Online magnetic bead dynamic protein-affinity selection coupled to LC-MS for the screening of pharmacologically active compounds.

The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.

Metal-complex formation in continuous-flow ligand-exchange reactors studied by electrospray mass spectrometry.

Electrospray ionization mass spectrometry was used to investigate complex formation of different metal complexes in a continuous-flow ligand-exchange reactor. A computer program was developed based on normal equilibrium calculations to predict the formation of various metal-ligand complexes. Corresponding to these calculations, infusion electrospray mass spectrometric experiments were performed to investigate the actual complex formation in solution. The data clearly show good correlation between the theoretically calculated formation of metal-ligand complexes and the experimental mass spectrometric data. Moreover, the approach demonstrates that the influence of the pH can be investigated using a similar approach. Indirectly, these infusion experiments provide information on relative binding constants of different ligands towards a metal-ion. To demonstrate this, a continuous-flow ligand-exchange detection system with mass spectrometric detection was developed. Injection of ligands, with different affinity for the metal-ion, into the reactor shows good correlation between binding constants and the response in the ligand-exchange detection system. Additional information on the introduced ligand, and the complexes formed after introduction of the ligand, can be obtained from interpretation of the mass spectra.

Selective detection and identification of phosphorylated proteins by simultaneous ligand-exchange fluorescence detection and mass spectrometry.

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.

Screening for metal ligands by liquid chromatography--ligand-exchange--electrospray mass spectrometry.

Electrospray ionization mass spectrometry is applied for the selective detection of metal ligands after a post-column continuous-flow ligand-exchange reaction. The detection is based on the specific release of a reporter ligand from a metal-reporter ligand complex by a high affinity ligand. Constant infusion and direct-injection experiments are performed to optimize the method. The on-line coupling of a liquid chromatographic separation prior to the continuous flow ligand-exchange reaction enables the screening for high affinity ligands in complex samples. The feasibility of the method is demonstrated by using several ligands with a different affinity for either Cu(II) or Zn(II) ions. The selectivity of the ligand-exchange detection method can be tuned by the choice of the reporter ligand. This is demonstrated by using either 2,2'-bipyridyl or 5-methyl-1,10-phenanthroline as reporter ligands.

Coupling of biological sample handling and capillary electrophoresis.

The analysis of biological samples (e.g., blood, urine, saliva, tissue homogenates) by capillary electrophoresis (CE) requires efficient sample preparation (i.e., concentration and clean-up) procedures to remove interfering solutes (endogenous/exogenous and/or low-/high-molecular-mass), (in)organic salts and particulate matter. The sample preparation modules can be coupled with CE either off-line (manual), at-line (robotic interface), on-line (coupling via a transfer line) or in-line (complete integration between sample preparation and separation system). Sample preparation systems reported in the literature are based on chromatographic, electrophoretic or membrane-based procedures. The combination of automated sample preparation and CE is especially useful if complex samples have to be analyzed and helps to improve both selectivity and sensitivity. In this review, the different modes of solid-phase (micro-) extraction will be discussed and an overview of the potential of chromatographic, electrophoretic (e.g., isotachophoresis, sample stacking) and membrane-based procedures will be given.

Evaluation of phytic acid as a buffer additive for the separation of proteins in capillary electrophoresis.

The use of phytic acid to improve protein analysis by capillary electrophoresis (CE) is becoming more and more popular. Due to its size and number of negative charges (up to 12) it provides a high ionic strength combined with a low conductance resulting in an efficient decrease of wall adsorption for proteins. Because of its twelve acidic groups, phytic acid can be used as a buffer over a wide pH range (pH 2-11). The limited wall adsorption of proteins using phytic acid-containing buffers is observed for buffers with a pH of 5.5 and higher. With a monoprotic buffer, most of the investigated proteins show wall adsorption at the pH values studied. In case of a phytic acid buffer, wall adsorption is reduced by a factor of 2-4. The use of phytic acid both as a modifier and as a pH buffer results in more pronounced differences between the various protein mobilities compared with the use of monoprotic buffers. As a result this feature can be used to improve resolution in protein separations.

Solid-phase extraction of polar pesticides from environmental water samples on graphitized carbon and Empore-activated carbon disks and on-line coupling to octadecyl-bonded silica analytical columns.

