Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays

Author Correction: Fabrication of microfluidic device for Aflatoxin M1 detection in milk samples with specific aptamers

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Labeling strategies for bioassays \ud

Different labeling strategies for enzymatic assays and immunoassays are reviewed. Techniques which make use of direct detection of a label, e.g. radioimmunoassays, are discussed, as are techniques in which the label is associated with inherent signal amplification. Examples of the latter, e.g. enzyme-linked immunosorbent assays or nanoparticle-label based assays, are presented. Coupling of the bioassays to chromatographic separations adds selectivity but renders the assays more difficult to apply. The advantages and drawbacks of the different analytical principles, including future perspectives, are discussed and compared. Selected applications from clinical, pharmaceutical, and environmental analysis are provided as examples

Sample preparation for peptides and proteins in biological matrices prior to liquid chromatography and capillary zone electrophoresis

The determination of peptides and proteins in a biological matrix normally includes a sample-preparation step to obtain a sample that can be injected into a separation system in such a way that peptides and proteins of interest can be determined qualitatively and/or quantitatively. This can be a rather challenging, labourious and/or time-consuming process. The extract obtained after sample preparation is further separated using a compatible separation system. Liquid chromatography (LC) is the generally applied technique for this purpose, but capillary zone electrophoresis (CZE) is an alternative, providing fast, versatile and efficient separations. In this review, the recent developments in the combination of sample-preparation procedures with LC and CZE, for the determination of peptides and proteins, will be discussed. Emphasis will be on purification from and determination in complex biological matrices (plasma, cell lysates, etc.) of these compounds and little attention will be paid to the proteomics area. Additional focus will be put on sample-preparation conditions, which can be 'hard' or 'soft', and on selectivity issues. Selectivity issues will be addressed in combination with the used separation technique and a comparison between LC and CZE will be made. © Springer-Verlag 2005