8

Analysis of Drug Residues in Food

SHERRI B. TURNIPSEED
U.S. Food and Drug Administration, Denver, Colorado, U.S.A.

I. INTRODUCTION
The use of antibiotics or other medications is common in modern agriculture because
animals are held together in dense populations where the potential for disease outbreak
is high. Drugs can be used therapeutically, to cure existing disease, or prophylatically, to
minimize the potential for disease threat across a population. Often, however, they are
used subtherapeutically as growth promotants to increase feed conversion. The possibility
of drug residues remaining in the edible product and the potential human health problems
associated with exposure to these residues is a concern because of the widespread drug
use in food animals. The actual public health significance of drug use in animal agriculture
and of their residues in food of animal origin is an area of much debate. Recently the
National Research Council convened a group to evaluate the benefits and risks of using
drugs in the animal food industry (National Research Council, 1999). They identified
antimicrobial resistance of disease-causing bacteria as the most serious risk associated
with the continued use of drugs in food animals. Animals fed low (subtherapeutic) levels
of antibiotics may develop bacterial infections that evolve to be impervious to these drugs.
Humans may be exposed to these bacterial populations in the environment or during prepa-
ration or consumption of food. A task force consisting of several U.S. government agencies
has formulated a public health plan to combat antimicrobial resistance, including ways to
limit the spread of drug resistance due to agricultural applications (Center for Disease
Control, 2001). In addition, some animal drugs may cause an immediate adverse reaction,
such as allergic response, in susceptible human populations. Therefore, it is important to
regulate the improper use of animal drugs by monitoring animal tissues or the resulting
food products for drug residues.

© 2003 by Marcel Dekker, Inc.

 The occurrence of drug residues is possible in any food of animal origin. This in-
cludes, but is not limited to, tissue (muscle, liver, kidney) or fat from bovine, swine,
poultry, fish, shrimp, or minor species (such as ratites, rabbits) in addition to animal prod-
ucts such as milk, eggs, and honey. Because this is such a diverse list, the procedures for
food processing, and therefore the sanitation guidelines as well as the regulations covering
these commodities, are also quite varied.
The governmental responsibility for reducing the incidence of animal drug residues
in the nation’s food supply is primarily shared between the United States Department of
Agriculture (USDA) and the Food and Drug Administration (FDA). The FDA regulates
animal drug residues in milk, eggs, and aquaculture products. The USDA is responsible for
monitoring meat and poultry products for animal drug residues. The Food Safety Inspection
Service (FSIS) of the USDA conducts the National Residue Program (NRP) to prevent
animals containing violative amounts of drug residues from being marketed (Food Safety
Inspection Service, 1998). The FDA is also responsible for approving new animal drugs,
setting tolerances, and conducting enforcement actions as a result of any FSIS findings.
In many cases the introduction of illegal residues occurs at the original producer or
farmer and not at the food processing establishment. In fact the most common causes for
the presence of illegal residues include not following proper withdrawal times, administra-
tion of improper dosage, etc. However, a recent review illustrates how contamination can
also be a cause of illegal animal drug residues (Kennedy et al., 2000). One possible source
of contamination includes mixing of nonmedicated feed with medicated feed during manu-
facturing, compounding, or transporting. In addition, these authors provide examples of
wild fish or other aquatic species harvested near aquaculture pens being contaminated
with drug residues from that facility. Another study cited in this review illustrates how
pigs held in the same housing structure as a previous population that had been given
sulfamethazine were found to be positive for this residue by immunoanalysis of their
kidney tissue (McCaughey et al., 1990). All of these possible sources of drug residue
contamination relate directly to food/feed sanitation procedures.
Even if the causes of illegal drug residues are more likely to originate with the food
producer, the introduction of hazard analysis critical control point (HACCP) programs
holds food processing plants responsible for minimizing this source of contamination. The
use of HAACP as a tool to ensure food safety extends to residue monitoring. Under the
HAACP system food processing plants must have systems in place to prevent hazards in
their products. These hazards include not only pathogenic organisms and physical hazards,
but also chemical residues such as illegal drugs or pesticides. The rule indicates that the
industry must evaluate significant residue hazards and develop a HAACP plan for control-
ling residues. An example of a HAACP plan designed to minimize the risk of drug residues
at the producer level is the Milk and Dairy Beef Residue Prevention Protocol (Boekman
and Carlson, 2000). Aspects of a HAACP plan for a food processing plant designed to
reduce the risk of illegal drug residues might include avoiding buying animals from prob-
lem producers and implementing live animal tracking and residue testing (Dey, 2001).
Monitoring the food supply for residues is important because of the potential health
risk from exposure to some animal drugs. However, the low levels and complex matrices
involved can make residue monitoring a real analytical challenge. Several broad types
of analytical methods for drug residues in food can be described, including screening,
determinative, and confirmatory procedures. These all play a role in monitoring the food
supply for chemical residues due to improper use of animal drugs.