The suitability of Empore-activated carbon disks (EACD), Envi-Carb graphitized carbon black (GCB) and CPP-50 graphitized carbon for the trace enrichment of polar pesticides from water samples was studied by means of off-line and on-line solid-phase extraction (SPE). In the off-line procedure, 0.5-2 l samples spiked with a test mixture of oxamyl, methomyl and aldicarb sulfoxide were enriched on EnviCarb SPE cartridges or 47 mm diameter EACD and eluted with dichloromethane-methanol. After evaporation, a sample was injected onto a C18-bonded silica column and analysed by liquid chromatography with ultraviolet (LC-UV) detection. EACD performed better than EnviCarb cartridges in terms of breakthrough volumes (> 2 l for all test analytes), reproducibility (R.S.D. of recoveries, 4-8%, n = 3) and sampling speed (100 ml/min); detection limits in drinking water were 0.05-0.16 microgram/l. In the on-line experiments, 4.6 mm diameter pieces cut from original EACD and stacked onto each other in a 9 mm long precolumn, and EnviCarb and CPP-50 packed in 10 x 2.0 mm I.D. precolumn, were tested, and 50-200 ml spiked water samples were preconcentrated. Because of the peak broadening caused by the strong sorption of the analytes on carbon, the carbon-packed precolumns were eluted by a separate stream of 0.1 ml/min acetonitrile which was mixed with the gradient LC eluent in front of the C18 analytical column. The final on-line procedure was also applied for the less polar propoxur, carbaryl and methiocarb. EnviCarb could not be used due to its poor pressure resistance. CPP-50 provided less peak broadening than EACD: peak widths were 0.1-0.3 min and R.S.D. of peak heights 4-14% (n = 3). In terms of analyte trapping efficiency on-line SPE-LC-UV with a CPP-50 precolumn also showed better performance than when Bondesil C18/OH or polymeric PLRP-S was used, but chromatographic resolution was similar. With the CPP-50-based system, detection limits of the test compounds were 0.05-1 microgram/l in surface water.

Derivatization in capillary electrophoresis.

In recent years capillary electrophoresis (CE) has been developed into a versatile separation technique, next to gas and liquid chromatography (LC), well suited for the determination of a wide variety of e.g., pharmaceutical, biomedical and environmental samples. The main advantages of CE over chromatographic separation techniques are its simplicity and efficiency. It is well recognized, however, that the sensitivity and selectivity of the detection are relatively weak points of CE. One way to overcome these limitations is the conversion (derivatization) of the analytes into product(s) with more favourable detection characteristics. Although, in principle, almost any detection mode can be combined with a derivatization procedure, in practice, fluorescence monitoring is favoured in most cases. This paper aims to give a short overview on the various reagents that can be used for pre-, post- and on-column derivatization in CE. First, a short introduction is given on CE as an analytical technique, followed by a discussion of the pros and cons of the various modes of derivatization, a comparison of derivatizations in CE with derivatizations in LC, the principles of fluorescence and prerequisites for a good fluorophore and the potential of using diode lasers in combination with a labelling procedure. With respect to the derivatization reagents the emphasis is on the labelling of amino, aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups.

Aspects of the chemical stability of mitomycin and porfiromycin in acidic solution.

Aspects of the degradations of mitomycin and porfiromycin were studied. The initial degradation processes of the compounds in an acidic medium were investigated. Influences of pH, buffers, and other additives such as halogenides and dioctyl sodium sulfosuccinate [sodium 1,4-bis(2-ethylhexyl)sulfosuccinate] were studied. The hydrogen ion catalyzes the degradation of both the uncharged and the protonated species. Anions also promote the degradation of the compounds in an acidic medium. Rate constants for all of the catalytic reactions could be determined. From the pH profiles, after correction for buffer influences, accurate pKa values for the aziridine nitrogens could be obtained. The protective influence of the dioctyl sulfosuccinate ion could be explained. From the data obtained a plausible mechanism for the initial acidic degradation reactions was developed.

Derivatization reactions in the gas-liquid chromatographic analysis of drugs in biological fluids.

Alkylation, acylation, silylation and other derivatization reactions applied to the gas chromatographic analysis of drugs in biological matrices are reviewed. Reaction conditions are discussed in relation to reaction mechanisms. Detector-oriented labelling of drugs, and derivatization with chiral reagents for the separation of enantiomers are surveyed. Data on the sample clean-up, derivatization and GLC analysis of more than 300 drugs and related compounds are listed.

On-line SPE-RP-LC for the determination of insulin derivatives in biological matrices.

