Analysis of Botulinum Neurotoxin A (BoNT/A) in Pharmaceutical Products based on Signature Markers

Yuk-king Cheung, Government Laboratory, Hong Kong, Hong Kong, Hong Kong (ykcheung@govtlab.gov.hk)
Yiu-chung Wong, Government Laboratory, Hong Kong, Hong Kong, Hong Kong
Ella Lai-ming Wong, Government Laboratory, Hong Kong, Hong Kong, Hong Kong

 

Botulinum neurotoxins (BoNT) are produced by the anaerobic bacterium Clostridium botulinum and are one of the most lethal known poisons (LD₅₀ = 0.8 microgram for a 70 kg human by inhalation). Despite its high lethal toxicity, BoNT have been used to treat spasms and other muscle problems. Most recently, BoNT serotype A (BoNT/A) gains its worldwide popularity in cosmetic surgery to prevent development of facial wrinkles. Clinical testing of BoNT/A is conventionally relied on in vivo mouse lethality assay in accordance with pharmacopeial and AOAC methods. In response to increased public pressure to the inhumane testing, sensitive non-animal assays are of urgent need.

Identification of BoNT/A in pharmaceutical products by chemical means is always a challenge due to its complex structure, ultra-trace level and the interference from excipients. This work reported a direct measurement of BoNT/A in pharmaceutical injection samples by mass spectrometry study using ten potential signature markers, seven from the light chain and three from the heavy chain. Pharmaceutical samples that claimed to contain BoNT/A were trypsin digested under optimal conditions and its identity was examined by the presence of the concerned signature markers. Detection limits were at 0.05 ppm level which is sensitive enough to detect registered BoNT pharmaceuticals in the market. The method has been validated across a few brands of BoNT/A pharmaceutical injections commonly available in the Asia Pacific region, and produced reliable results. In addition, the method is useful for the detection of BoNT in formal investigation cases related to fake pharmaceutical products.