The process of development and
validation of animal drug residue
methods for US Food and Drug
Administration regulatory use

Philip James Kijak

1

and Valerie B. Reeves

2

1

US Food and Drug Administration, Laurel, MD 20708, USA; and

2

US Food and

Drug Administration, Rockville, MD 20855-2745, USA

1

Introduction

The US Food and Drug Administration (FDA) evaluates methods to be used in
government regulatory laboratories for the determination and confirmation of drug
residues in food derived from animal products. The FDA Center for Veterinary
Medicine (CVM) oversees the validation (i.e., demonstration that the method is suit-
able for use) via a protocol known as a method trial. CVM ensures that the appro-
priate government laboratories have the tools needed to monitor the Nation’s food
supply.

In 1906, the original Food and Drugs Act was passed by Congress to prohibit

interstate commerce in misbranded and adulterated foods and drugs. The use of
poisonous preservatives and dyes in foods and cure-all claims for worthless and
dangerous patent medicines were major problems leading to the enactment of this
law. The Federal Food, Drug, and Cosmetic (FDC) Act of 1938 extended Federal
authority over cosmetics and therapeutic devices. The FDC Act also required that
new drugs be shown to be safe before marketing and provided for the establishment
of tolerances for unavoidable poisonous substances.

In 1958, the Food Additives Amendment was enacted, requiring manufacturers of

new food additives to establish safety and provide a description of practicable methods
for determining the quantity of such additive in or on food, and any substance formed
in or on food, because of its use. The Delaney Clause prohibited the approval of any
food additive shown to induce cancer in humans or animals. The Kefauver–Harris
Drug Amendments were passed in 1962 to ensure drug efficacy and greater drug
safety. Drug manufacturers were, for the first time, required to prove to the FDA the
effectiveness of their products before marketing them. The 1962 law also exempted
from the Delaney Clause animal drugs and animal feed additives that induce cancer

Handbook of Residue Analytical Methods for Agrochemicals.

C

2003 John Wiley & Sons Ltd.

The process of development and validation of animal drug residue methods

77

when ingested by man or animal but for which no residue of such drug will be found.
This is also known as the Diethylstilbestrol (DES) Proviso.

According to the DES Proviso, a carcinogenic animal drug, feed additive, or color

additive could be approved provided that, under the conditions of use specified in
proposed labeling and reasonably certain to be followed in practice, no residue of
such [substance] will be found (by methods of examination prescribed or approved
by the Secretary by regulations), in any edible portion of such animals after slaughter
or in any food yielded by or derived from the living animals [see FDC Act, Sections
409 (c)(3)(A)(ii), 512 (d)(1)(I)(ii), and 721 (b)(5)(B)(ii)]. Under 21 CFR 500.84 (c)(2),
no residue of a carcinogenic compound is operationally defined. Using a bioassay with
an animal model, the maximum lifetime risk of cancer to the test animal of one in
one million is determined. The conditions of use of the compound, including any
required preslaughter or milk discard time, are set to ensure that the concentration
of the residue of carcinogenic concern in the total diet of people does not exceed a
concentration calculated to correspond to the one in one million risk of cancer in the
animal model. As total diet is not derived from food-producing animals, the FDA
adjusts the concentration to account for food intake.

The 1968 Animal Drug Amendments consolidated the regulation of new animal

drugs in one section of the FDC Act, Section 512. Under Section 512 (b)(1), any
person may file with the Secretary an application with respect to any intended use
or uses of a new animal drug. Such a person shall submit to the Secretary as a part
of the application . . . (G) a description of practicable methods for determining the
quantity, if any, of such drug in or on food, and any substance formed in or on food,
because of its use. Sections 409 and 721 of the Act, addressing food additives and
color additives, respectively, contain similar language. Although Sections 409, 512,
and 721 of the Act and their implementing regulation vary slightly in wording, they
have a common purpose – ensuring the safety of residues that people will consume
from tissues of treated animals. Therefore, the FDA believes that the same testing
requirements should apply to a new animal drug used in, or a food additive or color
additive fed to, a food-producing animal.

The FDA requires [FDC Act, Section 512 (b)(1)(G)] that methods used for

the detection and confirmation of drug residues in animal products be practicable.
Overseeing the reliability of these methods is the responsibility of the FDA CVM. The
methods are corroborated using an interlaboratory evaluation of the method known as
a method trial. The method trial is used to demonstrate that the method is suitable for
use to detect and confirm drug residues and can be performed by a trained analytical
chemist.

