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U.S. Department of Health and Human Services

Animal & Veterinary

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CVM Researchers Use Latest Science to Develop Methods for Detecting Animal Drug Residues

by Richard L. Arkin
FDA Veterinarian Newsletter January/February 2005 Volume XX, No I

Along with its responsibility to be sure that drugs are safe when they go on the market, the Food and Drug Administration (FDA) also has responsibility to be sure drugs are used safely. FDA’s Center for Veterinary Medicine (CVM) has found that the latest developments in science and refinements in lab equipment significantly help in that effort.

Improper use of drugs in animals may leave residues in animal-derived edible tissues that could be hazardous to consumers. CVM is responsible for assuring that significant residues of drugs that have been used to treat the animals are not present in human foods such as milk or other dairy products that come from animals or in tissues that become human food after slaughter, such as meat from swine and cattle.

Drug tolerance, withdrawal times

CVM and the U.S. Department of Agriculture’s Food Safety Inspection Service (FSIS) participate jointly in a program to monitor the use of animal drugs, identify improper use, and protect the nation’s food supply from violative drug residues that can pose a potential health threat.

To ensure food safety, FDA sets a tolerance level for drugs used in food-producing animals. A tolerance is a level at which a substance may be present in a food that the Agency has determined is consistent with safety when the food is consumed by humans.

The tolerances that FDA establishes include a safety factor to assure that the drug will have no harmful effects on the human consumers of the food product. To do so, the Agency determines the level at which a drug does not produce any measurable effect in laboratory animals, then determines acceptable daily intake levels for humans, withdrawal times (the period necessary after administration of the drug to an animal for the animal’s metabolism to clear the drug so that any residue in meat or milk will be below the tolerance level), and drug tolerance so that the concentration of drug residues in edible tissues or milk will be below the acceptable daily intake level for humans when they are consumed. These tolerance levels are then used for monitoring, surveillance, and research.

In meat, seafoods, poultry, and milk, unsafe drug residues may result from a number of circumstances, such as illegal use of a drug in a food-producing animal, extralabel drug use in such an animal (permitted by law in certain circumstances) if excessive dosages have been administered, or if insufficient time has passed between administration and slaughter or harvest for the drug to clear the animal’s system.

A corporate or individual cattle farmer who repeatedly presents animals adulterated with illegal drug residues for slaughter creates a significant health risk to consumers, so investigating repeat violators is a top priority for FDA.

A growing need for fast, efficient test methods

The need for fast and efficient test methods has become greater since the enactment of the Animal Medicinal Drug Use Clarification Act, which allows for extralabel use of animal drugs by veterinarians under certain conditions. Occasionally, this can mean the presence of a drug residue when one might not ordinarily be expected.

The need for more efficient test methods has also become clearer with the increased availability of imported food products. The use of drugs in food-producing animals overseas has been increasing, giving rise to the potential that import products may contain residues of drugs not permitted for use in food-producing animals in the United States. Another concern is the potential use in food-producing animals of human drugs not approved for veterinary use, unapproved new animal drugs, or other substances.

FDA’s role focuses primarily on the protection of the food supply. For this purpose, test methods to determine whether harmful materials, such as drug residues, are present in the food supply are used both for initial screening tests in the field and in the more rigorous analysis that takes place in the laboratory.

Historically, field test methods have involved use of portable “test kits” in which chemical, bacteriological, or immunoassay assays are used. On-the-spot test results are made available through easily readable visual signals, such as color changes. These field tests have generally been effective as rough screening tests to detect the presence of a drug residue or family of residues, but generally have not provided much analytical detail by which a specific drug residue can be identified or quantified.

Fortunately, more informative in-laboratory residue methods can provide this specificity. Over the past 15 years there have been dramatic improvements in technology for combining liquid chromatography (LC) and mass spectrometry (MS). These improved techniques enable rapid, sensitive analysis of antibiotics and other veterinary drugs. Modern LC/MS techniques have largely supplanted older technologies for analyzing trace levels of bioactive compounds of the type regulated by CVM.

