would provide a specific assay for Cryptotime a highly sensitive assay for infecsporidium, ,ad ESI would dffer the eaditious Cryptosporidium. tional benefits of enhanced sensitivity and To their disappointment, repeated Although the mention of microorganisms reduced sample preparation. Burkhalter applications of this methodology recan conjure up images of biological weap- set out to find the endogenous phosphovealed that the unique fatty acid was, in lipid, which was previously thought to be ons or terrorist attacks, microorganisms actuality, an artifact added occasionally acylated at the sn-2 position of phosphatiare more likely to affect the average perfrom amber rubber bulbs in the extracson in food or water. The protozoan Cryp- dylethanolamine. However, parent ion tion/purification procedures. This ffndtosporidium has been particularly difficult scans revealed the presence of no signifiing was unfortunate because the identicant precursors. Neutral mass loss scan to detect with the sensitivity needed to fication oo fa nxtremely unusual fatty revealed that no phosphatidylethanolamine acid unique to species of Cryptosporidprotect human health. One of the challenges is in differentiating infectious from was present in the fraction containing ium would have allowed for the detecnon-infectious Cryptosporidium, espe- 10-OH 18:0. tion of this opporrunistic pathogen, cially when 20-30 oocysts has been such as a water distribution system's Burkhalter and co-workers eventually identified as an infectious dose. D. C. biofilms, without the prerequisite of determined mat 10-OH 18:0 did exist in White Rob Burkhalter, and co-workCryptosporidium samples, but as a sree fatty oocyst purification and identification. acid, which is extremely rare in nature. the University of Tennessee are Despite the artifactual nature of Low-energy collisionally induced dissociaworking on a mass spectrometric 10-OH 18:0, the research showed that it tion conditions for the selected reaction method for distinguishing infectious is possible to differentiate infectious and monitoring (SRM) of daughter ions of and non-infectious species. Their non-infectious Cryptosporidium parvum, 10-OH 18:0 were optimized for the produc- as well as differentiate C. parvum from search for a uniaue biomarker has tion of ions diagnostic of the site of hybeen interesting but so far unfruitful other species of Cryptosporidium and Eidroxyl substitution. It was found that under meria through hierarchical cluster analyHowever they have been able to use cluster analysis to differentiate species these low energy conditions, highly spesis of lipid patternsfrompurified oocysts. cificfragmentationprocesses were occurof Crvttos-boridium The current problem is the difficult purifiring, which provided in a much more sen- cation procedures required to isolate Earlier studies had seemed to link a Cryptosporidiumfromother waterborne novel fatty acid, 10-OH 18:0, to Crypto- sitive SRM assay because of the lesser microeukaryotes whose lipid patterns sporidium parvum. Burkhalter and dis extent of nonspecific fragmentation typical of high-energy CID studies. This reobscure those of infectious Cryptosporidco-workers had hoped to transfer the sulted in a highly sensitive and specific ium parvum. For these eeasons, efforts methodology for detection of the susfor the presence of 10-OH 18:0 in continue toward identifying unique biopected biomarker to HPLC/electrospray complex biological matrices and thus it marker patterns for Cryptosporidium and MS. Identification of the endogenous was thought to have provided for,the first other non-culturable pathogens phopholipids containing 10-OH 18:0
Going after Cryptosporidium
array detector for the simultaneous measurement of different mass ions. He has developed a miniature mass spectrometer of Mattauch-Herzog geometry, which is just 10 x 5 x 4 cm. .I measures ions up to 240 m/z, possesses a resolution of >300, and consumes only 1-2 W. Two kinds of array detectors have been developed. The first is based on an ion-electron-photon conversion process. The ion images are measured with a photodiode or a CCD array. The second approach employs 3. multianode system, which is fabricated along wiih pulsecounting electronics on a silicon chip. In addition, efforts are being made at other institutions to design mass spectrometers for cometary research. The International Rosetta Mission, sponsored by the European Space Agency and slated for launch in January 2003, is expected to deploy a spacecraft that will rendezvous with comet 46 P/Wirtanen and escort it for two more years. Hans Balsiger, Stefan Scherer, and co-workers at the University of Bern
(Switzerland), the Southwest Research Institute (Austin, TX), the Max-Planck Institut fur Aeronomie (Germany), and the University of Giessen (Germany) described a prototype reflectiontime-of-flight(RTOF) mass spectrometer that will be part of the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) package. The package wiil also include a double-focusing mass spectrometer and a "neutral dynamics monitor". The RTOF is ~ 1 ml with a flight tath oo 2 m when ions are reflected only once. The design also incorporates a "hard mirror" that can effectively double the flight path. The hard mirror consists of three cylindrical elements and a shaped backplane which be used to remove selected mass lines by applying a positive pulse to the backplane electrode Mass resolution of up to 5700 (132Xe) has been achieved and the mass accuracy is —10 oom The RTOF is compact only 1134 x 309 x 252 mm and 10 4 kg It consumes 13 4 W in the low nower mode and 23.0 W in the full operation mode.
