Effect-Directed Analysis: Using HPTLC with bioassays to determine biological activity of substances

Effect-Directed Analysis (EDA) is the process of combining fractionation techniques, analytical techniques and bioassays to study unknown active substances or contaminants and their biological effects. Combining a Bioassay with High Performance Thin Layer Chromatography (HPTLC) provides an efficient and inexpensive method of screening for bioactive compounds in complex matrices.

HPTLC fingerprints of the crude mushroom extracts and their fractions visualized under four detection conditions. (A) UV 366 nm prior to derivatization, (B) p-anisaldehyde post-derivatization, (C) naphthyl/FBS post-derivatization, and (D) UV 366 nm after derivatization with 4-MU acetate. Track 1: Agaricus silvicola; 2: Amanita muscaria; 3: Amanita phalloides; 4: Cortinarius orellanus; 5: Grifola frondosa; 6: Gymnopilus penetrans; 7: Hypholoma fasciculare; 8: Laccaria amethystine; 9: Lactarius blennius; 10: Lactarius subdulcis; 11: Tricholoma auratum; 12: Tricholoma saponaceum; 13: Tricholoma ustale; 14: Xerocomellus chrysenteron. Link to full CBS136 article below.

High Performance Thin Layer Chromatography (HPTLC) is an ideal coupling technique to use for EDA, especially when considering complex sample matrices. HPTLC utilises a single-use stationary phase, typically a thin layer of silica gel supported on a glass or aluminium plate. This has two major benefits; minimal sample preparation/clean-up and an extensive range of solvents can be used for the mobile phase. Thus making it an efficient and inexpensive technique with powerful separation capability.

Additionally, the planar surface of a HPTLC plate lends itself well to in-situ bioassays, up to 15 samples and standards can be applied side-by-side and reagents can be applied uniformly across the entire plate surface using the CAMAG Derivatizer. This means that all samples are in contact with reagents at the same time and the temperature and reaction conditions can be kept consistent across all samples.

Once the bioassay is complete, the biological response can be documented by capturing images under UV light using the CAMAG TLC Visualizer 3 and measuring the fluorescence using the CAMAG TLC Scanner 4.

Potential applications for HPTLC-EDA include:

• Identifying toxic pollutants in the environment

• Identification of new bioactive compounds in phytochemistry

• Linking environmental contaminants to disease in human and animals

  
  

For recent examples of HPTLC-EDA put into practice, check out these CAMAG CBS articles:  

• CBS134: Environmental impact of tyre and road wear particles assessed with HPTLC bioassays

• CBS136: A sensitive HPTLC-EDA method using a 4-MU fluorescent probe for AChE inhibitor screening