The challenge has been to apply these new techniques to analyze the complex and varied components of foodstuffs and veterinary samples.

CVM’s Office of Research has long been involved in studies to validate analytical methods. In the last several years, however, as new interfaces between LC and MS have moved from the experimental stage, through expensive scientific research, to less expensive commonly used techniques, the Office of Research has focused on refining this science and broadening its uses. Office of Research scientists have accepted the cutting-edge challenge of adapting newly available science into practical test methods that can analyze the multiple components of the molecules in foodstuffs and veterinary samples.

Test method development

The Division of Residue Chemistry in the Office of Research is charged with the development of test methodologies to address CVM’s post-market surveillance needs for analytical methods for drug residues in animal-derived foods. Drug sponsors are responsible for developing methods for new animal drugs, but normally these are single-compound methods. CVM has become involved in researching multi-compound test methods, which are more efficient and have more widespread applicability. A sample preparation for a multiple-compound test may be as time-consuming as one for a single-compound test, but in a multiple-compound test, only one sample preparation is required. Similarly, there are higher initial equipment costs and higher costs for training analysts to use the equipment for multiple-compound tests, but the time saved in running a single test operation instead of many separate ones should bring overall costs down. The result is greater efficiency that should bring real-world cost savings as they become widely adopted.

Regulatory methods that can detect and measure a broad range of drugs and other substances at very low concentrations, yet are rugged, fast, economical, and safe, are valuable to both FDA and FSIS. These would involve, ideally, minimizing sample preparation to the extent possible through use of more sensitive separation and detection technologies .

Liquid chromatography/mass spectrometry (LC/MS, see sidebar) has become an attractive and practical approach for the Office of Research to determine the presence and concentration of residues as LC/MS equipment has dropped in price and techniques have become more refined. In particular, LC/MS/MS (LC/tandem mass spectrometry) offers a high degree of specificity, extremely good sensitivity, potential for high throughput, and applicability to many compounds, giving it the ability to analyze multiple residues in a single procedure.

Multi-residue methods

Multi-residue methods are designed to look at one or several classes of compounds in a single analysis. Current single drug methods are often long, tedious, and provide only minimal information from an analysis. In the past decade, technological improvements have reduced the size of LC/MS/MS equipment so that it can fit on a laboratory bench, and improvements in personal computers have made it possible to link a bench-top LC/MS/MS set-up to a small PC. By utilizing improvements over the past decade in existing mass spectrometry and chromatography technology, practical laboratory methods can be developed that screen for many compounds in a single analysis.

Similarly, the Division of Residue Chemistry has been engaged in developing techniques to apply this science to the surveillance of multiple classes of drug residues in human foods. The first studies involved eggs and milk. Later studies have included meat, seafood, and honey. Some of the methods developed can identify up to three dozen compounds in a single extraction and analysis.

Methods to detect very low levels of banned drugs

Some veterinary drugs are potentially so dangerous that the FDA completely prohibits their use in food animals. Chloramphenicol and nitrofurans fall into this category.

When nitrofuran and chloramphenicol drug residues were first detected in export products from Southeast Asia to Europe, FDA had analytical methods for chloramphenicol, but they were not as sensitive as methods in use in the European Union with lower detection limits. In response, the Office of Research adapted and validated methods for these compounds to provide the Agency with improved regulatory analytical methods to better protect the U.S. food supply.

Using the new technology to detect residues

The Office of Research validates analytical methods used in FDA laboratories for compliance testing through a process known as a method trial.

A method trial establishes that a method performs as intended, that it is fit for its intended purpose, and that technology transfers successfully from laboratory to laboratory. This means that equipment is available and written procedures are developed that are clear, complete, and free of ambiguity so that the Agency and FSIS can use the methods with confidence.

It is only after a method is validated that the method can be considered to be acceptable for general use. Validated multi-residue methods allow both the Agency and industry to maximize resources by being able to screen for residues of several drugs in a single test process. The cost of the more sophisticated equipment and techniques can be more than balanced by the speed and efficiency of the multi-residue methods.

