conjugates are able to lyse liposomes effectively. Heterogeneous liposome immunoassays, performed either competitively or non-competitively, resemble more closely standard enzyme linked immunosorbent assays, with the enzyme being replaced by a liposome label. Washing steps are used to separate antigen-specifically bound liposomes from unbound liposomes. All bound liposomes are lysed with a detergent, giving an instantaneous amplification. Flow-injection liposome immunoassays and liposome immunosensors are also described as examples of other possible immunoassay formats.

A chemiluminescent substrate reagent for use in a sandwich immunoassay for the model antigen mouse interleukin-5 (IL-5) was developed using xanthine oxidase and luminol. Various parameters involved in this chemiluminescent reaction have been studied, including the substrate hypoxanthine, luminol and the Fe(II)-EDTA complex. Addition of the Fe(II)-EDTA complex enhances the chemiluminescence signal considerably. The xanthine oxidase-catalyzed chemiluminescent immunoassay was compared to horseradish peroxidase-linked immunoassays with luminol as chemiluminescent, and tetramethyl benzidine as colorimetric substrate. The detection limit of the xanthine oxidase-luminol assay was found to be about 0.6 pg/ml IL-5, whereas the peroxidase-catalyzed immunoassays have detection limits of about 1.3 (HRP-TMB) and 2.9 pg/ml (HRP-luminol) IL-5.

Flavonoids, a group of naturally occurring antioxidants and iron chelators, might be used as cardioprotective agents in doxorubicin-induced cardiotoxicity, which is believed to be caused by the formation of oxygen free radicals. To investigate the underlying molecular mechanism, we tested a large group of flavonoids from all major structural subclasses on their ability to inhibit doxorubicin (enzymatically)-induced and Fe2+/ascorbate (nonenzymatically)-induced microsomal lipid peroxidation (LPO) and to chelate Fe2+. In addition, we measured half peak oxidation potentials (Ep/2). LPO inhibition data gave a good qualitative correlation with the oxidation potentials. Most flavonoids tested chelated Fe2+, but there were large differences in the chelating capacity. For good scavenging activity, a catechol moiety on ring B is required. The 3-OH moiety can function as a chelation site and can also be oxidized. The 3-OH group in combination with a C2 C3 double bond, increases the scavenging activity. Fe2+ chelation only plays a role in the LPO inhibition by less active scavengers. Chelation can then raise the activity to the level of the most active scavengers, possibly by site-specific scavenging. It can be concluded that Ep/2 values and iron chelating activity can almost completely describe the LPO inhibiting behaviour of the flavonoids.

Seven 3,5-disubstituted analogues of paracetamol were synthesised in order to compare their physicochemical, pharmacological and toxicological properties with those of paracetamol (4'-hydroxyacetanilide, acetaminophen). Oxidation of the phenolic structure is likely involved in the analgesic action of paracetamol as well as in its toxification by cytochrome P450. The effect of disubstitution adjacent to the phenolic hydroxyl group was studied in order to establish possible structure-activity relationships. 3,5-Substituents with electron-donating capacities (R = -CH3, -OCH3, -SCH3) decreased the half-wave oxidation potential substantially by 0.07 V to 0.16 V, whereas electron-withdrawing substituents (R = -F, -Cl, -Br, or -I) increased the oxidation potential by 0.04 V to 0.06 V when compared to paracetamol. Electron-donating substituents (R = -CH3, -OCH3, -SCH3) increased the mouse brain cyclooxygenase inhibiting capacity of paracetamol. Electron-withdrawing halogen substituents (R = -F, -Cl, -Br or -I) decreased this inhibiting capacity. In agreement with this, the in vivo analgesic activity of the 3,5-dihalogenated analogues was lower when compared to paracetamol. Electron-donating substituents (R = -CH3, -OCH3, -SCH3) decreased the cytotoxicity of paracetamol, when measured as leakage of lactate dehydrogenase from freshly isolated rat hepatocytes, almost completely. Most 3,5-dihalogen substituents (R = -F, -Cl or -Br) diminished it slightly. The fourth electron-withdrawing substituent (R = -I) strongly lowered the cytotoxicity of paracetamol in this test system. In conclusion, a higher cycylooxygenase inhibitory potency of 3,5-disubstituted analogues of paracetamol seemed to correlate with a lower cytotoxicity. 3,5-Disubstituted analogues with electron-donating substituents might therefore be safer analgesics than paracetamol itself. The opposite probably applies to analogues of paracetamol with electron-withdrawing substituents at the 3- and 5- positions of the aromatic nucleus.

