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The Science and Art of Measurement: The Work of CVM’s Division of Residue Chemistry

A Brief History of CVM’S Division of Residue Residue Chemistry

The components of today’s Division of Residue Chemistry (DRC) of the Center for Veterinary Medicine (CVM) have existed for more than four decades.

In 1963 the Food and Drug Administration’s (FDA) Veterinary Research facility opened in rented space at the U.S. Department of Agriculture’s Beltsville Agricultural Research Center, in Maryland, just outside Washington, DC. This facility was part of the Veterinary Medical Branch in the Bureau of Medicine.

By the late 1970s, residue method development and testing was being performed by chemists in the Bureau of Foods (now FDA’s Center for Food Safety and Applied Nutrition) in downtown Washington. This methods group transferred to Bureau of Veterinary Medicine (the forerunner of the Center for Veterinary Medicine [CVM]) in the early 1980s and became part of the Chemistry Division of the Office of Human Food Safety (now CVM’s Office of New Animal Drug -Evaluation).

Then, in the mid 1980s, the methods group relocated to Beltsville, MD, and later merged with chemists from Veterinary Medical Research to form a new analytical branch. The analytical staff was increased in the early 1990s and the branch achieved division status within CVM.

In 1996, CVM’s Office of Research opened at the FDA laboratory and research facilities at “MOD2” at the Muirkirk Road Campus in Laurel, MD. This facility, dedicated in October 1996, was the first new construction of the FDA consolidation.

CVM’s MOD2 facilities include more than 165 acres of pastures and other land for animals, and contain large animal research buildings, specialized laboratories, pastures, feed mixing facilities, and quarantine facilities.

by David H. Heller, Research Chemist
FDA Veterinarian Newsletter 2006 Volume XXI, No III

Science and research depend on measurement. If a thing cannot be measured, it cannot be studied via science. Measurements are an essential part of scientific experiments, which are controlled situations in which the measurements provide evidence of relationships among various forces and parameters. In a well-designed experiment, some aspects are held constant while others are varied in a controlled manner. The data may reveal a cause-and-effect relationship between certain variables.

The Food and Drug Administration’s (FDA) Center for Veterinary Medicine (CVM) relies on measurement science to evaluate animal drugs and ensure the safety of food from animals. But measuring for residues is not simple. Analytical chemists at CVM’s Office of Research, Laurel, MD, employ highly sophisticated systems to measure residues.

Here’s a tour of the field of residue chemistry and FDA’s mission to protect animal and human health.

Measurements are integral to all of FDA’s work

The need for measurement is written into all of FDA’s laws and regulations; measurements are integral to our work in several fundamental ways.

  1. The heart of the FDA’s mission is to ensure that products are effective and safe. Effectiveness may be demonstrated with controlled experiments that relate a product’s dosage to some beneficial effect, and this relationship is established with measurements.
  2. Other experiments evaluate product safety by measuring the dosage at which negative effects may occur. These experiments often show that a product is healthful at a certain level, but not at a higher level.
  3. Products are safe and effective only when used in the approved manner, so other measurements are also needed to verify that approved products are used in the approved manner.

For these different cases, CVM’s mission requires the development and evaluation of two different kinds of methods: For 1 and 2 above, Research Methods to help establish conditions for a compound’s proper use, study the compound’s distribution in various tissues, or track its depletion rate from those tissues; and for 3, Regulatory Methods to provide surveillance data on usage patterns and support legal action against violations, such as excessive or unapproved uses.

These two types of methods can be differentiated in another way, according to who will make use of the results and how they are discussed. The customers for data acquired with Research Methods are typically other CVM scientists, but the customers for data acquired with Regulatory Methods are CVM’s compliance officers and legal counsel (lawyers).

Residue chemistry fundamentals

Residue chemistry means both identifying the presence of a compound that has been administered to an animal and measuring its concentration in the animal’s tissue.

At CVM, tissues might refer to samples drawn from living animals, such as blood, milk, or eggs. It could also mean food products derived from animals, such as muscle or liver. Or it could be by-products from animals, such as fat or skin.

The residue can be the compound itself, a metabolite (a form of the compound that has been modified by the body), or a contaminant that was inadvertently administered to or consumed by the animal.