FDA chemist David N. Heller led the Office of Research team that developed two broad-scan analysis techniques for drug residues in eggs. One method can identify residues of 29 drugs, including tetracyclines, fluoroquinolones, and sulfonamides. Another can identify residues of nine other drugs, including ionophores and macrolides.

Methods developed by the Office of Research have begun to be used in surveillance of retail eggs in the United States. For example, surveillance has shown the occasional presence of the polyether ionophore, lasalocid, although at levels not considered to be a human health risk.

According to Heller, LC/MS/MS “opens a lot of doors” for techniques that “apply instrumental capabilities in new ways.”

FSIS has also successfully implemented a procedure developed by an Office of Research team under the leadership of chemist Mary Carson, Ph.D., for identifying residues of nine aminoglycoside drugs, including gentamicin and neomycin, in cattle, swine, horse, chicken, and rabbit tissues. As a result, regulatory enforcement action has been expedited.

Another team under the leadership of Philip J. Kijak, Ph.D., and Hui Li, Ph.D., Office of Research chemists, has developed a multi-class method for 18 veterinary drugs in shrimp. The instrumental analysis time for screening 18 drugs in one sample is only about 19 minutes.

The Office of Research, FDA field laboratories, and the Florida Department of Agriculture and Consumer Affairs jointly validated methods for chloramphenicol in shrimp and crabmeat. The new methodology improved FDA’s detection capability for this banned substance from 5 parts per billion to less than 0.3 parts per billion.

An LC/MS/MS method for determining and confirming residues of furazolidone, nitrofurazone, nitro-fura-n-toin, and furaltadone in shrimp has been validated by a group led by Pak-Sin Chu, Ph.D., and is currently being adapted to other species, including channel catfish. The shrimp method has been transferred to FDA field labs that are responsible for analyzing imported seafood.

Michael H. Thomas, Division of Drug Chemistry Director, noted that the division’s research was showing that the higher capital costs associated with LC/MS/MS multi-residue methods are likely to be outweighed by lower operational costs, because a single sample preparation and a single analysis process can take the place of separate preparation and traditional analyses.

Overall costs were being further reduced, he added, because “computer-based analysis has made LC/MS/MS more automated and more miniaturized.” As a result, Thomas explained, “what had once been an esoteric research tool has now become commonplace.”

(Related information follows in [originally] green background.)

Equipment Used by CVM in Methods for Residue Detection

The Center for Veterinary Medicine’s Office of Research has added new technology as it becomes available. Here is a description of some of the equipment the researchers use for developing methods for -detecting animal drug residues.

by Richard L. Arkin


Chromatography is a process by which a complex mixture is separated into its component compounds. Two principal types of chromatography are used at the Office of Research, Gas Chromatography (GC) and Liquid Chromatography (LC).

GC is used when the compounds are fairly volatile. Among these are some hormones and pesticides. Most veterinary drugs are not volatile and cannot be analyzed by GC.

Most studies at the Office of Research use LC, which only requires that the compounds be soluble in liquid. LC has been available as a separation tool since the late 1960s-early 1970s. However, its usefulness was limited by the detectors that were then available.

Separated compounds in the gaseous or liquid effluent from a chromatograph are detected by a variety of means. The least expensive and most widely available detectors for LC depend on ultraviolet (UV) or visible light (vis) absorbance of the compounds. These detectors are very reliable and easy to use, but not very selective. They are routinely used by most analytical laboratories, but samples analyzed by LC-UV/vis must be fairly clean, meaning the extraction and cleanup is usually long, tedious, and focuses on one or a few compounds. Fluorescence detectors are slightly more expensive, and offer greater selectivity, but the compounds must either have a native fluorescence or be chemically modified to fluoresce.

The most versatile detectors at the Office of Research, and the most expensive, are mass spectrometers. In the late 1980s, practical and effective interfaces became available to couple LC to MS for maximum separation/information about samples.

