Microfractionation revisited: a 1536 well high resolution screening assay

Area of Science:

• Analytical Chemistry
• Biochemistry
• Natural Product Discovery

Background:

• Existing online and offline systems for bioactivity screening have limitations.
• There is a need for integrated methods that combine separation, detection, and identification.
• High-performance liquid chromatography (HPLC) and plate reader technologies offer complementary strengths.

Purpose of the Study:

• To develop a novel "at-line" method integrating HPLC with plate reader technology.
• To enable simultaneous bioactivity determination and analyte identification from complex mixtures.
• To overcome drawbacks of existing online and offline screening approaches.

Main Methods:

• Coupling of microbore HPLC with post-column addition of biochemical reagents.
• Simultaneous bioactivity assay and online QTOF MS data acquisition.
• Microfractionation of the screening assay mixture into a 1536-well plate.

Main Results:

• Successful screening of a natural extract spiked with known Protein Kinase A inhibitors.
• Identification of a bioactive inhibitor within a complex natural extract.
• Demonstrated applicability for detecting low-concentration bioactive compounds.

Conclusions:

• The developed "at-line" approach effectively hyphenates high-resolution separation with biochemical screening.
• This method fully utilizes modern plate reader capabilities for enhanced screening efficiency.
• The approach overcomes challenges associated with high-density well formats and complex sample analysis.