Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum.

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 a validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, 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 and 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 valid alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, and speed and that it does not require experimental animals.

Mass spectrometric detection of protein-based toxins.

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.

Bioanalytical investigation of asarone in connection with Acorus calamus oil intoxications.

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.

Bioanalytical and clinical evaluation of 103 suspected cases of intoxications with psychoactive plant materials.

INTRODUCTION: Problems associated with the increasing abuse of plant-derived psychoactive substances have recently attracted attention. This study involved bioanalytical and clinical examinations of intoxication cases suspected to be linked to such plant materials. METHODS: Urine samples were collected at emergency wards in Sweden from patients who either admitted or were suspected of ingestion of psychoactive plant materials. The bioanalytical investigation employed a liquid chromatography-tandem mass spectrometry multicomponent method covering 10 plant-derived substances (atropine, dimethyltryptamine, ephedrine, harmaline, harmine, ibogaine, lysergic acid amide, psilocin, scopolamine, and yohimbine) and a gas chromatography-mass spectrometry method for asarone. Routine testing for illicit drugs was also performed. RESULTS: Over a 4-year period, 103 urine samples collected from mainly young people (age range 13-52 years, median 19) were studied. Among 53 cases where ingestion of any of the 11 plant-derived substances covered in this study was admitted or suspected, 41 (77%) could be confirmed bioanalytically. Nine of the 11 substances tested for were detected, the exceptions being ibogaine and yohimbine. Psilocin, originating from ingestion of hallucinogenic mushrooms, was the most frequent drug accounting for 54% of the cases. The most common means of drug acquisition (56%) was purchase over the Internet. CONCLUSION: 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.

A multi-component LC-MS/MS method for detection of ten plant-derived psychoactive substances in urine.

A sensitive and specific LC-MS/MS method for simultaneous detection of 10 plant-derived psychoactive substances (atropine, N,N-dimethyltryptamine, ephedrine, harmaline, harmine, ibogaine, lysergic acid amide, psilocin, scopolamine and yohimbine) in urine was developed. Direct injection of urine diluted with 3 deuterated internal standards allowed for a readily accessible method suitable for application in clinical intoxication cases. Separation was achieved using reversed phase chromatography and gradient elution with a total analysis time of 14 min. Electrospray ionization was used and ions were monitored in the positive selected reaction monitoring mode. The calibration curves were linear (r(2)>0.999) and the total imprecision at high (1000 microg/L) and low (50 microg/L) substance concentrations were 4.9-13.8% and 8.3-26%, respectively. Infusing the analytes post column and injecting matrix samples showed limited influence by ion suppression. The multi-component method proved to be useful for investigation of authentic cases of intoxication with plant-derived psychoactive drugs and was indicated to cover the clinically relevant concentration ranges.

Development and clinical application of an LC-MS-MS method for mescaline in urine.

Mescaline (3,4,5-trimethoxyphenylethylamine) is an hallucinogenic psychoactive substance present in several species of cacti. Mescaline has a documented use dating back 5700 years. In more recent years, the interest in hallucinogenic designer drugs such as ecstasy has also triggered interest in the naturally occurring mescaline. This study was undertaken to develop a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the screening and confirmation of mescaline in human urine samples and to apply this method to routine testing in patient samples. For the screening procedure, chromatographic separation was achieved on a 5-microm HyPURITY C(18) column, using a methanol gradient in ammonium acetate buffer. The MS-MS analysis was performed using selected reaction monitoring; the transitions monitored were m/z 212.3 --> m/z 180.3 for mescaline and m/z 221.3 --> m/z 186.3 for the deuterated internal standard (mescaline-d(9)). The detection limit for mescaline in urine matrix was 3-5 microg/L, the upper limit of quantification was 10,000 microg/L, and the total coefficient of variation for spiked samples containing 10 to 1025 microg/L was < 8.5%. The confirmation procedure included a sample clean-up by solid-phase extraction on a C(18) cartridge, and one extra transition for mescaline (m/z 212.3 --> m/z 195.2) was monitored. The LC-MS-MS method was found to be sensitive and specific for the routine detection of mescaline in urine. Among 462 urine samples collected from young people with alcohol or drug problems, 32% were positive for illicit drugs, but none for mescaline.