© 2003 by Marcel Dekker, Inc.

 II. ANALYTICAL METHODS FOR DRUG RESIDUES
A. Screening Methods
Screening methods are designed to be rapid, easy-to-use tests which provide a positive
or negative response for a drug at a given concentration level in a matrix. Samples
taken at food processing plants are often analyzed initially using a screening test to de-
termine if there may be a problem with drug residues. Traditionally, microbial inhibi-
tion tests (MITs) have been used to screen large numbers of samples for antimicrobials,
and these tests are still widely used. All MITs are based on the inhibition of bacterial
growth by antibacterial residues present in a matrix that results in zones of inhibition on
bacterial plates. These tests are relatively simple to use and detect many classes of anti-
bacterial compounds. Selective sensitivity for specific classes of antibacterials can be
obtained by changes in the culture medium, indicator bacteria, or pH. However, these
methods often lack the specificity and sensitivity required for residue detection at maxi-
mum residue limits. They may be affected by nonspecific inhibitors and do not detect
microbiologically inactive metabolites. In addition, they often require a 20–24 hr incu-
bation period.
Microbial inhibition tests that have been used by the FSIS for screening red
meat and poultry tissues for antibacterial residues include the Swab Test On Premises
(STOP) and the Calf Antibiotic and Sulfa Test (CAST) (Sundlof, 1989). The Fast Antibi-
otic Screen Test (FAST) is a test used by FSIS that provides results within 6 hr. There
are several other MITs used globally, including the New Dutch Kidney Test, the German
Three Plate Assay, the European Four Plate Test, and the Charm Farm Test.
Although MITs can be very useful, there are some inherent problems with these
tests, including the FAST test. Imprecision occurs as a result of zone of inhibition size
differences between replicate plates. Zone size may vary as a result of differences in agar
layer thickness, agar quality, uneven seeding of bacterial spores on the agar surface, or
incubator temperature variation (Brady and Katz, 1987). Additionally, bacteriostatic drugs
such as sulfonamides may result in a diffuse zone, while bactericidal drugs provide a
sharply defined zone of inhibition, and this may complicate the interpretation. In fact,
studies indicate that the sensitivity to the FAST test for different classes of drugs can vary
widely (Korsrud et al., 1998).
In addition to MITs, new rapid test kits, generally based on bacterial cell recep-
tor or enzyme immunoassay, are being used to screen samples for specific drugs. The
Charm II test is a proprietary competitive microbial receptor binding assay that can de-
tect residues of seven classes of antibiotics. Although this test can detect a number of
drugs within a class, the relative sensitivity of the test to individual drugs varies. It is
commonly used for monitoring antibiotics in milk, but has also been tested for use in
bovine muscle and kidney samples (Korsrud et al., 1994). Immunoassays are widely used
on dairy farms and processing plants to screen milk samples for veterinary drug residues.
With appropriate extraction methodology many of these assays may also be used for
residue analysis of food animal tissues. Recent examples of the use of immunoassays in
meat analysis include the analysis of aminoglycosides in porcine kidney (Haasnoot et al.,
1999) and tetracyclines in pork and chicken meat (De Wasch et al., 1998). These rapid
tests are well suited for in-plant use due to the limited amount of sample manipulation,
simple analytical equipment and procedures required, and the relatively fast analysis
time.