The principles of chemiluminescence and the application of chemiluminescent labels and substrates in immunoassays are reviewed. Various immunoassay formats and all known classes of chemiluminescent molecules, including 1,2-dioxetanes, luminol and derivatives, acridinium esters, oxalate esters and firefly luciferins are described as well as the many sensitizers and fluorescent enhancers. Recent promising developments are discussed.

Injection of a low dose of mercuric chloride into Brown Norway (BN) rats caused a marked decrease in the concanavalin A (ConA)-induced generation of interferon-gamma-producing cells (IFN-gamma pc) in spleen cell cultures prepared 1 h after mercury administration. A second injection 48 h later caused a further diminution of IFN-gamma pc down to 30% of the number generated in splenocyte cultures of phosphate-buffered saline (PBS)-injected controls. Injection of Lewis rats with either one or two doses of HgCl2 revealed no inhibitory effect on splenic IFN-gamma production. The presence of the reduced form of glutathione (GSH) in the culture medium was found to be essential in these experiments. In the absence of GSH there was an overall 20-fold reduction of the number of IFN-gamma pc in splenocyte cultures of normal or PBS-injected rats, which was further reduced to a 60- to 70-fold-lower level in cultures of rats exposed to HgCl2. This mercury-mediated extra reduction could be fully reversed with an excess (2 mM) of GSH in Lewis but not in BN splenocyte cultures. Since the bivalent Hg2+ ion is known to bind to and inactivate sulfhydryl groups of proteins and low molecular weight thiols, most notably GSH, we investigated a possible role for thiols in IFN-gamma production. It was found that the generation of IFN-gamma pc in normal BN and Lewis splenocyte cultures was strongly dependent on GSH or its precursor cysteine in the culture medium. Other thiol compounds were also effective but disulfides were completely inactive. Depletion of intracellular GSH in ConA-stimulated splenocytes by buthionine sulfoximide (BSO), an inhibitor of de novo GSH biosynthesis, strongly inhibited the generation of IFN-gamma pc. The inhibitory effect of BSO was not abolished by the addition of interleukin-2 (IL-2), but was mimicked with antibodies directed to the IL-2 receptor. The data stress the importance of GSH in the enhancement of IL-2-mediated IFN-gamma production and are most consistent with a model in which mercury interferes with T cell IFN-gamma production by affecting the intracellular availability of GSH. The strain-specific susceptibility to mercury-mediated inhibition of IFN-gamma production is discussed.

A study of the on-line phase-transfer catalysed dansylation of phenolic compounds is presented. The extraction-dansylation is performed in the extraction coil of a home-made flow-injection extraction unit. After phase separation, the organic phase is fed to a normal-phase liquid chromatographic system with fluorescence detection. Ethynyloestradiol, oestradiol and paracetamol are used as test compounds. The influence of temperature on the reaction is examined. Calibration graphs showed good linearity (r greater than 0.996) and limits of detection are satisfactory (8 x 10(-7) M for ethynyloestradiol, 2 x 10(-6) M for oestradiol and 5 x 10(-7) M for paracetamol). The method is not applicable for the assay of oxychinoline, phenylephrine and morphine.

An automated assay for the determination of penicillin in formulations suitable for use in pharmaceutical quality control is presented. The method is based on the classical iodometric penicillin assay which is incorporated in a flow injection analysis (FIA) system. The required hydrolysis is performed on-line by using an immobilised penicillinase reactor. Packed-bed and single-bead-string enzyme reactors are compared. It turns out that a packed-bed penicillinase reactor (10 cm x 1.5 mm i.d.) provides complete hydrolysis within short residence time, while only little back-pressure is generated. This enzyme reactor is stable for at least 9 months. Enzymatic hydrolysis of the beta-lactam ring results in the formation of the corresponding penicilloic acid, which consumes iodine. The iodine consumption is determined colorimetrically by measuring the decrease of the absorbance of the blue coloured iodine/starch complex. The optimum reactor length and flow rate for the colourimetrical detection reaction are determined. The optimised method is applied to the assay of penicillin in formulations and the results are compared with the "true" results obtained with a reference method: a mercurimetric titration. The reliability of the flow injection method is evaluated quantitatively by determining the maximum total error (MTE). The reliability is shown to be highest when measuring at a 0.3-mM level. Eight formulations including capsules, tablets and injectables containing penicillin G, amoxicillin or flucloxacillin are assayed. The MTE does not exceed the 6% level and the most probable MTE is between 1.5 and 3.5%.

Benzalkonium chloride was assayed by on-line extraction of the benzalkonium ion with picrate to chloroform. The absorbance of picrate was measured. The extractions were performed with a home-made flow injection extraction unit. Calibration curves (1.5-180 x 10(-4)% w/v) were straight lines (r = 0.9993) and the relative standard deviation of a series of injections was less than or equal to 2%. Pharmaceutical benzalkonium preparations, containing xylometazoline, timolol, phenylephrine or carbachol could also be assayed. The method was compared with a modified HPLC assay.

