Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines.

An on-line liquid chromatography-immunochemical detection (LC-ICD) system for the quantification of cytokines in cell extracts has been developed using a post-column continuous-flow reaction detection system using fluorescence labelled antibodies. Cytokines eluting from the micro-HPLC column react with antibodies to form fluorescent complexes. In a second step the excess of free antibody is trapped on a cytokine bound support prior to fluorescence detection. The concentration detection limit of the flow injection-ICD system was 50 pM (20 microl injection volume) for interleukin 4 (IL-4). An absolute detection limit of 1 fmol was obtained for IL-4. Similar to ICD systems for small non-protein analytes developed earlier, reaction times were in the order of 1 minute. The immobilised cytokine affinity columns can easily be regenerated and used for months. The present ICD system for interleukins 4, 6, 8 and 10 was coupled to ion exchange-, size exclusion- and reversed phase chromatography. Important parameters (reaction times, reaction conditions) were investigated to get a better understanding of post-column ICD systems for macromolecules.

Protein mapping by two-dimensional high performance liquid chromatography.

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 microm polymeric beads bonded with diethylaminoethyl and sulfonic acid groups, respectively, were applied as ion exchangers and operated at a flow-rate of 1 ml/min. To achieve very high-speed and high-resolution separations in the second dimension, short columns of 14 x 4.6 mm I.D. with 1.5 microm n-octadecyl bonded, non-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/min. Two reversed-phase columns were used in parallel in the second dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins, which were not completely resolved in the first dimension can, in most cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less than 1% (n = 15), while the RSDs of the peak areas were less than 15% (n = 15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimension.

Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol.

Miniaturized supported liquid membrane device for selective on-line enrichment of basic drugs in plasma combined with capillary zone electrophoresis.

A hollow fiber miniaturized supported liquid membrane (SLM) device for sample preparation is connected on-line with capillary electrophoresis and used for determination of a basic drug, bambuterol, in human plasma. The analyte is extracted from the outside of the hollow fiber (donor) through the liquid membrane (pores of the fiber impregnated with organic solvent) into the acceptor solution in the fiber lumen. The process is driven by differences in pH between the donor and acceptor solution. The whole volume of the acceptor solution can then be injected into the CZE capillary by using the double-stacking procedure for large volume-injection. Very clean extracts of low ionic strength are obtained from the SLM treatment, making this sample pretreatment method compatible with the CZE double-stacking procedure, which in turn makes it possible to inject large volumes of sample onto the separation capillary. Good performance of the whole procedure is demonstrated, and detection limits in the low nanomolar range were obtained in spite of the relatively weak UV absorbance of bambuterol. Extractions through the miniaturized SLM unit can be performed for 5-6 h without regenerating the fiber. The regeneration procedure was tested, and no relevant changes in the performance of the extraction could be found after seven regenerations, allowing the same fiber to be used for a week.

Liquid chromatography-mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection.

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with liquid chromatography-mass spectrometry (LC-MS), for the analysis of biological samples is described. The system was tested with cortisol and prednisolone for plasma analysis and arachidonic acid for urine analysis. A precolumn packed with a 25-micron C18 alkyl-diol support is used for direct plasma or urine injection. Using column-switching techniques, the analytes enriched on the precolumn are eluted to the analytical column without transfer loss. An on-line heart-cut technique was employed and only the analyte-containing fraction eluting from the LC column is directed to the MS to protect the LC-MS interface and ion-source from contamination. The whole system is operated in a parallel mode, that is, sample pre-treatment and LC-MS analysis are performed simultaneously to provide the shortest possible analysis time. The only off-line sample pre-treatment step required was centrifugation to remove particulate matter. With the fully automated system, total analysis times of 5 and 9.5 min were achieved for cortisol in serum and arachidonic acid in urine, respectively. Cortisol and related compounds were quantitatively recovered from plasma with a detection limit for prednisolone (direct injection of 100 microliters on restricted-access precolumn) of 2 ng/ml.

Determination of a basic drug, bambuterol, in human plasma by capillary electrophoresis using double stacking for large volume injection and supported liquid membranes for sample pretreatment.

