MALDI points to the origin of anthrax spores - ACS Publications

Jun 1, 2004 - MALDI points to the origin of anthrax spores. Methods for detecting the nerve agent. VX, one of the most toxic chemicals known, are need...
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Extracting the nerve agent VX from soil

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Methods for detecting the nerve agent VX, one of the most toxic chemicals known, are needed to track any illegal preparation, use, or stockpiling of it. In the May 15 issue of Analytical Chemistry (pp 2791–2797), Bruno Bellier and his colleagues at the Centre d’études du Bouchet (France) point out a flaw in commonly used procedures, such as those recommended by the Organization for the Prohibition of Chemical Weapons, for extracting VX from soil. The results suggest that VX is more persistent in soil than previously thought. VX has long been thought of as an unstable compound that only leaves degradation products in soil. Bellier and his co-workers, however, show that VX is relatively stable and can be recovered from soil up to three months after contamination. Bellier says, “The [formerly described] methods did not recover VX not because it degraded, but because the [extraction procedures] were not suitable.” Solvents in the previously described methods generally can’t dislodge VX from soil unless the contamination level is high (0.1–10 mg/g). In contrast, the technique developed by Bellier and colleagues can extract VX from soil at spik-

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The nerve agent VX can be successfully recovered from neutral silt loam (red) and acidic sandy silt loam (blue) soils for up to 3 months following contamination.

ing concentrations as low as 1–10 µg/g. The soil is mixed with a buffer with a pH close to VX’s pKa of 8.8. When the soil is mixed with an alkaline buffer, the nitrogen atom in the VX loses its positive charge and interacts less with the soil particles. The buffered soil is then mixed with hexane/dichloromethane, allowing VX to dissolve into the organic phase. The organic phase is concentrated and introduced into a GC column for separation and detection. Bellier and his colleagues have successfully applied the technique on three different types of soils—a neutral silt

loam, an acidic sandy silt loam with high organic content, and a commercially available sea sand. They have also shown that their technique can extract the former Soviet Union’s VX analogue from soil. Most importantly, Bellier and his colleagues have demonstrated that VX can be isolated from soils that have been contaminated for up to 3 months. As Bellier points out, “When you send inspectors to a suspected area, it’s generally a long time after an event has occurred. It’s very important to know whether you are able to find traces of a past activity.” a —Rajendrani Mukhopadhyay

MALDI points to the origin of anthrax spores In light of the anthrax-laced letters that have sorted their way into post offices, mailboxes, and legislative offices across the United States since 2001, the use of Bacillus anthracis spores as a biological weapon has become a common threat to the American public. Although the combination of analytical and forensic techniques has allowed scientists to better detect and characterize anthrax and its production from other biological agents, the challenge remains to find the culprit of the terrorist act. In the May 15 issue of Analytical Chemistry (pp 2836–2841), Jeffrey 194 A

Whiteaker and Catherine Fenselau of the University of Maryland, College Park, and colleagues at the Federal Bureau of Investigation describe a MALDI-TOFMS method that can determine the environment that was used to grow Bacillus spores. Forensic detectives are hoping that this type of determination will ultimately be an important step in leading them to the terrorists. Whiteaker and colleagues hypothesized that because spores are often grown on a blood-supplemented medium known as blood agar, analysis of the spores could contain information about how they were

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prepared. The researchers focused on the detection of the hydrophobic compound heme (ferriprotoporphyrin IX), which is released from red blood cells during the spore-forming process. “We’re not using MALDI to identify the biological agent; our method is aimed at characterizing a sample forensically to determine if blood agar was used in preparation,” says Whiteaker. There were several reasons, say the researchers, for using MALDI. The technique offers a fast and sensitive method for heme analysis. In addition to requiring little sample handling and preparation,

