Robert Barker/Cornell University

Toxicologists Jack Henion, left, and Joseph Ebel with an LC/MS/MS instrument in Cornell's Analytical Toxicology Laboratory.

Substances that are poisonous to animals can now be subjected to the same chemical scrutiny given to materials in high-profile human cases – including the O.J. Simpson murder trial. That's because the Cornell University College of Veterinary Medicine has equipped its Analytical Toxicology Laboratory for the 'gold standard' in forensic testing: LC/MS/MS, or liquid chromatography tandem mass spectrometry.

The state-of-the-art instrumentation already has proved its worth by detecting and confirming the presence of a horse-killing chemical that other established analytical techniques could not positively identify, according to Jack Henion, director of the laboratory and a professor of toxicology at Cornell. With his colleagues, Joseph Ebel, director of laboratory operations, Kerry Manzell, research support specialist, and a research group headed by Timothy Wachs, the team is responsible for detection and characterization of toxic substances that cause poor health or death in animal cases brought to the New York State College of Veterinary Medicine.

"LC/MS/MS can tell us – with scientific certainty that stands up in court -- whether even the smallest amount of a particular chemical is present in animal tissue or other biological samples. And just as important, the technique can determine if a chemical is not present," Henion explains, displaying a gleaming white machine that resembles a 21st century vacuum cleaner. "This is truly the gold standard of forensic toxicology."

The pride of the Cornell Analytical Toxicology Laboratory is an API 2000 LC/MS/MS system, on loan to Cornell from PE Biosystems of Foster City, Calif. The $200,000 machine in the toxicology laboratory, a unit of the Department of Population Medicine and Diagnostic Science, incorporates patented technology (an atmospheric pressure ionization LC/MS interface) that was perfected at Cornell's toxicology research-and-development laboratory.

Henion and his research colleagues have worked to develop better analytical tests for everything from illegal drugs in racehorses' urine to a telltale substance in a retired football player's blood. Henion's expertise makes the toxicologist a sought-after consultant and expert witness in court.

The combination of liquid chromatography (LC) with tandem mass spectrometry (MS/MS) provides a significant advance in analytical capability, Henion explains. "The LC component of this combination provides online separation of chemicals in a complex mixture such as a urine or blood samples, followed by subsequent online transfer of these separated chemicals directly to the tandem mass spectrometer for detection and confirmation. This combined LC/MS/MS system readily provides both identification of chemicals as well as an indication of how much chemical is present – which we call quantitative analysis. Both of these pieces of information can be very valuable in a forensic or toxicology case."

An important feature of this new system is the Cornell-developed "ion spray" LC/MS interface. A variant of so-called "electrospray," this has revolutionized the use of atmospheric pressure ionization mass spectrometers during the last decade, according to Henion. He notes that the device allows scientists at Cornell and a few other laboratories to measure intractable chemicals that are not possible to characterize with established instrumentation.

Recent advances at Cornell and elsewhere are allowing laboratories to conduct LC/MS/MS tests with greatly increased sensitivity and accuracy, as well as in much greater volume -- a goal called "high throughput analysis." In July 1999, Henion's laboratory published a record-breaking achievement when it successfully tested 1,152 samples of human urine for traces of benzodiazepine-type drugs on one LC/MS/MS machine in 12 hours. More recently, the Cornell lab has raised its testing output to more than 2,000 samples per machine daily.

But selectivity and sensitivity, not rapidity, was the issue recently when the Cornell toxicology lab tested its latest LC/MS/MS system on a case that occurred in 1998 involving 100 horses at a neighboring institution. Several animals went off their feed and many became ill with severe colic in a 12-hour period after eating a newly delivered batch of feed. These symptoms were consistent with ionophore antibiotic toxicity in horses. The Cornell department is authorized by the State of New York as the official laboratory for these tests, but without the latest equipment, the analytical chemists could not unequivocally confirm what had affected the horses.

Using the new API 2000 LC/MS/MS instrument, they positively identified the suspect culprit: the antibiotic compound, monensin, which is routinely added under various trade names to poultry and cattle feeds as an antibiotic and a growth promoter. Monensin has no business being in horsefeed, Henion explains, because horses are extremely sensitive to very small amounts of the chemical, even though is tolerated by chickens and cows.

It is entirely appropriate, Henion comments, that a horse-poisoning case should be solved by technology that was developed, in part, to serve the species. These developments will help forensic toxicology for humans, too, Henion says, pointing to a couple of notorious cases:

• The journal Analytical Chemistry reported one dramatic incident under the headline, "A Quest for Oleandrin in Decayed Human Tissue" (the case also was recounted by another author in the book A Family Business). The story detailed experimental results from the Cornell lab that might have resulted in the death penalty. A California mortician had suffered cardiac arrest and, although young, he was overweight and overstressed so the medical examiner's ruling of death by "natural causes" closed the case.

But then a former employee of a crosstown funeral director claimed that his boss had killed his competitor with oleandrin, an extract of the deadly oleander plant. The mortician was arrested, and authorities ordered an analysis of tissue samples. Exhumation was easy enough, Cornell's Henion recalls, because the body was entombed above-ground in glass. But embalming had failed to halt decomposition, and the samples were badly decayed when they reached Ithaca. Cornell toxicologists found no oleandrin and no sign of a related compound, oleandrigenin. The accused man went free, and forensic scientists had learned the value of modern analytical methodology applied to the analysis of decayed tissue, Henion notes.

• Followers of the O.J. Simpson trial learned the terms "liquid chromatography" and "mass spectrometry" from lead prosecutor Marcia Clark. She tried to persuade the jury that blood in socks and elsewhere at the crime scene came directly from Simpson and not from police, whom the defense accused of tampering with evidence. The telltale chemical was EDTA (ethylenediaminetetraacetic acid), an anticoagulant often found at very low levels in human blood.

EDTA also is found in much greater quantities in blood collected by police in special tubes that contain the anticoagulant as a preservative chemical. If blood at the scene contained comparatively high levels of EDTA, the defense could claim that police had planted previously collected blood to frame the suspect. Tests performed by the FBI lab found only trace levels of EDTA in Simpson's blood, a fact that bolstered the prosecution's case, and Henion prepared to fly to Los Angeles and explain these results in court.

But at 2 a.m. Ithaca time, Henion's phone rang. Don't bother, he was told. Judge Ito had heard enough science. So, apparently, had the jurors, who ultimately disregarded DNA evidence, analytical chemistry findings and other high-tech science.

For the foreseeable future, LC/MS/MS research in support of human and animal forensic cases will continue to be conducted in Cornell's research laboratory also under the direction of Henion. The veterinary college's toxicology laboratory complements these research efforts by employing the latest techniques in support of veterinary forensic toxicology. This analytical toxicology service laboratory is one of the few public-service veterinary labs in the United States with the "gold standard" of analytical toxicology, Henion says. "And now poisoned animals get the best that modern science and technology can offer."