In this work we show the potential of using a double stacking procedure based on field enhancement as a means to increase the concentration sensitivity in CZE analysis of human plasma extracted by the supported liquid membrane (SLM) technique. A basic drug, bambuterol, was used as a model substance. The low ionic strength of the SLM extract makes this pretreatment technique compatible with the double stacking sequence. No significant loss of separation performance was observed when 3 microliters of SLM extract was concentrated by the CZE double stacking sequence. Almost no visible difference was seen between the electropherograms after enrichment of a plasma blank and an aqueous blank. Good performance of the whole procedure was demonstrated and detection limits in the low nM range were obtained in spite of the relatively weak UV absorbance of bambuterol. The developed procedure was evaluated for both achiral and chiral separation. In the latter approach chiral selectivity was obtained by adding cyclodextrin to the separation electrolyte.

Competitive flow injection enzyme immunoassay for steroids using a post-column reaction technique.

Two types of flexible, sensitive and rapid competitive flow injection enzyme immunoassay were developed and evaluated for their potential use in the bioanalysis of steroids. Instead of the more typical approach where the signal is generated from antibody-bound hapten-enzyme conjugate, the non-bound fraction passing through the affinity column was allowed to react with an enzyme substrate from a merging channel in a post-column reaction system. The enzyme product (p-aminophenol or 4-methyl umbelliferol) was amperometrically or fluorometrically detected. Several parameters known to affect signal generation in the immunoassay were evaluated, including flow rate through the affinity column and through the reaction coil, the length of the reaction coil and of the affinity column. In the pre-incubation approach, where samples were mixed with enzyme conjugate and antibodies before injection, a sample throughput as high as 20 h(-1) was possible. The signal precision was about 1% (RSD) for cortisol (0.6-80 pmol) and 2% (RSD) for budesonide (0.02-12.5 pmol). In the displacement assay for cortisol, enzyme-labelled analyte was displaced from the affinity column when the sample was injected into the flow. A standard curve was obtained with a signal precision of 4-20% for 12.5-1250 pmol injected. The same instrumental set-up was used in both types of immunoassay, and thus a highly flexible system was obtained. A simple replacement of the affinity column from protein G in the pre-incubation approach to a column containing primary antibodies in the displacement assay was needed.

Design of non-competitive flow injection enzyme immunoassays for determination of haptens: application to digoxigenin.

A theoretical model immunoradiometric assay (IRMA) was adapted to provide a non-competitive flow injection enzyme immunoassay for haptens and used as a guide in studying the effects of different parameters on the sensitivity, precision and dynamic range of the assay. As well as the concentration of the antibody-enzyme conjugate, the affinity constant, the run time through the affinity column, the homogeneity of the antibody population and purity of the antibody-enzyme conjugate were all shown to be important parameters in the optimisation of the assay. The findings were used to design an optimised enzyme flow injection immunoassay for the model compound digoxigenin in standard solutions. A linear calibration curve was established in the range 0.38-7.7 fmol of digoxigenin, resulting in a precision of 14.8% RSD at 1 fmol and 3.7% RSD at 7.7 fmol. Antibody fragments reacting with digoxigenin and labelled with alkaline phosphatase, (Fab-AP) were used to convert 4-methyl umbelliferyl phosphate to a fluorescent product measured downstream. The sample throughput was 15 h-1 and over 60 injections were possible before regenerating the affinity column.

Micro-CLC as an interface between SLM extraction and CZE for enhancement of sensitivity and selectivity in bioanalysis of drugs.

This work demonstrates the high selectivity and sensitivity obtainable in bioanalysis using the supported liquid membrane (SLM) technique coupled on line with capillary zone electrophoresis (CZE) through a micro-column liquid chromatography (CLC) interface. The system utilizes two selective, sequential enrichment steps before the third analyte focusing and separation step with double-stacking CZE. The enantiomers of bambuterol in human plasma can be concentrated about 40,000 times (approx. 6 times by the SLM treatment, approx. 17 times by micro-CLC focusing, and approx. 400 times by double-stacking CZE) on their way through the system, and extremely high selectivity is obtained. Determinations in the subnanomolar region are achievable for the enantiomers despite relatively weak UV absorbance. Good performance of the entire procedure is demonstrated and a method to increase the sample throughput is presented.

Biocompatible sample pretreatment for immunochemical techniques using micellar liquid chromatography for separation of corticosteroids.

