No abstract

Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an enzymatic and mass spectrometric assay called Endopep-MS. However, no comprehensive validation of the method has been presented. The main purpose of this work was to perform an in-house validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, E, and F in serum. The limit of detection (LOD), selectivity, precision, stability in matrix and solution, and correlation with the MBA were evaluated. The LOD was equal to or even better than that of the MBA for BoNT-A, B, D/C, E, and F. Furthermore, Endopep-MS was for the first time successfully used to differentiate between BoNT-C, D and their mosaics C/D and D/C by different combinations of antibodies and target peptides. In addition, sequential antibody capture was presented as a new way to multiplex the method when only a small sample volume is available. In the comparison with the MBA, all the samples analyzed were positive for BoNT-C/D with both methods. These results indicate that the Endopep-MS method is a good alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, speed, and that it does not require experimental animals.

Preparations of the plant Acorus calamus (calamus or sweet flag) (A. calamus) are available via internet trade and marketed as being hallucinogenic. In 2003-2006, the Swedish Poisons Information Centre received inquiries about 30 clinical cases of intentional intoxication with A. calamus products. The present investigation aimed to identify alpha- and beta-asarone, considered active components of A. calamus, and metabolites thereof in urine samples collected in seven of these cases. To further aid the identification of asarone biotransformation products, a calamus oil preparation was incubated with the fungus Cunninghamella elegans, which is used as a microbial model of mammalian drug metabolism. Using gas chromatography-mass spectrometry (GC-MS) analysis in selected ion monitoring mode, alpha-asarone was detected in five urine samples at concentrations ranging between approximately 11 and 1150 microg/L and beta-asarone in four of those at approximately 22-220 microg/L. A previously identified asarone metabolite, trans-2,4,5-trimethoxycinnamic acid (trans-TMC), was detected in the fungus broth by liquid chromatography-tandem mass spectrometry whereas cis-TMC was tentatively identified in the human urine samples. Using GC-MS, a hydroxylated asarone metabolite was identified both in fungus broth and urine samples. However, this study demonstrated no evidence for the presence of 2,4,5-trimethoxyamphetamine, claimed as a hallucinogenic component of A. calamus. The main clinical symptom reported by the patients was prolonged vomiting that sometimes lasted more than 15 h.

The patients using psychoactive plant materials were mainly young and commonly used the Internet for drug acquisition. Having access to bioanalytical methods for detection of plant-derived psychoactives is therefore considered important, when providing clinical toxicology service.

This review focuses on mass spectrometric detection of protein-based toxins, which are among the most toxic substances known. Special emphasis is given to the bacterial toxins botulinum neurotoxin from Clostridium botulinum and anthrax toxins from Bacillus anthracis as well as the plant toxin ricin produced by Ricinus communis. A common feature, apart from their extreme toxicity, is that they are composed of 2 polypeptide chains, one of which is responsible for cell uptake and another that has enzymatic function with the ability to destroy basic cellular functions. These toxins pose a threat, both regarding natural spread and from a terrorism perspective. In order for public health and emergency response officials to take appropriate action in case of an outbreak, whether natural or intentional, there is a need for fast and reliable detection methods. Traditionally, large molecules like proteins have been detected using immunological techniques. Although sensitive, these methods suffer from some drawbacks, such as the risk of false-positives due to cross-reactions and detection of inactive toxin. This article describes recently developed instrumental methods based on mass spectrometry for the reliable detection of botulinum neurotoxins, anthrax toxins, and ricin. Unequivocal identification of a protein toxin can be carried out by mass spectrometry-based amino acid sequencing. Furthermore, in combination with antibody affinity preconcentration and biochemical tests with mass spectrometric detection demonstrating the toxin's enzymatic activity, very powerful analytical methods have been described. In conclusion, the advent of sensitive, easily operated mass spectrometers provides new possibilities for the detection of protein-based toxins.