**7. Mass spectrometry-based methods to detect toxins**

Mass spectrometry (MS) has been used as a method to dissect components of botulinum toxin complexes [37–39]. The MS-based method, called ENDOPEP-MS, uses antibodies to concen‐ trate and extract BoNT from test samples [38]. The concentrated toxins are then subjected to an endopeptidase activity–based assay to generate target cleavage products. Finally, MS is used to identify these products. This approach has been successful in identifying BoNT serotypes A, B, E, and F in various food and clinical matrices with greater sensitivity than the mouse bioassay.

Morineaux et al. [40] recently described a MS method that employs immunocapture enrich‐ ment by antibodies specific for BoNT/A-L chains. The enriched analyte is then analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) on a triple quadrupole mass spectrometer (QqQ) in multiple reaction monitoring (MRM) mode. Peptides from BoNT LC specific to the subtypes BoNT/A1–A3 and BoNT/A5–A8 could be identified. BoNT/A subtypes were correctly identified in culture supernatants, water, and orange juice samples with a LOD of 20–150 mouse lethal doses (LDs), but there was a lower sensitivity in serum samples.

Kalb et al. [41] have described the development of a quantitative enzymatic method for the detection of four BoNT serotypes using matrix-assisted laser desorption/ionization—time of flight (MALDI-TOF) MS. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were carefully examined, including the concentration of the substrate and internal standard, the length of time for the cleavage reaction, and the components present in the reaction solution. Longer incubation time produced more sensitive results but was not capable of determining higher toxin concentrations, whereas a shorter incubation time was less sensitive. To address these limitations, a novel two-step analysis was developed [41]. By combining the results from a two-stage quantification, four or five orders of magnitude in dynamic range are observed for detection of BoNT serotypes A, B, D, and F. To minimize the number of cleavage reactions and analytical samples, the assay can be multiplexed using mixtures of different neurotoxin substrates. Numerous different research groups (including Kalb et al. [42], Björnstad et al. [43], and Hines et al. [37]) have used MS to dissect the components of the BoNT/G complex, revealing BoNT/G as well as other toxin protein components, namely NTNH, HA-17, HA-33, and HA-70. Overall, the use of MS can provide rapid and definitive results.