Mass spectrometry

Mass spectrometry, as used at Office of Research and other state-of-the-art laboratories, is an instrumental method in which the chemical makeup of a substance is identified by its molecular mass. A mass spectrometer is an instrument that measures the masses of individual molecules that have been converted into gaseous ions—in other words, molecules that have been both vaporized and electrically charged. Mass spectrometers use magnetic fields, electric fields, or both to separate a stream of charged particles or gaseous ions according to their mass and charge. The results are recorded onto a computer drive and may be displayed in a graph-like output called a mass spectrum.

The technique of mass spectrometry had its beginnings in the early 20th century. Originally, the spectrometer was confined almost entirely to the world of physics, where the tool was used to discover a number of isotopes and measure their atomic masses. As the cost of mass spectrometry has plummeted, mass spectrometers have become increasingly available in well-equipped laboratories.

Today, a mass spectrometer ranges in size from about the size of an ordinary home oven to large instruments that can fill whole rooms.

The mass sorting and detection processes that occur in a mass spectrometer require samples to be a gas. However, modern developments allow liquid samples to be introduced because liquids can be volatilized at the inlet to the device’s vacuum chamber.

Instead of a single substance or family of substances, the mass spectrometer can provide information from a single sample simultaneously on as many as four dozen substances or more, allowing the mass spectrometer, particularly when used in conjunction with a liquid chromatograph, to be used both as for screening and detailed analysis.

Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known compounds, and to reveal the structure and chemical properties of molecules. In simpler terms, a mass spectrometer electronically “weighs” molecules by determining their molecular mass. This means compounds can be identified at extremely low concentrations in chemically complex mixtures.

[Page 9 photo caption] Michelle Smith, a CVM Staff Fellow from the Oak Ridge Associated Universities, and CVM chemist David N. Heller examine samples prepared for LC/MS/MS analysis in an Office of Research laboratory at CVM’s facilities in Laurel, MD.

Liquid chromatography with tandem mass spectrometry

When a mass spectrometer is connected to the end of a chromatographic column in a manner similar to the other detectors, the result is a powerful analytic instrument. Use of liquid -chromatography with mass spectrometry is increasingly common at Office of Research and other laboratories. The power of this technique is in the production of multiple readouts instead of a simple electronic signal that measure the amount of a specific substance from each of the substances detected. Thus, this technique can rapidly determine both the identity and quantity of a number of unknown components.

Structural information for a substance can be read with a high degree of specificity when specialized tandem mass spectrometers are used. A tandem mass spectrometer can be thought of as two mass spectrometers in series connected by a chamber or collision cell in which a molecule is broken into its component parts. A sample is “sorted” and “weighed” in the first mass spectrometer, then broken up in the collision cell, where its components are sorted and weighed in the second mass spectrometer. So the tandem mass spectrometer is able to fragment or separate the substances in a sample and analyze the products that are generated.

Another way of looking at how tandem mass spectrometers work is the analogy of sorting pocket change. Each coin in a pocketful of change has a unique weight and size—quarters weigh more and are bigger than dimes. A person can sort the coins by weight and size without even looking; then the quantity of each type of coin can be counted quickly. Tandem mass spectrometers can take the sorting even further, like adding vision to the coin sorting process. For example, a person who looks at quarters can, by their specific designs, identify ordinary quarters and each of the new State quarters, and separate them further by their slightly different structures, even though each has a similar weight and size. A tandem mass spectrometer can quickly sort biochemically important molecules of similar weight just as a person can look at the pocket change and sort out how many of the different state coins the person has.

Accordingly, LC/MS/MS techniques can be automated and give high throughput in new analytical and diagnostic methods. Many of the procedures used in human clinical diagnostics also find applications in food and veterinary diagnostics (clinical chemistry, bacteriology, enzyme immunoassays, molecular diagnostics) in meat and fatty tissues, seafood, milk, honey, and processed materials such as feeds.

[Page 10 photo caption] Mary C. Carson (center), Ph.D., a CVM chemist, discusses LC/MS/MS analytical results with Staff Fellow Michelle Smith (left) and CVM chemist David N. Heller in an Office of Research laboratory in Laurel, MD.