© 2003 by Marcel Dekker, Inc.

 B. Determinative Methods
These screening tests, however, are not always accurate at or below the test threshold. They
are often class, not compound, specific, and may or may not give quantitative information.
Therefore, additional analytical tests may be needed to determine if a sample is actually
violative for an animal drug residue. Determinative methods are designed to separate,
quantitate, and perhaps provide some qualitative information on the analyte of interest.
These tests may require more specialized laboratory equipment and experienced personnel
to perform than the screening tests discussed previously.
A determinative method involves several stages including sample preparation, ex-
traction of the analyte from the food matrix, separation of the drug residue from any
remaining matrix components, and detecting (measuring) the amount of residue present.
Sample preparation, isolation, and cleanup are major rate-limiting factors in sample analy-
sis. The classical approach to isolation of drugs from tissues involves tissue homogeniza-
tion followed by liquid–liquid partitioning of the homogenate, with or without additional
cleanup or concentration steps. These methods may provide adequate separation of the
drug from the matrix but are often expensive in terms of time, labor, material use, and
organic solvent disposal costs. Such approaches also tend to be highly nonspecific in their
isolation of the target drug(s).
Recent advances in the field of residue analysis offer several promising techniques
as possible solutions to the problems caused by outmoded and complex analytical methods.
Three techniques—solid-phase extraction (SPE), matrix solid-phase dispersion (MSPD),
and immunoaffinity—are receiving particular attention because they have the potential to
greatly reduce analytical costs and reduce analysis-generated waste and pollution. The
SPE process is a type of chromatographic separation designed to isolate the analyte of
interest from the rest of the food matrix. Before SPE can be used with solid tissue (e.g.,
muscle and liver), a separate homogenization step and often additional steps are required.
The most common use of SPE is to develop conditions whereby the analyte adheres to
the solid-phase material while the other components are washed through, then the solvent
system is changed to elute the analyte separately. Because selection of the SPE column
depends on the matrix and on the particular compound of interest, a wide range of solid-
phase extraction columns of differing polarities have been used for drug extraction from
tissue and include silica, alumina, C 18 , NH 2 , and ion exchange resins.
Matrix solid-phase dispersion (MSPD) is a variation of the SPE isolation technique.
In general terms, MSPD involves blending a tissue sample (0.1–1.0 g) with lipophilic
polymer-derivatized silica particles, which simultaneously disrupts and disperses the sam-
ple. The resulting slurry is then packed as a ‘‘column’’ which can be eluted with appro-
priate solvent to give an extract containing the residue of interest. Matrix solid-phase
dispersion has been used in the analysis of furazolidone (Long et al. 1990), penicillins
(McGrane et al., 1998), and sulfamethazine (Shearan et al., 1994) in swine tissue. This
technique has also been used to analyze milk samples for chemical residues (Schenck and
Wagner, 1995).
The simplest methods of extraction, however, require minimal or no sample manipu-
lation. These are the methods that extract the drug directly from the sample matrix by
means of specific or selective antibodies or receptors. Immunoaffinity isolation techniques
can be used for sample cleanup. Recent examples where immunoaffinity techniques have
been used in animal drug residue analysis include the determination of 19-nortestosterone

© 2003 by Marcel Dekker, Inc.