In most cases the residues we at CVM’s Division of Residue Chemistry deal with are of antimicrobial compounds, such as tetracycline, penicillin, or neomycin, which are administered to keep the animal healthy and to promote growth.

Residue chemists have five major areas of -concern:

  • Fitness: Did we measure the right thing in the right way? Success in the laboratory results from addressing these concerns in order of importance. Before beginning laboratory work, the fitness of a technical solution depends on defining the technical problem jointly between the residue chemist and the customer for the data. It is also important to know if there will be scientific or legal evaluation of the results.
  • Uncertainty: How sure are we? What is the degree of bias and uncertainty? There will always be some uncertainty associated with the results of a residue analysis. The existence of uncertainty is unavoidable; scientists strive to assess and control uncertainty, not to eliminate it. When non-scientists hear results that are qualified by a discussion of potential error and imprecision, this is a good thing, not a reason to question their validity. There is no such thing as a perfect method that always gives the exact same answer every time. The closest we can come is a measured value obtained with a known degree of confidence. Measurement error, or bias, is the difference between a “true” or known value and the value found by actual analysis. Measurement precision describes how consistent the method is when run repeatedly on the same sample. Uncertainty is controlled by setting limits for accuracy, precision, concentration range, and identification confidence, and by not adopting methods that don’t meet the acceptance criteria.
  • Quality: Could we have made a mistake? Quality management plays a critical role in establishing the validity of results. Laboratory quality is built from many individual steps that are carried out according to standardized procedures and acceptance criteria. There are procedures to test, control, and double-check critical steps as they are carried out, then to audit the results afterwards. Data must be shown to have been acquired when the method was under control. Only if known samples give proper results when analyzed alongside unknown samples can the values for unknowns be -acceptable.
  • Quantitative: How much is present? Are these results consistent with proper use? Is the level sufficient to call for legal action? And 
  • Qualitative: Is a particular product present in tissue?

Maintaining laboratory quality and assessing method uncertainty are major elements of our day-to-day lab-ora-tory work. Methods are tested for ruggedness in a variety of ways after they are developed. No method is used for critical analyses without extensive testing known as a method validation. Nearly every laboratory activity, from weighing chemicals, mixing standard solutions, or calibrating instruments to conducting entire research studies and documenting results are covered by standardized procedures. Once these procedures are in place, laboratory work can begin.

There are two technical disciplines that must be grasped to make sense of modern analytical laboratory practice: separation science, or extraction, and instrumental analysis.

Separations at the molecular level

How do you remove the proverbial needle from a haystack? Residue analysis poses a similar problem. Drug residues occur in complex biological tissues consisting of proteins, fats, fibrous connective tissue, carbohydrates, and an amazing variety of other small molecules. The residues of concern have to be separated from this matrix by any means possible. For an analogy, think of a haystack which is doused with gasoline and set on fire; this is a chemical reaction that converts the hay to gases while leaving minerals behind (such as a metal needle). If the hay is doused with water and allowed to rot, this is a biochemical digestion (carried out by microbes). If the hay is probed with a giant electromagnet, magnetic metal needles may be recovered by a physical process of attraction.

Other separation steps can be carried out by manipulating solution chemistry. For example, a mixture of salt and pepper can be separated with water, by dissolving the salt to take advantage of its differential solubility. You could use a form of residue chemistry yourself if extra-hot BBQ sauce burns your lips. It is more effective to drink milk than water to ease the burning sensation, because the spicy components are more fat-soluble than water-soluble. Tarnished metal can be treated with acid (a lower pH value) to change the solubility of the oxidized metal surface. Juice is removed from apples by grinding, pressing, and -filtering.

Basic techniques such as these are now supplemented by more sophisticated approaches, such as solid phase extraction, or SPE. Think “tea bag” when visualizing the SPE process. A small amount of specially treated particles (tea) is loaded inside a permeable container (the bag) and chemical components (caffeine) are extracted with a solvent (boiling water). SPE particles come in various “flavors” based on their chemical affinity: lipophilic (fat-loving), hydrophilic (water-loving), or ionic (possessing electrostatic -attraction).

In summary, the initial step in separation science is a lab-scale extraction based on a combination of chemical, physical, or biochemical processes, where the goal is to recover 100% of the compound of interest in a more purified form.

