Immunomagnetic bead-based sample preparation method and immunoassay for detecting higenamine in herbal products

Area of Science:

• Analytical Chemistry
• Biochemistry
• Pharmacology

Background:

• Higenamine (HM) is an S3 β2 agonist prohibited by the World Anti-Doping Agency (WADA).
• HM is found in various supplements and traditional medicines, posing a risk of accidental doping for athletes.
• Existing detection methods lack the speed and simplicity required for daily monitoring.

Purpose of the Study:

• To develop a rapid, quantitative detection method for Higenamine (HM).
• To incorporate efficient sample preparation for practical, daily use.
• To analyze HM presence in diverse products like lozenges, beverages, syrups, and herbal extracts.

Main Methods:

• Development of a novel method combining immunomagnetic beads for sample enrichment and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for detection.
• Generation of a highly sensitive anti-HM monoclonal antibody (1E9-mAb).
• Optimization of assay conditions and validation of sensitivity and detection limits.

Main Results:

• Achieved high sensitivity with an IC50 of 0.23 ± 0.02 ng/mL and a detection limit (IC15) of 0.04 ± 0.01 ng/mL.
• Established low detection limits for various matrices: 3 μg/kg for lozenges, 4 μg/kg for beverages, and 2 μg/kg for syrups.
• Successfully detected HM in 28 commercial products, including the first-time identification in grass coral extract and its derivatives.

Conclusions:

• The developed immunomagnetic bead-based ic-ELISA offers a simple, efficient, and rapid method for daily detection of trace HM.
• This method addresses the need for practical monitoring of HM in food supplements and traditional medicines.
• The study provides the first evidence of HM in grass coral products.