An automated and on-line solid-phase extraction (SPE)-liquid chromatography (LC) procedure is described for the determination of insulin in biological matrices. The total procedure consists of two SPEs in series, followed by RP-LC separation. During the first SPE a strong anion-exchange (SAX) cartridge (ISOLUTE, 40-90 microm, 10 x 4 mm i.d.) is used, followed by a RP-cartridge (Luna C(8), 4 x 2.0 mm i.d.). The second SPE cartridge contains the same material as the LC column and is used to transfer the sample from the SAX cartridge to the LC column. The developed system can detect 100 nmol/l insulin in aqueous samples and 200 nmol/l insulin in spiked plasma samples using UV. When electrospray ionization (ESI)-mass spectrometry (MS), was coupled with the developed system, the LODs were lowered by a factor two to 50 and 100 nmol/l for aqueous and spiked plasma samples, respectively.

Chiral ligand-exchange chromatography as the screening method for proposed modifications in exametazime synthesis to enhance diastereoselectivity.

99M Tc (V)-d,l-HM-PAO complex is well-known radiopharmaceutical for regional cerebral blood flow imaging. The proposed modifications in exametazime, hexamethylpropyleneamine oxime (HM-PAO) (4,8-diaza-3,6,6,9-tetramethylundecane-2,10-dione bisoxime) synthesis, for reduction of intermediary reactant diiminebisoxime (DI) (4,8-diaza-3,6,6,9-tetramethylundecane-3,8-diene-2,10-dione bisoxime) concerned two reductants (NaBH(4) and KBH(4)), two solvents (ethanol and 2-propanol), and three mole ratios of reactant/reductants (1:1, 1:1.5, and 1:2). The simultaneous analysis of diastereo-enantiomeric HM-PAO content, as well as the content of starting DI, in different reduction mixtures were performed using chiral ligand-exchange chromatography (CLEC). The separation of the samples of investigated reduction mixtures, obtained in the second step of HM-PAO synthesis, has been accomplished by using an achiral sorbent (RP-18) and a chiral mobile phase (CMP) containing copper(II) complex with N,N-dimethyl-l-phenylalanine (l-DM-PhA) as initial complex for CLEC. With 12 different reduction conditions, the obtained ratios of diastereoisomers d,l-HM-PAO: meso-HM-PAO varied from 69.2:30.8 to 15.9:84.1, in comparison to the reduction in routine synthesis of HM-PAO which gives an equal mixture of diastereoisomers. The ternary mixed complexes formation recorded spectrophotometrically on addition of HM-PAO or DI to the mobile phase with binary complex Cu(l-DM-PhA)(2), due to the evidence of bathochromic shift of 46nm for lambda(max) with significant difference in absorptivity contributes to separation mechanism.

On-line SPE-CE for the determination of insulin derivatives in biological fluids.

An on-line SPE-CE system is described for the determination of insulin derivatives in urine, serum and plasma. By combining techniques based on different separation mechanisms, in this case reversed-phase SPE and CE, a more selective sample clean-up is obtained. The described on-line SPE-CE procedure is able to desalt and clean biological samples, resulting in more repeatable electrophoretic results as well as a good linearity for urine, serum and plasma samples spiked with insulin derivatives, thus proving the elimination of detrimental effects caused by the sample matrix. The on-line SPE-CE system was linear for urine, serum and plasma samples spiked with insulin derivatives between 5 and 80 mg/l. The repeatability in migration time was below 1% relative standard deviation (R.S.D.). The repeatability of the peak was better (<2.4% R.S.D.) when no off-line precipitation reaction (<6.2% R.S.D.) was used, proving the beneficial characteristics of on-line sample pretreatment procedures over off-line sample pretreatment procedures which are prone to sample losses and contamination.

Chiral ligand-exchange chromatography for diastereo-enantio separation of exametazime.

The diastereo-enantio separation of isomeric mixtures of exametazime (HM-PAO) by liquid chromatography is described using an achiral sorbent (RP-18). A chiral eluent with the initial complex of Cu(II) and the optically active selector N,N-dimethyl-l-phenylalanine (l-DM-PhA), based on the ligand-exchange principle, has been applied. The separation is based on the presence of the immobilized binary complex Cu(l-DM-PhA)(2) and formation of mixed ternary complex. The optimal mole ratio of Cu(II):l-DM-PhA is 1:4, the pH should be between 4.1 and 4.2 and up to 0.8 mM of triethylamine is added for column presaturation with the initial complex. The elution order has been defined using isolated l-HM-PAO via l-HM-PAO L(+)tartrate and meso-HM-PAO obtained by repeated recrystallization from the isomeric mixture of HM-PAO. Complete resolution between all isomers (R(S) from 2.14 to 3.91) and partial resolution for meso(EE)/l-HM-PAO (R(S)=0.83) has been obtained. This means that the proposed chiral ligand-exchange chromatography (CLEC) can be used for determination of the isomeric purity of HM-PAO. This as an alternative method for resolution measurements with chiral columns.