GOVERNMENT AND SOCIETY
Eggsacting standards A review by U.K. government scientists of techniques used to estimate veterinary drug residues, such as ionophores for preventing coccidiosis in poultry, has shown there to be no reliable way of correlating residue levels with duration or amount of medication or whether there has been any cross contamination or misuse of the drugs. Christopher Elliott and colleagues at the Veterinary Sciences Division in Belfast, a European Union drug-residues monitoring unit, say that with the current analytical methods there is no satisfactory way to determine residue levels of all of the widely used ionophore drugs in the poultry industry. "MRLs [maximum residue limits] do not exist for these drugs [the ionophores]
Analytical Chemistry News & Features, August 1, 1998 5 0 5 A
News
LABORATORY PROFILE Nothing fishy at FDA Seafood Laboratory Approaching the FDA Seafood Laboratory in Bothell, WA, the clearest landmark is a red brick Microsoft building. Across the way, the FDA building is nondescript by comparison. It's fitting. Despite the northwest's historical reliance on the seafood industry, high tech and computers dominate the local economy today. While Microsoft captures the spotlight, its neighbor quietly works to protect and promote public health while encountering what all federal agencies face—reduced resources. A boon in the late 1970s led to the establishment of numerous FDA field centers, bringing the total to seven, according to Marleen Wekell, director of the Seafood Products Research Center. But that number is now down to two, and by the year 2000, field research could be managed differently than it is today. The FDA seafood center's research lab complements the regulatory lab that was established in 1937, which will become one of five FDA megalaboratories. Other changes are also likely on their way. For example the research lab could be eliminated as a free-standing research center. That wouldn't elimiate research entirely says Susan Hutchcroft director of public relations but it could reduce the amount of effort put into it A mega-regulatory lab with the additional responsibility of conducting research may not trive it the full attention that a stand-alone research lab wnnld she contends T h e refriilatory lnh can hanrllp- outbreakc. of known toxinc. a n r l m i c t - o f i p c u n t i l tVie w i r l e ranrYP l\{ rt\t*cA
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animal model methods. But when unrecognized symptoms signal a novel outbreak, a research team must quickly locate and identify the culprit. It is important mat the FDA maintains a strong research base, says Weckell. "You need those kinds of brains in research in the government. It gives the agency credibility when it publishes in journals and can hold its own in scientific meetings," says Weckell. The greatest challenge facing the research center is taking leading-edge analytical technology and making it robust enough to stand up in court. Also, the matrix of the sample—not the sensitivity of the measurement—raises the biggest barriers, says Wekell. In one project, the research lab is applying PCR techniques to determine whether toxin genes are present in particular isolates of E. coli. But the food being tested often contains inhibitors to the PCR enzymes, requiring extensive pretreatment with columns or even additives such as dried milk according to Karen Jinneman a microbiologist in the research center. What difference the latter makes is unclear but it seems to do the trick Ironically, just as the research center faces potential cutbacks, one of its projects was honored by vice-president Al Gore in his campaign to reinvent government. Inspired by a NASA hammer that carried a $400 price tag, Gore's Hammer Award recognizes quality federal projects produced on the cheap. Seafood lab research chemist Brad Tenge, together with a team of FDA scientists from across the agency, initiated the regulatory fish encyclopedia—available online at http://vm.cfsan..da.gov/~frf/ rfe0.html—in 1993 to give inspectors and fishing industry officials a tool to prevent economic fraud. The site includes photographs, safety information, and gel electrophoresis isoelectric focusing data of muscle proteins that can be used to identify various species. FDA officials can also use specially developed pattern-recognition software to compare their own isoelectric focusing data to that in the database. Tenge says this feature will soon be available for public use. On the short list to add to the site: DNA restriction fragmentation length polymorphism (RFLP) data. Muscle protein analysis works well for fresh or frozen fish, but the proteins denature during cooking, clouding the analysis. In contrast, DNA weathers the frying pan, making it a good
Analytical Chemistry News & &eatures, August t, 1199
Preparing a PCR analysis for an unknown pathogen.
complement to the muscle protein data already in place. Tenge would like to see the resource grow to include up to 150 species of fish but expects the addition of new fish to come at a slower pace. New species will be added with an emphasis on those that carry greater health hazards—such as ones with a higher likelihood of carrying a natural toxin or a species prone to mishandling. That change in emphasis reflects a larger trend at FDA 'We have been told to focus more on public health and to de-emphasize economic fraud," says Jim Ahlrep, supervisory chemist in the regulatory lab. A primary focus is known, and emerging hazards such as toxins and microbes, which could place additional stress on reduced research resources. That, in turn, could reduce expenditure on innovative projects such as the fish encyclopedia, which is partially supported by the research budget. In any event, don't expect a tome the size of Microsoft's Encarta to appear overnight. "That's the advantage of a Web resource^—we can update it over a period of time," says Tenge. Al Gore—and even Microsoft—would no doubt approve. Jim Kling
Monensin, a common treatment to prevent coccidiosis.