Micellar liquid chromatography (MLC) using Tween 20 as surfactant was evaluated as a biocompatible sample pretreatment preceding immunoassay in order to obtain an increased selectivity of the assay and a simplification of the sample pretreatment procedure. Different stationary phases and chromatographic conditions were studied for the separation of budesonide and cortisol and some steroids known to interfere in immunoassay of these compounds. The separation was dependent on several parameters, for example, temperature, the concentration of Tween 20, pH and ionic strength of the mobile phase, and nature of the stationary phase. A precolumn venting system was used, which allowed for 140 direct injections of 25 microliters of human blood plasma, without loss of chromatographic performance. Results obtained from the coupling of MLC to an immunoassay for cortisol illustrates the selectivity which can be obtained, and that simplification of the sample pretreatment is possible using this technique.

Supported liquid membrane technique for selective sample workup of basic drugs in plasma prior to capillary zone electrophoresis.

The potential of using the supported liquid membrane (SLM) technique for pretreatment of plasma samples before analysis with capillary zone electrophoresis (CZE) has been investigated. A basic drug, bambuterol, was used as a model substance in a system, where either 6-undecanone or a mixture of di-n-hexyl ether (DHE) and tri-n-octyl phosphine oxide (TOPO) was used as membrane liquids. It was found that the electropherograms obtained after SLM enrichment of bambuterol in plasma samples were as clean as when aqueous samples containing the same substance were processed in the same way. The low ionic strength of the SLM treated blood plasma samples permitted subsequent sample stacking in the CZE step. The linearity of the detector signal for different concentrations of bambuterol in plasma was satisfactory from 50 to 1000 nmol/L with regression co-efficients of 0.997 using 6-undecanone as membrane liquid and 0.999 with the other liquid. In both systems, the confidence interval of the intercept included the origin. The detection limit was about 50 nmol/L. The long-term stability of the two membrane liquids proved adequate as a membrane lasted at least through the working day.

Determination of drugs in biosamples at picomolar concentrations using competitive ELISA with electrochemical detection: application to steroids.

A competitive ELISA with electrochemical detection in a flow injection system (FIA) has been developed for determinations of the steroid drug budesonide in biological samples. Plasma samples were cleaned from interfering and cross-reacting compounds by two pretreatment steps consisting of a solid-phase extraction and a liquid chromatography fractionation. The enzyme label was alkaline phosphatase, which was used with p-aminophenyl phosphate (PAPP) as a substrate. The product, p-aminophenol, was detected electrochemically at a glassy carbon electrode at 250 mV (vs Ag/AgCl). The limited stability of both the substrate and the product influenced the performance of the method and had to be taken into account in the procedure by a normalization with time. Budesonide could be quantified in plasma samples down to 10 pM. The major sensitivity-limiting factor was the amperometric background response, probably due to spontaneous hydrolysis of PAPP to p-aminophenol.

Coupled-column chromatography on immobilized protein phases for direct separation and determination of drug enantiomers in plasma.

Columns packed with immobilized alpha 1-acid glycoprotein and albumin were used in coupled-column chromatography to increase their utility for determining low concentrations of enantiomers in biological samples. The two enantiomers eluted from the protein columns were trapped and compressed on two separate columns, packed with hydrophobic stationary phase, and subsequently transferred to a fourth column for final separation. The overall effect was an increase in efficiency and selectivity. Examples are given of separations of the enatiomers of terbutaline, metoprolol, oxazepam and bupivacaine in plasma. For quantitative determination a single calibration can be used for both enantiomers.

Determination of terbutaline enantiomers in biological samples using liquid chromatography with coupled columns.

The purpose of this work was to develop and validate a method for the separation and determination of the enantiomers of terbutaline in plasma and intestinal juice. Terbutaline was extracted from plasma and intestinal juice by liquid-solid extraction on small C18 cartridges. The extract was then analyzed by coupled column liquid chromatography with amperometric detection. For chiral separation a beta-cyclodextrin phase was used. The within-day variation (Cv) on spiked plasma samples was in the range 0.8-6.4% at 3.8-33.8 nmol/liter for the (-)-enantiomer, and 2.6-23.0% at 1.3-11.3 nmol/liter for the (+)-enantiomer. The between-day variation on spiked plasma samples was 5.5% at 10.7 nmol/liter and 13.6% at 4.3 nmol/liter for the (-)-and (+)-enantiomers, respectively. The within-day variation for intestinal juice was in the range 0.7-1.5% at 5.6-30.0 mumol/liter for the (+)-enantiomer.

