Ray C T Hung
Government Laboratory, China
Ray Chi-tat HUNG did Bachelor of Science in Chemistry and a Master of Philosophy in Chemistry, both conferred by the Hong Kong University of Science and Technology. He has been employed as a Chemist from June 2002 in the Analytical and Advisory Services Division of the Government Laboratory. His current major responsibilities include the determination of veterinary drug residues, chemical analysis in pharmaceutical products and microbiological tests on pharmaceutical products and environmental samples.”
Neurotoxins consisting mainly of botulinum neurotoxin A (BoNT/A) produced by anaerobic bacterium Clostridium botulinum are the most lethal known poisons. Although BoNT/A has high lethal toxicity (LD50=0.8 µg for a 70 kg human by inhalation), it has been used as a versatile substance for muscle spastic disorder treatments in clinical settings. BoNT/A also gains its worldwide popularity in cosmetic surgery to relieve facial wrinkles in recent years. The potency of therapeutic BoNT/A is usually tested using in vivo mouse bioassays as stated in British Pharmacopoeia and European Pharmacopoeia, or alternatively, in vitro enzymatic or immunoassay methods. Botulinum toxin itself comprises a single heavy chain of ~100 kDa linked to a ~50 kDa light chain by a disulfide bond. The breaking of the disulfide bond can release the light chain, which can target a soluble N-ethylmaleimide-sensitive attachment protein (SNARE protein)- either synaptosomal-associated protein-25 (SNAP-25) or vesicle-associated membrane protein 2 (VAMP2), leading to a series of biological processes for its medicinal and cosmetic effects. Different botulinium toxins (serotype A-H) cleave uniquely on a specific site of SNAP-25 or VAMP2. In this paper, we attempt to develop a sensitive chemical method to analyse BoNT/A in pharmaceutical products and the detection mechanism is based on the unique BoNT/A neurotoxin proteolytic activities on the synthetic peptide SNAP-25. The specifically cleaved peptide fragments from SNAP-25 are then determined using liquid chromatography coupled with high resolution tandem mass spectrometry.