Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin’s activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices.     

Toxicokinetics of soman in cerebrospinal fluid and blood of anaesthetized pigs

The toxicokinetics of the four stereoisomers of the nerve agent C(±)P(±)-soman was analysed in cerebrospinal fluid (CSF) and blood in anaesthetized, spontaneously breathing pigs during a 90-min period after injection of soman. The pigs were challenged with different intravenous (i.v.) doses of C(±)P(±)-soman corresponding to 0.75–3.0 LD₅₀ (4.5, 9.0 and 18 μg/kg in a bolus injection and 0.45 μg/kg per min as a slow infusion). Artificial ventilatory assistance was given if, after soman intoxication, the respiratory rate decreased below 19 breaths/min. Blood samples were taken from a femoral artery and CSF samples from an intrathecal catheter. The concentrations of the soman isomers were determined by gas chromatography coupled with high resolution mass spectrometry. All four isomers of soman were detected in both blood and CSF samples. The relatively non-toxic C(±)P(+) isomers disappeared from the blood stream and CSF within the first minute, whereas the levels of the highly toxic C(±)P(−) isomers could be followed for longer, depending on the dose. Concurrently with the soman analyses in blood and CSF, cholinesterase (ChE) activity and cardiopulmonary parameters were measured. C(±)P(−) isomers showed approx. 100% bioavailability in CSF when C(±)P(±)-soman was given i.v. as a bolus injection. In contrast, C(±)P(−) isomers displayed only 30% bioavailability in CSF after slow i.v. infusion of soman. The ChE activity in blood decreased below 20% of baseline in all groups of pigs irrespective of the soman dose. The effect of soman intoxication on the respiratory rate, however, seems to be dose-dependent and the reason for ventilatory failure and death. Artificial ventilation resulted in survival of the pigs for the time-period studied. Received: 3 March 1998 / Accepted: 5 May 1998     

Characterization of Ricin and R. communis Agglutinin Reference Materials

Ricinus communis intoxications have been known for centuries and were attributed to the toxic protein ricin. Due to its toxicity, availability, ease of preparation, and the lack of medical countermeasures, ricin attracted interest as a potential biological warfare agent. While different technologies for ricin analysis have been established, hardly any universally agreed-upon “gold standards” are available. Expert laboratories currently use differently purified in-house materials, making any comparison of accuracy and sensitivity of different methods nearly impossible. Technically challenging is the discrimination of ricin from R. communis agglutinin (RCA120), a less toxic but highly homologous protein also contained in R. communis. Here, we established both highly pure ricin and RCA120 reference materials which were extensively characterized by gel electrophoresis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS), and matrix-assisted laser desorption ionization–time of flight approaches as well as immunological and functional techniques. Purity reached >97% for ricin and >99% for RCA120. Different isoforms of ricin and RCA120 were identified unambiguously and distinguished by LC-ESI MS/MS. In terms of function, a real-time cytotoxicity assay showed that ricin is approximately 300-fold more toxic than RCA120. The highly pure ricin and RCA120 reference materials were used to conduct an international proficiency test.     

Species-dependent variation in algal sensitivity to chemical compounds

Nineteen miscellaneous chemical compounds were tested on thirteen freshwater algal species grown in 250-microliter liquid cultures on plastic microtitration plates. It was demonstrated that the species-dependent variation in algal sensitivity (EC100) may reach over three orders of magnitude, the degree of variation depending on the chemical tested. No generally sensitive or generally insensitive species could be identified. An effort was made to quantify the predictive value of algal test batteries, at a given confidence level with respect to chemicals. The predictive value of small test batteries was low. Although it did increase with size it took the presence of more than nine members in the test batteries to improve the value to 0.1. This means that a nine-membered test battery might underestimate the sensitivity of the most susceptible algae by a factor of 0.1. A predictive value of 0.01 requires a three-membered test battery when accounting for 95% of the chemicals and a five-membered battery at the 99% confidence level. Implications for toxicity test strategies are discussed and it is suggested that algal test batteries, using simple test techniques, replace single unialgal cultures in routine work.