Determination of drug enantiomers in biological samples by coupled column liquid chromatography and liquid chromatography-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) and coupled column chromatography can be used to overcome problems likely to occur in direct separation and determination of drug enantiomers in biological samples. This is exemplified here with the direct separation and determination of terbutaline in human plasma at the nmol/l level. A beta-cyclodextrin column with an aqueous mobile phase was used for chiral separation. For coupled column chromatography, the concentration of each enantiomer was calculated from the enantiomeric area ratio and the racemate concentration. A deuterium-labelled internal standard was used in the LC-MS experiments.

Coupled columns in bioanalytical work.

The bioanalytical laboratory giving a service in the development of new drugs has to provide flexibility as well as routinely assay a large number of samples, preferably with automated procedures. Liquid chromatography with coupled columns can be most useful for this purpose, as exemplified in the present paper, where a coupled-column configuration, which has been used for automation, screening and method validation, is described.

Combined actions of bronchodilators in guinea-pigs depend on the severity of cholinergic airway obstruction.

The effects of theophylline (THEO), given alone and in combination with terbutaline (TER) or ipratropium bromide (IPRA), were investigated on the dose-related methacholine (MeCh)-induced bronchoconstriction in anaesthetized guinea-pigs. MeCh increased lung resistance (RL) relatively more than it decreased dynamic lung compliance (CDYN), which indicates obstruction of mainly large airways. THEO counteracted MeCh effects on CDYN more than those on RL, in comparison with the effects of TER or IPRA. Combined treatment with THEO + TER or THEO + IPRA usually antagonized the MeCh-induced rise in RL more than did the individual drugs, but the decrease in CDYN was not prevented more than by THEO alone. The actions of the bronchodilator combinations on RL depended on the degree of airway obstruction, being additive on mild or moderate states and synergistic on the most severe bronchoconstriction. Subtherapeutic doses of TER antagonized rather than enhanced the effects of THEO on RL. These results suggest that THEO has a more peripheral apparent site of action than TER or IPRA. A sufficient dose of a beta 2-agonist seems decisive in order to produce an enhanced bronchodilator effect with THEO on large airways.

Multidimensional column liquid chromatography with electrochemical detection for the analysis of terbutaline in human plasma.

Because of low drug concentrations and complex nature of the sample, analytical methods with sensitive and selective detection are necessary for pharmacokinetic studies of terbutaline. Up to now, for non-radiolabelled drugs, usually only methods based on gas chromatography plus mass spectrometry have met these requirements. As an alternative, we have developed an automated method based on liquid chromatography. The necessary sensitivity and selectivity were obtained by using electrochemical detection and a microprocessor-controlled column switching system. The accuracy of the method was compared with a method based on gas chromatography plus mass spectrometry. The overall precision expressed as per cent of the mean was +/- 3.5% and +/- 2.2% at 5 and 50 pmol/mL, respectively. The total absolute recovery for terbutaline and internal standard at these concentration levels were in the range 85-106%.

Enprofylline kinetics in healthy subjects after single doses.

The kinetics of enprofylline, a novel antiasthmatic xanthine derivative, were studied. Eight healthy subjects received three different single enprofylline doses, 0.5, 1, and 1.5 mg/kg, injected as an infusion over 10 min. Plasma and urine levels of unchanged enprofylline were observed 3 to 7 and 21 to 24 hr after dosing. Plasma t 1/2 varied among individuals from 1.2 to 1.9 hr. Volume of distribution (V beta or area) and volume of distribution at steady state (V ss) averaged 0.57 and 0.511 X kg-1. Total clearance averaged 0.25 l X hr-1 X kg-1. Renal clearance ranged from 200 to 400 ml X min-1, indicating a large contribution by active tubular secretion. The mean recovery of unchanged drug in the urine was 89%. Thus, unlike theophylline, enprofylline was eliminated mainly by the kidney.