Chromatography further separates the compounds in a mixture from one another. Chromatography is carried out in a specially lined tube through which liquid or gas flows. Different compounds have different affinities for the stationary lining of the tube compared to the moving liquid or gas, so they move through the tube at different rates. Imagine a large crowd lining a busy street. An agile jogger can maneuver fairly quickly by avoiding contact and “diffusing” quickly to the far side of the crowd. However, a candidate for public office might stop to talk with each person, and thereby take many times longer to emerge from the crowd. If the two had arrived at the edge of the crowd at the same time, the jogger would always emerge first. This “retention time” is a feature that both separates and helps characterize the components of a mixture.

Instrumental analysis

Advances in technology and computers have steadily changed the way residue analyses are conducted. In earlier years, the most common approach to antibiotic detection was to measure their inhibition of bacterial growth in laboratory cultures. Now, instrumental detectors based on chemical and physical principles can provide direct analysis of specific chemical entities with amazing sensitivity.

Physico-chemical detectors provide a response that is proportional to the amount of compound present; the more response, the higher the concentration must be. A calibration curve is prepared from solutions of certified standards at known concentrations. When the response of an unknown sample is compared against the calibration curve, the sample’s concentration can be computed.

There are two primary types of instrumental detectors used in today’s residue laboratories. Spectroscopic detectors are based on the absorbance of light by the compound. Mass spectrometric detectors respond directly to the molecules themselves, after they have been ionized and separated according to mass in a specialized vacuum chamber. In fact, the development of electrospray ionization mass spectrometry has become so critical in pharmaceutical and other applications that its developer, John Fenn, was a co-recipient of the Nobel Prize in Chemistry in 2002.

These detectors are sophisticated instruments that are heavily dependent on electronics, computer control, and automated digital data processing. You can’t see what is happening inside, and the operator doesn’t necessarily have to understand every internal process to obtain valid data. Mass spectrometers are normally inside heavy boxes with noisy vacuum pumps. Instrumental laboratories require good ventilation and temperature control, so they tend to be noisy and filled with computers connected to large boxes with flickering lights.

The power of mass spectrometry can be illustrated with an example based on a familiar compound. A typical soda contains about 0.1 mg of caffeine per ml. One quarter of a liter (about 8 ounces) contains about 25 mg caffeine, which corresponds to about 75,000,000,000,000,000,000 molecules. If that 8 ounces is diluted by 100,000 times, say, by pouring it into a tanker truck, electrospray tandem mass spectrometry could still detect the caffeine. Detection limits might be on the order of 10 picograms, or 30,000,000,000,000 molecules. This extreme sensitivity puts pressure on CVM’s toxicologists and regulators to determine at what point detectable residues begin to create a health risk to consumers.

Mass spectrometers can also identify a particular compound with a high degree of confidence. These instruments respond directly to signals from the intact molecule and its constituent pieces. The resulting “mass spectra” are highly specific, much like fingerprints or bar code tags.

Traceability

Traceability refers to comparing the response of an unknown sample against that of a certified standard.

The calibration process depends on a detector response that is proportional to the amount of compound present. Calibration standards are prepared at a series of concentrations using a standard whose amount is certified by the manufacturer.

Mass spectrometry can be used to confirm the presence of a suspect compound by comparing the specific mass values from a mass spectrometer against the corresponding signals from standards.

Every physical parameter we can measure has some ultimate benchmark to which measured values can be traced back. That benchmark is the basis for the validity of results.

Conclusion: The Critical Eye – What to Look for In Evaluating Measurements

A significant part of a regulatory chemist’s time is spent evaluating the work of others, whether internal or submitted by animal drug sponsors or other government laboratories. Over time one develops a process for checking the most critical aspects of measurements and methods. Here are some important questions that can be asked of measurements in any context.

  • Qualitative: How selective is the separation and detection? Could the signals arise from any other compound?
  • Quantitative: What are the upper and lower perform-ance limits? How much of the analyte is recovered by the extraction?
  • Quality: Did the quality assurance samples give the correct result? Have the data been audited by an independent expert?
  • Uncertainty: What is the method’s degree of bias and uncertainty?
  • Fitness: What is at stake? How sure do you have to be?

In the final analysis (so to speak), regulatory analysts provide a service in support of regulatory decision-makers. We respond to method needs that are determined by toxicology studies and risk assessment. We provide methods and data that can be relied upon by those who determine what action to take on the basis of the numbers.