A review of physical, chemical, analytical and pharmacological properties of nitroprusside is presented. In view of the pharmaceutical applications of nitroprusside special attention is given to the discussion of the (photo)degradation, the stability of the pharmaceutical formulations, the application as a reagent in pharmaceutical analysis and the redox behaviour.

The performance of a number of normal phase and reversed-phase systems, with ultraviolet detection at 360 nm, has been investigated with respect to their applicability to pharmacokinetic studies of mitomycin C (MMC). The reversed-phase system developed was also combined with a polarographic detector in order to compare the sensitivity and selectivity of ultraviolet and electrochemical detection. A simple isolation procedure, based on the adsorption of MMC on a non-ionogenic resin, has been developed. The developed assay is applied to a pharmacokinetic study from which some examples are given.

Penicillins and cephalosporins belong to the most prescribed antibiotics. Despite the relatively extended knowledge of these drugs, the qualitative and quantitative analysis of the compounds still gives rise to many problems. These difficulties are due to the chemical instability of the common beta-lactam nucleus, the minor differences in chemical structures between the analogues, and the complex and relatively fast degradation of the compounds in aqueous solutions. In this review a compilation of the physicochemical properties, the degradation routes and methods for analysis of these substances in biological and other matrices is presented.

A systematic theoretical treatment of the two-phase titration based on ion-pair and metal-complex formation is presented. Equations for the titration curve, accuracy, equivalence point and choice of the indicator are derived. Much attention is paid to the parameters determining the 'titratability' viz. extraction constant, distribution constant, phase volume ratio and the concentration of the analyte. Special attention is given to the role of intermediate ion-pair and metal-complex formation in the aqueous phase. The ion-pair formation in aqueous solution is examined closer by means of the relationship between the association constant in water, the degree of dissociation of the ion pair (alpha) and the concentration of the counter ion. The merits of this theoretical treatment are illustrated with literature examples.

The extraction constant of three organic compounds containing nitrogen as a picrate ion pair in a water-chloroform system were determined by following polarographically the picrate concentration in the aqueous phase as a function of the added amount of reagent. The extraction constants were also determined spectrophotometrically (via batch extraction). The obtained results are in good agreement. The proposed method is applicable for extraction constants with values between 10(3) and 10(9).

A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow-injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h(-1) and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow-injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, intercept = 0.6, n = 216, r = 0.977). The main advantages of the proposed method are high sample throughout, high sensitivity, and good reproducibility.

The metal complexation behaviour of several beta-lactam antibiotics and derivatives is explained, based on the results of potentiometric titrations. The (organo)metal ions used were (organic derivatives of) transition elements and elements with a filled d-subshell. The emphatic class b (organo)metal ions Ag(I), Hg(II) and C6H5Hg(I) form the most stable complexes with the studied ligands: Hg(II) is the most suited ion. The alkaline degradation products and hydroxamic acid derivatives of penicillins and cephalosporins are very similar to penicillamine in their complexation behaviour. This emphasizes the dominant role of the thiol group as site of complexation. A scheme for stepwise complex formation with Hg(II) and Ag(I) is presented. The availability of the thiol group is used to explain small differences in complexation behaviour between penicillin derivatives on the one hand, and cephalosporin derivatives and penicillamine on the other.

An automated high-performance liquid chromatographic method for the plasma assay of two neutral drugs, etoposide and teniposide, involving direct plasma injection is presented. The problematic nature of protein precipitation has been circumvented by adding the anionic surfactant sodium dodecyl sulphate to the plasma at a final concentration of 38 mM. Plasma samples are loaded on to a clean-up column with an aqueous mobile phase with which the analyte(s) is (are) retained, whereas the solubilized plasma proteins are flushed to waste. Next, the retained compounds are eluted from the clean-up column on to the analytical column by using the chromatographic mobile phase with a higher elution capacity. The column-switching technique is used to achieve an automated assay. At least 10 ml of plasma, representing 100 repeated injections of 100 microliters or five repeated injections of 2 ml, can pass through the clean-up column without increasing the back-pressure. The recovery increased considerably from 10-30% to 90-95% on adding surfactant to the plasma samples prior to the analysis. The relative standard deviation of the proposed clean-up procedure is 3.5% (n = 6) for both drugs measured at the 2 micrograms/ml level without using an internal standard. The limit of determination with 100-microliters injections is 0.10-0.15 microgram/ml for ultraviolet detection and is seven times lower with electrochemical detection. Teniposide was determined in patients' plasma and the results agreed well with those obtained by the conventional procedure involving manual liquid-liquid extraction prior to chromatographic analysis.