 and trenbolone in animal tissue (Stubbings et al., 1998) and avermectins in cattle meat
(Li and Qian, 1996).
Once a drug residue has been extracted from the food matrix, the amount of residue
can be measured using the physical characteristics of the molecule after separation from
any remaining food components. Many of the drugs used in animal agriculture can be
separated from any remaining food matrix using liquid chromatography (LC). In LC,
separation occurs when a compound is isolated from a liquid sample based on its relative
solubility in the liquid mobile phase compared to its solubility in a solid support-bound
liquid stationary phase or its affinity to a solid support stationary phase. Reverse-phase
LC chromatography columns generally consist of a liquid phase (C 18 , C 8 , phenyl, etc.)
covalently bound to a spherical (particle size 3–10 µm) silica support. The degree of free
silanol groups remaining unbound can greatly affect the selectivity of the column for any
given analyte. There is a great variety of LC columns commercially available based on
principles of polymer chemistry, chiral selection, ion exchange, and size exclusion, as
well as the traditional reverse-phase chemistry. Ideally the compound of interest can be
eluted from the chromatographic column isolated from any other material and then intro-
duced into a detector to measure the amount of compound present. For LC the most com-
mon detector is a UV/VIS spectrophotometer using a variable wavelength or diode array.
Liquid chromatographs using fluorescence, chemiluminescence, or postcolumn reaction
detectors are also available and have been successfully used to determine drug residues
in animal tissue.
Gas chromatography (GC), in which separation is based on the relative partitioning
of an analyte into a liquid coated onto a fused silica capillary as it travels through the
column in the gas phase, can also be used to analyze drug residues in food matrices. GC
is very suitable to volatile analytes such as organophosphates. Many animal drugs have
a large molecular weight and are relatively nonvolatile and thermally labile. To overcome
these characteristics, chemical derivatization is generally required to obtain sufficient
volatility and stability for GC analysis. Detection for GC is generally done using flame
ionization or electron capture techniques. More selective detectors for nitrogen-, phospho-
rus- and sulfur-containing compounds are also available, and mass spectrometry (MS)
detection is commonly used as a detector for GC.
Several reviews have been written on the determinative methods in use today (Oka
et al., 1995; Turnipseed and Long, 1998). In addition, the European Union has established
a database of veterinary drug determinative methods (Van Eeckhout et al., 1998). Section
III of this chapter lists the common types of animal drugs that might be found in food,
typical extraction and analytical parameters, as well as some references for more recent
specific procedures or reviews.

C. Confirmatory Methods
Confirmatory methods are meant to provide absolute identification of the drug residue in
question. Because of its sensitivity and specificity, mass spectrometry is the preferred
method for confirmation. Guidelines as to what should constitute a positive identification
with mass spectral data have been discussed (Sphon, 1978; FDA, 2001). In most cases,
confirmation is achieved using an instrument that interfaces the MS with a chromatograph
(GC or LC). There are many examples in the reference section of this chapter of methods

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 that contain not only determinative procedures, but also give information on qualitative
confirmatory analysis.

III. SUMMARY OF ANALYTICAL METHODS FOR DRUG RESIDUES

IN FOOD
A. Sulfonamides
Uses: pneumonia and other respiratory diseases, mastitis, diphtheria, diarrhea
Examples: sulfamethazine, sulfadimethoxine, sulfamerazine, sulfathiazole
Foods of concern: milk; bovine, swine, and poultry tissue (muscle, liver, kidney);
fish tissue
Typical extraction: liquid–liquid extraction and/or SPE isolation
Typical detection: reverse-phase (RP) LC with UV (270 nm) or LC/fluorescence
after derivatization
Alternative methods: LC/MS/MS, supercritical extraction
Comments: multiresidue methods available
References: Parks and Maxwell (1994), Tsai and Kondo (1995), Gehring et al.
(1997), Stoev and Michailova (2000), Van Eeckhout et al. (2000a)

B. Beta-Lactams
Uses: colibacillosis, bacterial enteritis, salmonellsis, etc.
Examples: penicillin, ampicillin, amoxicillin, cloxacillin, cephapirin, ceftioflur
Foods of concern: milk; bovine, swine, and fish tissue
Typical extraction: liquid–liquid extraction (sometimes using tungstic or trichloro-
acetic acid) with SPE (usually C 18) cleanup
Typical detection: RPLC/UV penicillins: 210–230 nm; cephalsosporins: 260–295 nm
Alternative methods: derivatization to penicellenic acid mercuric mercaptide, deri-
vatization with fluorescamine, LC fractionation, LC/MS
Comments: difficult to analyze for all types simultaneously
References: Boison and Keng (1998), Moats and Romanowski (1998), Luo and Ang
(2000), Hong and Kondo (2000)

