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LC-ESI-MS/MS draws the line on doping The thought of having to pedal 3408 km—17,300 m—uphill over 21 days is enough to make any cyclist gasp for air. That’s why some Olympic riders resort to using illegal or banned performanceenhancing drugs for that extra burst of energy. But in this issue of Analytical Chemistry (pp 2955–2961), Mario Thevis and Wilhelm Schänzer at the German Sport University (Germany) and his colleagues Rachel and Joseph Loo at the University of California–Los Angeles introduce LC-ESI-MS/MS as a new method for detecting some performance enhancers such as Hemopure. Hemopure, which is banned by the International Olympic Committee, rode into the national spotlight with the doping scandal at the 2001 Giro d’Italia cycling marathon. The drug is based on bovine hemoglobin, a hybrid of natural hemoglobin (Hb) intra- and intermolecularly cross-linked with glutaraldehyde, with an average molecular weight of
~250,000 (normal Hb has a molecular weight of ~64,000). The cross-linked structure maintains the hybrid Hb’s stability, and its presence increases oxygen transport capacities, thus giving the athlete longer endurance and strength. Although cyclists routinely take urine tests, “the problem,” says Thevis, “is that urine tests do not uncover Hemopure misuse.” Previous detection methods for cross-linked Hb relied heavily on size exclusion chromatography. Australian researchers identified cross-linkers in Hemopure but lacked an analysis from plasma samples. “Until the introduction of our LC-ESI-MS/MS procedure, no mass spectrometric assay for the detection of cross-linked hemoglobins was available,” says Thevis. Hemopure can be distinguished from human Hb because the amino acid sequence of its subunits differs by ~15%, resulting in a unique and easily identifiable set of peptides. The method can iden-
tify peptides originating from bovine Hb at concentrations much lower than those expected in human plasma after a recommended dose of Hemopure, says Thevis. According to the researchers, the new application also promises to be more accurate than previous methods. The scientists tested 100 plasma samples using LC-ESI-MS/MS methods and positively identified bovine Hb in all samples containing Hemopure; no interfering signals were observed. The ability to reliably detect cross-linked hemoglobins such as Hemopure is sure to flatten the tires of athletes who use such banned drugs to get their “second wind”. a —Wilder D. Smith
MEETING NEWS Knowledge Foundation’s 3rd International Symposium on Biodetection Technologies—Arlington, Va.
Wilder D. Smith reports from the
Commercial HHAs fail the test In light of the 2001 anthrax attacks, the analytical instrument market has been flooded with the demand for smaller, hand-held commercial biodetection products. However, according to a joint study released by the FBI and the Centers for Disease Control and Prevention (CDC), today’s commercial hand-held immunoassays (HHAs) are hindered by a 3–83% false-positive rate when detecting virulent strains of Bacillus anthracis, the causative agent of anthrax. Lawrence Kerr, director of bioterrorism, research, and development at the Office of Science and Technology of the
Department of Homeland Security, says, “This type of information is critical to first responders and key decision makers who must understand the limitations of the devices they use.” In the FBI–CDC study, the sensitivity, specificity, repeatability, robustness, and stability of six HHAs were judged to determine their ability to identify four different infectious strains of B. anthracis and two strains of Yersina pestis, which causes pneumonic plague (Forensic Science Communications 2003, 5, www.fbi.gov/hq/lab/fsc/backissu/ jan2003/fsru.htm). The detection limit for washed anthrax spores—washing is a step in preparing the spores as a biological
weapon—varied from 105 to 108, whereas only 8000–50,000 spores for inhalation anthrax are needed to cause disease. The detection limit by the HHAs for the two Y. pestis strains fared better with a detection limit of 104–105 (100–500 microorganisms are needed to cause pneumonic plague). However, results also revealed a major problem in that the HHAs could detect Y. pestis grown at 37 °C but not at room temperature. Some alternatives to HHAs give fewer false positives. Microbiological cultures are considered to be the “gold standard” for bacterial identification. “The limitation is that it requires many hours, sometimes days, to culture the organism,” says Kerr.
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MEETING NEWS
51st American Society for Mass Spectrometry Conference—
Katie Cottingham reports from the
Montreal, Canada
New LC-MALDI interfaces drop onto the scene Most automated systems for depositing LC fractions onto MALDI plates touch a tip onto the surface of the target, which can cause cross-contamination and damage to both the surface and the tip. Now, two research groups have developed technologies to avoid touching the tip to the plate after separating samples by LC. Ansgar Brock and colleagues at the Genomics Institute of the Novartis Research Foundation use a pulsed electric field to polarize droplets at a micro- or nano-LC column outlet and transfer them to the plate surface. Brock says, “We thought, ‘What’s the simplest way of getting a droplet to drop down from a tip to a plate?’” They remembered that, at the initial stages of electrospray, droplets at the spray tip jump to a counter electrode, and they adapted this idea for MALDI plate deposition. “There are no mechanical parts, which makes it very robust,” says Brock. In addition, the plate is coated with a hydrophobic/hydrophilic surface that helps concentrate the sample (1). Liang Li and co-workers at the University of Alberta (Canada) wanted to use a higher flow rate LC method, such as microbore- and nanobore-column HPLC. More sample can be loaded onto these columns, which extends the dynamic range, but there’s a lot of solvent, too. Li’s group created an interface in 338 A
charged ions that react with multiply charged positive product ions in the trap. Every product ion then becomes singly charged, which makes the (a) (b) job of deducing masses from the measured m/z (a) A non-contact deposition device and (b) a heated droplet values much easier. interface for spotting MALDI plates. Detlev Suckau and Anja which a capillary tube connected to the Resemann of Bruker Daltonik (GerLC column is encased within a stainless many) address the problem of analyzing steel heated tube. They heat the interthe amino- and carboxy-termini of proface to evaporate some of the solvent in teins with a new top-down method the hanging droplet just before it falls called T3 sequencing. They ionize a onto the MALDI plate, which is also whole protein and fragment it twice heated “to rapidly evaporate any remain- using a TOF-TOF mass spectrometer ing solvent,” says Li. The temperature is in a “pseudo MS3 experiment” to anacarefully controlled, something Li says is lyze modified terminal ends and obtain important for drop deposition. “If the structural information. temperature of the heated MALDI plate “The idea came up when we realized is too low, the droplets just accumulate that we have two types of fragmentain one area, forming a big spot,” he says. tions that we can use on the MALDI But if the plate is too hot, “the liquid TOF-TOF: a fast process on the nanodroplet slides away!” With this method, second timescale inside the source [inLi’s group can produce spots of