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MALDI is tolerant to a high level of contaminants, allows quantitative analysis, and can provide structure-specific information for identification. One important factor in the quantitative analysis of heme is having an internal standard that is chemically identical to the analyte, or as close as possible, says Whiteaker. “We evaluated several protoporphyrin compounds with chemical structures similar to heme and found a cobalt (III) protoporphyrin IX compound to give the best performance,” he says. The researchers used the MALDITOFMS method to analyze spore sam-

ples grown on blood and nonblood agar. The samples were prepared using a modified sandwich deposition technique, in which a layer of matrix crystals was placed on the target spot and dried via fast evaporation. A mixture of the sample and matrix was then placed on top of the matrix layer. The resulting mass spectra from spores grown on blood agar showed a peak at m/z 616.2, but this peak was absent in spores prepared without blood agar. The results show that heme can be detected in sporulated samples prepared

on blood agar but not in those prepared without blood. The researchers estimate that 0.5–1.0 mg of purified spores are needed for the qualitative determination of heme. For quantitative purposes, they estimate that ~5 mg of spores are needed. Whiteaker believes this is only the first step in the forensic use of MS for biological agent detection. “While our method uses a marker for the use of blood agar, it may be possible to find other markers that tell us more about how a sample was prepared,” he says. a —Wilder D. Smith

Budapest, Hungary. He left Hungary in 1956 and worked for a few years at Farbwerke Hoechst AG in Frankfurtam-Main. He later did his doctoral research on porous-layer open-tubular GC columns under the supervision of István Halász. He then moved to the United States where, under the direction of S. R. Lipsky in the School of Medicine of Yale University, he began working on the development of analytical methodologies for lunar samples. This led him to build the first instrument for HPLC. After becoming a professor in the chemical engineering department of Yale University, Csaba devoted all of his attention to the development of the theory and the applications of reversed-phase LC. His landmark paper, “Solvophobic interactions in liquid-chromatography with nonpolar stationary phases” (J. Chromatogr. 1976, 125, 129), has been cited 985 times (ISI Web of Knowledge). He also is the author or coauthor of 300 publications (10% of them have been cited more than 100 times since their publication) and has presented hundreds of lectures at meetings and seminars all over the world. He was deeply interested in philology and forged numerous words. Some (e.g., isocratic and multimodal) have become so common that their origin is lost; others still delight his friends, such as the four ailments of analytical biologists—lithophobia, sidero-

phobia, barophobia, and adiaphanophobia— the fear of silica, the fear of steel, the fear of pressure, and the fear of devices that are not transparent. Among the numerous honors that Csaba received are the Dal Nogare Award of the Forum of the Delaware Valley (1978), the Humboldt Award for Senior American Scientists (1982), the Chromatography Awards of the American Chemical Society (1983) and the Eastern Analytical Symposium (1986), the Separation Sciences Award of the American Chemical Society (2001), and the I. Halász Medal Award of the Hungarian Separation Science Society (1997). The Halász Award was particularly dear to his heart, as was the Honorary Doctorate received earlier from his alma mater, the Technical University of Budapest (1986). In January 2004, Csaba was elected a member of the U.S. National Academy of Engineering. While his work has had a profound influence on the development of analytical biochemistry, advancing the progress of the life sciences for the past 30 years, his influence on an immense number of scientists has been even greater. The grief of the “Csabaites” community is heartbreaking, and we share it. Csaba Horváth’s memory will live forever in all of us. a —Georges Guiochon and Lois Ann Beaver

PEOPLE Csaba Horváth (1930–2004) Csaba Horváth, the Roberto C. Goizueta Professor of Chemical Engineering at Yale University and one of the founders of HPLC, died on April 13 in New Haven, Conn. He was 74 years old. Csaba invented porous-layer open-tubular GC columns and designed and built the first high-performance liquid chromatograph and the first microbore HPLC columns (for ion-exchange separations of biological compounds). Csaba pioneered the use of reversedphase HPLC, the most widely applied chromatographic method of analysis; the use of displacement chromatography for preparative HPLC; and innumerable applications of HPLC to the separation of samples of biological origin. He developed the solvophobic theory of retention in reversed-phase LC, the use of entropy–enthalpy compensation in the study of retention mechanisms, and the fundamentals of electrochromatography. Csaba graduated as a chemical engineer from the Technical University in

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