within the European Union, yet [the drugs] are used globally," he says. He suggests that without stringent regulations and enforcement there is a very real possibility that residues might reach appreciable concentrations in produce. Legislation is expected to come into play later this year, which should help avoid this circumstance, but the lack of a fast and effective analysis could preclude effective implementation of the law. The protozoal infection coccidiosis is widespread in intensively farmed poultry and can cause bloody diarrhea, reduction in feed and water consumption, emaciation and loss of egg production and eventually death. The damage is often done before symptoms are spotted. So, a precautionary approach rather than a treatment is used, with farmers applying a blanket drug regime to their stock with various coccidiostatic preparations such as carboxylic acid ionophores like monensin and lasalocid. Rotation is used to minimize the risk of resistance. European Union and U.S. legislations strictly limit drug residues allowable in produce and, according to Elliott, for most compounds a two-tier screening and confirmatory testing system provides an efficient cost-effective approach. The European Union is soon to include ionophores in its guidelines, and, in anticipation, Elliott and his team set out to assess the available analytical procedures. The original methods for monensin are in vitro assays, which require lengthy incubation periods and so are not viable for lowcost routine analysis. Although currently used chromatographic techniques provide detection limits an order of magnitude lower than needed (at some 25 ug/kg), HPLC methods have deficiencies in detecting the numerous ionophores used in poultry farming.
Elliott's team also canvassed opinion from the regulatory laboratories involved in coccidiostats and found virtually unanimous agreement that the current analytical methods need improvement. There has been a recent move towards ELISA techniques for the initial screening, whereas LC/MS can be used in the confirmatory step of the two-tier testing; but there is, says Elliott, "a lack of fast and reliable multiresidue methods available." There is an urgent need to develop suitable tests. Currently it is very difficult to determine if ionophore residues detected in pig exactly the significance of such residues to the consumer. David Bradley
Laboratories that wish to participate in SETOC pay a fee of 1500 Dutch Guiiders (—730 USD) and select a code name. Every three months, participants receive four dried sediment samples, which they can analyze for the analytes of interest by any method they choose. The analytes evaluated so far have included polycyclic aromatic hydrocarbons, polychlorinated biphenyls, organochlorine pesticides, mineral oil, organic and inorganic carbon, and some heavy metals. Beginning this year, samples with detectable amounts of dioxins and furans will be included. The organizers at Wageningen calculate the median value and the median of the absolute deviation of the submitted analyses. Results are published every three months with codes to indicate the performInternational ing laboratory and the techniques used. round-robin for SETOC is only one of the programs run dioxins and furans by Wageningen. The three other international round-robins are the International Soil and plant analysis is a demanding task Plant Analytical Exchange Program (222 because of the heterogeneity of the materiparticipants), the International Soil Analytials, the large number of analytes of interest, and, in many cases, the low concentrattons. cal Exchange Program (300 participants), and the International Manure and Refuse Analytical results obtained by different laboratories may vary widely, and all labora- Exchange Program (54 participants). tories can benefit from some sort of outside Despite the participation of many facilievaluation. The need is even greater if diox- ties in SETOC, only approximately 1% oo the ins and furans must be analyzed reliably. participating laboratories are located in the United States. Programs run by the Council Currently, no international round-robin for these classes of compounds exists. How- of Soil and Plant Analysis and by AOAC Inever, that will change by autumn 1998 with a ternational may reduce participation by U.S. laboratories. Another problem is the existing program at the Agricultural University of Wageningen (The Netherlands), where one restrictions by the U.S. Customs on the imof the largest international programs of labo- port of plants and soils. ratory evaluation is located. The possibility Veronika R. Meyer of analyzing dioxins and furans is offered through the already existing international Sediment Exchange for Tests on Organic Contaminants (SETOC) program. The current push for a dioxin and furan round-robin was initiated by the Institute of Environmental Protection and Agriculture (Switzerland). The Swiss government requires that laboratories perform accurate analyses of these toxins; SETOC was chosen as the best platform to assess competence in dioxin and furan analysis. Victor Houba, the coordinator of SETOC and three other round-robins conducted at Wageningen, claims proudly: "We have accees to soil lamples with detectable amounts of these compounds!" The cost of the first or "reference" analyses of these soils will be paid by the Swiss government. Although 101 laboratories participate in SETOC Houba expects 15 laboratories from around the world to participate in the dioxin and furan round-robins. Making representative samples. Analytical Chemistry News & &eatures, August 1, 1998 5 0 7 A
