H. Irth

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellmans reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.

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.

On-line immunochemical detection in liquid chromatography using fluorescein-labelled antibodies

Abstract A postcolumn immunochemical detection system for on-line coupling to HPLC is described. The effluent from a reversed-phase LC column is mixed with fluorescein-labelled antibodies that are added via a mixing union. Antigenic analytes react with the antibodies to form strongly fluorescent immunocomplexes. In a second step, free antibodies are removed prior to fluorescence detection via passage through a small column packed with an antigen-bound support. The performance of the immunochemical reaction system was investigated using digoxin and its metabolites as analytes and fluorescein-labelled Fab fragments of polyclonal anti-digoxigenin as immunoreagent. This system tolerates up to 95% methanol or 45% acetonitrile in the LC eluent, allowing the separation of digoxin and its metabolites. The immunoreaction sequence is in equilibrium after ca. 1 min resulting in peak broadening comparable to that in standard postcolumn derivatization systems. The detection limits obtained for digoxin and digoxigenin after separation on a C 18 column are 200 and 50 fmol, respectively. The applicability of the method is demonstrated for the bioanalysis of digoxin and digoxigenin. Owing to the high selectivity of the immunodetection system, sample pretreatment can be reduced to deproteination and dilution of plasma and urine samples. Detection limits in both matrices (100-μl injections) are 1 · 10 −9 M for digoxigenin and 4 · 10 −9 M for digoxin.

Bioanalysis of some anthracyclines in human plasma by capillary electrophoresis with laser-induced fluorescence detection

A rapid method for the determination of daunorubicin, doxorubicin and epirubicin in human plasma is described. Samples are pretreated and concentrated by liquid-liquid extraction with chloroform and back-extraction into phosphoric acid, respectively. This pretreatment results in a sample matrix of low ionic strength in comparison with the electrophoresis buffer, permitting a 20-30-fold increase in the injected amount by zone sharpening when electrokinetic injection is applied. Analyte interaction with the capillary wall is prevented by using high acetonitrile contents in the electrophoresis buffer, which results in reproducible migration times and highly efficient separations. Laser-induced fluorescence detection provides an extremely sensitive and selective method without detectable biological interferences. The limit of determination of daunorubicin, epirubicin and doxorubicin in plasma ranges from 125 to 250 pg/ml.

Combined liquid-liquid electroextraction and isotachophoresis as a fast on-line focusing step in capillary electrophoresis

Abstract Combined liquid-liquid electroextraction and isotachophoresis as a fast on-line focusing step in capillary electrophoresis is described. Owing to the limited conductivity of the organic phase, high electric field strengths can be applied, resulting in high migration rates. Liquid-liquid electroextraction is used to focus the analyte ions from a large organic sample into a small volume in the terminating buffer zone, just above the liquid-liquid interface. Simultaneously, isotachophoresis is used to focus the analyte ions between the terminating buffer and the leading buffer. A steady state is reached within a few minutes, as isotachophoresis starts with a small sample volume. On-line capillary zone electrophoresis is used for the separation of the analytes. Propantheline, neostigmine, salbutamol and terbutaline were used as model compounds, and crystal violet was used to visualize the process for tuning the system. Concentration detection limits of pure solutions down to 10−9-10−10 mol l for the model compounds were obtained using simple UV absorbance detection.

Combined liquid-liquid electroextraction-isotachophoresis for loadability enhancement in capillary zone electrophoresis-mass spectrometry

Abstract Combined liquid-liquid electroextraction (EE) and isotachophoresis (ITP) as a fast on-line focusing step in capillary zone electrophoresis (CZE) prior to electrospray mass spectrometric (ESP-MS) detection is described. Very high electric field strengths can be applied owing to the low conductivity of the organic phase, which results in high migration rates. Liquid-liquid electroextraction enables the fast extraction of analyte ions into a small buffer volume, whereas ITP is used to focus the analytes away from the liquid-liquid interface. As a result, ITP starts with a small sample volume containing the extracted ions. After reaching the steady state within several minutes, CZE separation follows. Clenbuterol, salbutamol, terbutaline and fenoterol were used as model compounds. Concentration detection limits of pure solutions down to 2·10 −9 mol/l for clenbuterol, salbutamol and terbutaline, and 5·10 −9 mol/l for fenoterol have been achieved using on-line EE-ITP-CZE-ESP-MS.

Bioanalysis of digoxin and its metabolites using direct serum injection combined with liquid chromatography and on-line immunochemical detection

An automated dual-column liquid chromatographic assay for digoxin is described. Serum samples are directly injected onto a restricted-access solid-phase extraction support. After liquid chromatographic (LC) separation on a C18 analytical column, antigenic analytes are detected by means of post-column immunochemical detection (ICD) using fluorescein-labelled antibodies against digoxigenin. The detection limit of this assay is 160 pg/ml (preconcentration of 1.0 ml serum). With the present method digoxin and three of its cross-reactive metabolites were determined in serum taken from patients which were orally administered a 1-mg dose of digoxin. The results obtained with LC-ICD were compared with data provided by a batch immunoassay.

Development of a needle device for on-line electroextraction-liquid chromatography

The development of a needle device enabling on-line electroextraction coupled to high-performance liquid chromatography in an autosampler is described. This electroextraction needle meets all the demands set to automated liquid handling and can fully replace the conventional autosampler needle. In addition, the system has been fully automated, and can be used as an interfacing technique between solid-phase extraction and liquid chromatography. A detailed description of the electroextraction needle hardware, as well as some typical examples of its use in on-line electroextraction-liquid chromatography are presented.

Strategies for on-line coupling of immunoassays to high-performance liquid chromatography

Abstract On-line coupling of reversed-phase high-performance liquid chromatography (HPLC) with bioassays, such as immunoassays, allows a combination of the separation power and automation potential of LC with the high selectivity and sensitivity of biochemical detection principles. In this way important problems related to LC and batch immunoassays, i.e., the lack of selective and sensitive detection methods on the one hand and the interference of cross-reactive matrix components on the other hand, can be overcome. This article presents different strategies to implement immunochemical interactions in on-line post-column detection techniques: either labelled antibodies or labelled antigens can be used to detect the presence of antigens eluting from the reversed-phase LC column.

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.