C. Aminoglycosides
Uses: used as broad-spectrum antibiotics, also to enhance growth efficiency
Examples: streptomycin, apramycin, dihydrostreptomycin, gentamicin, neomycin
Foods of concern: milk; bovine, poultry, and swine tissue (especially kidney)
Typical extraction: liquid extraction with aqueous buffers, ion exchange columns
Typical detection: RPLC/fluorescence with derivatization or GC/electron capture
Alternative methods: LC/MS
Comments: usually need ion pair reagents for LC separation
References: Heller et al. (2000), Isoherranen and Soback (1999)

D. Tetracyclines
Uses: to treat enteritis, pneumonia, and anaplasmosis, also to promote weight gain
and increase feed efficiency
Examples: tetracycline, oxytetracycline, chlortetracycline
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 Foods of concern: milk; bovine, poultry, and swine tissue; shrimp; eggs; honey
Typical extraction: mild acid with chelating agents, metal chelate affinity columns
Typical detection: RPLC or ion exchange with UV at 270–350 nm
Alternative methods: LC/MS/MS, LC/fluorescence of metal complexes
Comments: oxalic acid or chelating agent used in LC mobile phase to avoid com-
plexing with metals and adsorbing on silanol sites of column
References: Oka et al. (2000), Moats (2000), Van Eeckhout et al. (2000b), Posyniak
et al. (1999)

E. Macrolides
Uses: to treat gram positive organisms and some strains of Listeria and Mycoplasma,
promote growth efficiency
Examples: erythromycin, tylosin, oleandomycin, spiramycin, tilmicosin
Foods of concern: milk; bovine, poultry, swine, and fish tissue; eggs
Typical extraction: liquid–liquid extraction and/or SPE isolation
Typical detection: RPLC with UV or LC/fluorescence after derivatization
Alternative methods: LC/MS
Comments: many have weak UV chromophores
References: Kiehl and Kennington (1995), Chan et al. (1994), Leal et al. (2001),
Stobba-Wiley et al. (2000)

F. Quinolones
Uses: broad-spectrum effective against gram positive, gram negative (fluoroquino-
lones) and mycoplasma
Examples: oxolinic acid, naladixic acid, sarafloxacin, enrofloxacin, ciprofloxacin,
flumequine
Foods of concern: milk; bovine, poultry, swine, and fish tissue; eggs
Typical extraction: liquid–liquid extraction and/or SPE cleanup using C 18 or cation
exchange
Typical detection: RPLC with fluorescence and/or UV detection
Alternative methods: on-line dialysis, LC/MS
Comments: high level of concern regarding antibiotic resistance to these drugs
References: Munns et al. (1995), Maxwell et al. (1999), Roybal et al. (1997), Rose
et al. (1998)

