N.C. van de Merbel

Membrane-based sample preparation for chromatography

Abstract The use of membrane-based systems for sample preparation prior to chromatographic analysis is reviewed. A categorization of membrane separation techniques (dialysis with porous and with non-porous membranes, electrodialysis and ultrafiltration) for sample preparation purposes is presented and the theoretical background of membrane separation is shortly discussed. The applicability of these techniques is demonstrated by presenting examples of the use of membrane-based devices, on-line coupled to gas or liquid chromatography, for sample preparation in the biomedical, environmental and biotechnological field. Finally, the relative merits of the various membrane separation techniques are compared, with special emphasis on speed, recovery, selectivity and robustness.

Membrane-based sample preparation coupled on-line to chromatography or electrophoresis

A review on the use of membranes for on-line sample preparation prior to chromatographic and electrophoretic analysis is provided. The current state-of-the-art of four membrane-based techniques (dialysis, electrodialysis, filtration and membrane extraction) is described by reviewing their principles and applications. Possible future developments are discussed.

The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium.

BACKGROUND The European Bioanalysis Forum dried blood spots (DBS)/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. This paper is focused on the impact of hematocrit changes on DBS analyses. RESULTS The hematocrit can have an effect on the size of the blood spot, on spot homogeneity and on extraction recovery in a compound-dependent manner. The extraction recovery can change upon aging in an hematocrit-dependent way. Different card materials can give different outcomes. CONCLUSIONS The results from the conducted experiments show that the issues of DBS in regulated bioanalysis are real and that the technology will need improvements to be ready for use as a general tool for regulated bioanalysis.

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.

Sampling and analytical strategies in on-line bioprocess monitoring and control

The use of on-line systems for the determination of small organic molecules during biotechnological processes is reviewed. Since sampling of part of the culture medium is an inherent and crucial part of the use of on-line systems, several sampling systems are described and compared and a categorization of sampling techniques - non-membrane, dialysis and ultrafiltration - is provided. Further, several analytical approaches - based on flow-injection or chromatographic analysis - and their relative merits for on-line measurements are discussed. Selected examples from the recent scientific literature serve to illustrate the applicability for on-line monitoring and control of bioprocesses.

Determination of aflatoxin M1 using a dialysis-based immunoaffinity sample pretreatment system coupled on-line to liquid chromatography. Reusable immunoaffinity columns

A liquid chromatographic column-switching system containing a dialysis unit and an anti-aflatoxin immunoaffinity precolumn (immuno precolumn) is described for the automated determination of aflatoxin M1 in milk samples. Both a flat membrane dialysis unit working according to the flowing donor-flowing acceptor principle and a laboratory made hollow-fibre dialysis unit working according to the stagnant donor-flowing acceptor principle were evaluated. The hollow-fibre unit is superior with respect to repeatability (3% relative standard deviation) and detection limit (10 ng/l for aflatoxin M1 in milk), in spite of the fact that the overall recovery is only 6%. Interfering compounds, which would destroy the activity of the immuno precolumn, are efficiently removed from the system by the dialysis step; a single immuno precolumn can then be used for over 70 milk analyses. No decrease in the performance of either the immuno precolumn or the hollow-fibre dialysis unit is observed.

On-line monitoring of fermentation processes by ultrafiltration and column liquid chromatography

SummaryAn automated on-line monitoring system for low-molecular-weight compounds (e.g. sugars) during fermentation processes is described. The applicability of cross-flow ultrafiltration, coupled on-line with high-performance liquid chromatography, is evaluated. Various detection modes for the determination of the test compounds lactose, glucose and fructose) in a complex fermentation broth are compared. In addition, the influence of some system parameters on the performance of several ultrafiltration modules for the removal of cellular and macromolecular broth constituents is investigated. A method for the fully automated determination of sugars during anE. coli batch culture in a brain-heart infusion medium is presented. With the present system five analyses can be performed every hour, using only 35μl of sample, without significant deterioration of the ultrafiltration process. Contamination by external micro-organisms was not observed and good correlation with an off-line method was found.

LC phases improve, but not all assays do: Metformin bioanalysis revisited

SummaryTwo assay methods for the antidiabetic metformin, one developed and validated in 1990 and one developed and validated in 1996, are compared. The first method, using an octadecyl phase and an ion pairing agent in the eluent, could not be reproduced some five years later, but another method, using a phenyl phase and no ion pairing agent, could be successfully applied. This paper shows that the retention mechanism of the positively charged analyte is not due to ion-pair formation, as originally assumed, but to interaction with free silanol groups in the LC phase. It is suggested that the number of free silanol groups in octadecyl phases was strongly reduced between 1990 and 1996, whereas for phenyl phases this was not the case. For the second method, validation results on linearity, specificity, accuracy, precision, recovery and stability as well as application of the method to samples from a clinical trial are shown. The validated calibration range is from 20.0 to 2000 ng.mL−1, with accuracy (bias) and precision (coefficient of variation) being below 15% at all levels. Using automated solid-phase extraction for sample preparation, a sample throughput of typically 100 per day can be achieved.

Automated column liquid chromatographic determination of amoxicillin and cefadroxil in bovine serum and muscle tissue using on-line dialysis for sample preparation

A fully automated method is described for the determination of amoxicillin and cefadroxil in bovine serum and muscle tissue. The method is based on the on-line combination of dialysis and solid-phase extraction for sample preparation, and column liquid chromatography with ultraviolet detection. In order to enhance the UV detectability of the analytes, post-column addition of 0.1 M sodium hydroxide is performed. The method shows good linearity and repeatability for both analytes in serum as well as in muscle tissue; the limits of detection in these samples are 0.05 microgram/ml and 0.2 microgram/g, respectively. The method has a sample throughput of 30 samples per 24 h.

On-line dialysis as a sample-preparation technique for column liquid chromatography

Abstract The use of dialysis as an on-line sample-preparation technique for column liquid chromatography is briefly reviewed by discussing the influence of the construction of the dialysis block, the different modes of performing dialysis, the properties of the membrane, and the nature of the sample. on the speed of dialysis and the analyte recovery. Relevant examples from the recent scientific literature serve as illustrations.