Conventional and laser induced fluorescence detection of glucuronic acid conjugates after derivatization and liquid chromatographic separation.

Pre-chromatographic derivatisation of the carboxylic acid function of glucuronic acid conjugates is a suitable method for the selective and ultra-sensitive analysis of these compounds in urine and plasma samples. This goal is achieved by applying an indirect derivatisation procedure and laser induced fluorescence detection with a homemade detection system equipped with a continuous-wave argon-ion laser. The minimum detectable amounts for the analytes, after derivatisation, are about 3 amol using the fluorescein fluorophore. In comparison with conventional induced fluorescence detection a gain in sensitivity of over four orders of magnitude is obtained.

Mitomycin antitumour agents: a review of their physico-chemical and analytical properties and stability.

The review enumerates the physico-chemical and analytical properties of mitomycin antitumour antibiotics, of which mitomycin C is the most important representative. After a short overview of the position of the compounds in oncology the following subjects will be discussed: structural features, prototropic properties, spectroscopy (UV-VIS, ORD, CD, IR, NMR, MS), chromatography and electrochemistry. The chemical stability and aspects of the mechanism of action of the compounds are also discussed. The last part of the review surveys the analysis of mitomycin C in biological fluids.

Determination of the anticancer drug metabolite WR1065 using pre-column derivatization and diode laser induced fluorescence detection.

A liquid chromatographic (LC) procedure using alumina as stationary phase in both the pre- and the analytical column, is reported for the determination of WR1065, the active metabolite of the amino- and thiol-containing anticancer drug WR2721. After pre-column derivatization of the thiol group, the analyte is determined by LC with diode laser induced fluorescence detection in the near-infrared. Selective removal of excess label is achieved by means of column switching; it allows the detection of 5 x 10(-9) M WR1065 in water and 10-fold diluted, deproteinated plasma samples. The detection limit is determined by the derivatization reaction and not by the fluorescence detection of the labelled analyte. Endogeneous thiols do not interfere.

Dialysis as an on-line sample-pretreatment technique for column liquid chromatography: influence of experimental variables upon the determination of benzodiazepines in human plasma.

An evaluation is provided of dialysis, coupled on-line to column liquid chromatography, as a sample pretreatment procedure for macromolecule-containing biological samples. The influence of parameters such as acceptor phase flow rate, temperature, hydrophobicity of the analytes, pH, ionic strength and viscosity of the sample on the recovery and rate of dialysis is studied. In addition, methods to reduce the degree of drug-protein binding and thereby improve the recovery are reported. Diazepam, nitrazepam and oxazepam are used as model compounds. A method is reported for the fully automated determination of these compounds in human plasma using only 100 microliters of sample. Data on repeatability, linearity and detectability are given.

Determination of phenprocoumon in plasma and urine using at-line solid-phase extraction-capillary electrophoresis.

The use of capillary electrophoresis (CE) for the analysis of biological samples is rather problematic because of the large number of interferences present in the matrix. One of the possibilities to solve such problems is to couple solid-phase extraction (SPE) at-line with CE, a technique developed in our laboratory. In this study at-line SPE-CE is performed for the determination of the anticoagulant phenprocoumon in biological fluids. Plasma samples are injected after the addition of 1 vol.% of formic acid to release the drug from binding proteins, while urine samples can be directly injected. The procedure is linear between 0.2 and 30 microg ml(-1) with a correlation coefficient, r2, of 0.9996. The detection limit in plasma is 0.1 microg ml(-1), which is fully adequate in view of the concentrations, that have to be dealt with in practice. The phenprocoumon concentration in a plasma sample of a patient treated with the anticoagulant was 3.8 microg ml(-1).

Determination of roquefortine C in blue cheese using on-line column-switching liquid chromatography.

A method is described for the determination of roquefortine C in (blue) cheese. After liquid liquid extraction with a mixture of hydrochloric acid and methanol, and filtration, an aliquot is analysed using column-switching reversed-phase liquid chromatography. The recovery of roquefortine C in Fetta cheese is about 85%, the calibration curve is linear from 10 to 2500 ng g(-1) (r2 = 0.998), and the detection limit is about 10 ng g(-1). In different batches of Danish Blue concentrations of 1000-2000 ng g(-1) of roquefortine C are found. As regards the stability of roquelfortine C its half-life in diffuse daylight is ca. 50 min, while after irradiation with ultraviolet light, it is about 10 min.