G. Phenicols
Uses: infections, bovine respiratory disease
Examples: chloramphenicol, florfenicol, thiamphenicol
Foods of concern: milk; bovine, poultry, swine, and fish (including shrimp) tissue;
eggs
Typical extraction: liquid–liquid extraction and/or SPE isolation
Typical detection: RPLC with UV or GC/electron capture after derivatization
Alternative methods: GC/MS or supercritical fluid extraction
Comments: multiresidue methods available, florfenicol metabolized to amine
References: Pfenning et al. (2000), Pensabene et al. (1999)
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 H. Ionophores
Uses: coccidiostats, growth efficiency
Examples: monensin, lasalocid, salinomycin
Foods of concern: poultry, eggs, beef tissue
Typical extraction: liquid–liquid extraction and/or SPE cleanup
Typical detection: RPLC with visible or fluorescence detection after derivatization
Alternative methods: LC/MS
Comments: present as sodium salts, weak chromophore
References: Matabudul et al. (2000), Gerhardt et al. (1995)
I. Benzimidazoles
Uses: anthelmintics; growth efficiency
Examples: albendazole, fenbendazole, oxfendazole, thiabendazole
Foods of concern: bovine and swine tissue, milk (also fruits and vegetables)
Typical extraction: liquid–liquid extraction and/or SPE isolation, MSPD
Typical detection: RPLC with UV (290 nm)
Alternative methods: LC/MS/MS
Comments: multiresidue methods available
References: Long et al. (1990), Sorensen and Hansen (1998), Balizs (1999)
J. Avermectins
Uses: anthelmintics
Examples: ivermectin, eprinomectin, doramectin, moxidectin
Foods of concern: milk; bovine, swine, and fish tissue
Typical extraction: liquid–liquid extraction and/or SPE isolation
Typical detection: RPLC/fluorescence after derivatization
Alternative methods: LC/MS, postcolumn photochemical reaction
Comments: multiresidue methods available
References: Rupp et al. (1998). Roybal et al. (2000), Schenck and Lagman (1999),
Danaher et al. (2001)
K. Hormones
Uses: increasing weight gain
Examples: estradiol, progesterone, and testosterone melengestrol acetate, trenbolone
acetate, zeranol, and diethylstilbestrol (DES)
Foods of concern: bovine and swine tissue
Typical extraction: liquid–liquid extraction and/or SPE, LC fractionation
Typical detection: GC/MS after derivatization
Alternative methods: LC/MS
Comments: approval for these drugs varies, DES banned worldwide
References: Marques et al. (1998), Daeseleire et al. (1992, 1998). Hori and Naka-
zawa (2000)
L. Corticosteroids
Uses: anti-inflammatory and gluconeogenic agents
Examples: dexamethasone, prednisolone, betamethazone, flumethasone, prednisone,
triamcinolone
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 Foods of concern: bovine and swine tissue
Typical extraction: liquid–liquid extraction (sometimes with the addition of NaOH),
SPE
Typical detection: GC/MS after derivatization, LC/UV (240 nm)
Alternative methods: LC/MS, LC/chemiluminescence
Comments: multiresidue methods available
References: Mallinson et al. (1995), Stolker et al. (2000), Iglesias et al. (1999), Van
Den Hauwe (2001)

IV. FUTURE TRENDS

The most significant trend in drug residue analysis is to increase the numbers of samples,
residues, and matrices that can be monitored simultaneously. A higher throughput of sam-
ples being analyzed for more types of residues obviously increases the efficiency of moni-
toring. In addition to the rapid screening methods already discussed, there has also been
an emphasis in developing determinative methods that will monitor more compounds more
rapidly. For example, the development of generic, universal extraction methods that will
extract many classes of drugs from a single sample (Rose et al., 1998; Heller and Cui,
2001). High throughput processing of samples using arrays of SPE columns developed
for combinatorial chemistry applications in drug discovery may have relevance to the
analysis of animal drug residues as well (Harrison and Walker, 1998).
As mass spectrometry, particularly modern LC/MS, becomes more practical and
affordable it has also been used to screen and perform quantitative analyses as well as
just provide qualitative confirmation. There are now several examples of methods where
MS is used for multiresidue screening and analyses (Volmer, 1996; Heller and Cui, 2001;
Stolker et al., 2000; Tarbin et al., 1998)
Technological advances made in other areas of analytical, process, and food chemis-
try will have an effect on the analysis of drug residues in animal tissues. For example,
the Biocore company in Switzerland has developed optical biosensors which have been
used for the analysis of animal drug residues including the detection of sulfonamides in
meat (Crooks et al., 1998; Bjurling et al., 1999). This technology has been used on-site at
a slaughterhouse. Biochip array technology using immunoassay with chemiluminescence
detection has been developed for the detection of growth promoters and sulfonamides
(McConnell et al., 2000). All of these new technologies will have an impact on how animal
drug residues are monitored and regulated in the future.

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