Generally a new animal drug is considered to be unsafe and edible animal products

containing residues of the drug are considered adulterated unless an approved New
Animal Drug Application (NADA) is in effect. Before approving a carcinogenic
or noncarcinogenic compound for use in food-producing animals, CVM asks that
the sponsor provide an acceptable analytical method (either chemical or biological)
capable of reliably measuring the drug residue of concern to ensure that the total
residue of toxicological concern is not exceeded. Methods to detect and confirm drug
residues are reviewed as part of the application process for food animal drugs and feed
additives. Methods that are developed and validated as part of a NADA are referred
to as NADA methods, and the process of validating these methods is referred to as

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Regulatory and scientific consideration for residue analytical methods

the NADA method trial. This process is more fully explained in Section V of FDA
Guideline for Approval No. 3.

1

The NADA method approval process consists of three phases: (1) method devel-

opment by the sponsor and generation of information to establish that the method
satisfies acceptability criteria; (2) FDA review of the sponsor’s data to determine
suitability of the method; and (3) the ‘method trial’, an inter-laboratory study, which
determine whether the method meets performance criteria when used in multiple
laboratories. The inter-laboratory method trial procedure provides an indication of a
method’s ability to be used as a practicable and reliable regulatory tool. Sponsors are
urged to develop methods that are rugged and exceed rather than meet the minimal
standards of acceptability. Those methods that appear marginally acceptable after
review often do not pass the inter-laboratory method trial.

NADA methods should be capable of reliably measuring an analyte (i.e., the marker

residue) that has a defined quantitative relationship to the total residues of toxicolog-
ical concern in the tissues of interest, namely the target tissue and muscle. The target
tissue is generally the last tissue in which total residues deplete to the permitted
maximum safe concentration. When the marker residue is at the tolerance, a defined
unique concentration, the total residues have depleted to the respectively established
safe concentrations in the target tissue and muscle.

The FDA often develops methods to detect drug residues to support other regulatory

needs. In certain instances, new animal drugs may be used legally for unapproved
uses, i.e., extra-label use. For example, an approved new animal drug may be used
to treat a disease in a species other than the approved species. In addition, animal
drugs are sometimes used in an illegal manner in food animals (e.g., clenbuterol
or phenylbutazone in food-producing animals). The FDA may need to develop a
method to detect unsafe levels of drugs resulting from extra-label or illegal uses.
Furthermore, methods may become obsolete with the rapid changes in technology.
For many species, the number of approved drugs is very limited. For example, only five
drugs are approved for use in aquaculture. Drug residue methods developed for use by
the FDA outside of the drug approval process are known as non-NADA methods. As
with sponsor-developed methods, FDA developed methods require validation. These
FDA methods are validated using a process know as the non-NADA method trial.

The non-NADA method trial process mirrors the NADA process. Methods are

developed, reviewed for scientific and technical soundness, and validated in multiple
laboratories, and the data generated are analyzed to determine if the method is suitable
for its intended use.

2

The method

A method should be able both to quantify the amount of marker drug residue present
in the sample and to identify the compound unambiguously. Historically, this required
two distinct procedures: a determinative procedure used to quantify the analyte, and
a confirmatory procedure used to unequivocally identify the analyte. The need for
two procedures was driven by the limitations of available technology. Most determi-
native methods over the last two decades have been based on liquid chromatography,
usually with ultraviolet (UV)/visible or fluorescence detection. Limitations of cost,

The process of development and validation of animal drug residue methods

79

availability, and technology prevented the regulatory use of mass spectrometry for
quantitation. Even with recent advances in liquid chromatography/mass spectrometry
(LC/MS), the need for separate determinative and confirmatory procedures has not
been totally alleviated. Often two separate LC/MS analyses are required, one opti-
mized for quantitation and the other for confirmation.

2.1

Determinative procedures

By definition, the determinative procedure must be able to quantify the concentration
of the marker residue. For compounds with a tolerance, it is critical that the analysis
be able to determine accurately if the concentration of the marker residue is above
or below the tolerance in the target tissue. The CVM guidelines

1

for determinative

procedures call for an average recovery

≥80% with a coefficient of variation (CV)

of

≤10% for marker residue tolerances of 100 µg kg

−1

or greater and an average

recovery of

≥60% with a CV of ≤20% for marker residues with a tolerance below

100 ppb.

Most determinative procedures are based on chromatographic techniques. Because

of the amphoteric nature of most animal drugs, derivatization is necessary for analysis
by gas chromatography. Therefore, liquid chromatography with UV or fluorescence
detection is usually the method of choice. Although practical quantitative LC/MS
has been available since the mid-1990s, sponsors have only recently been proposing
quantitative procedures based on this technology. Two factors have contributed to
the slow adaptation of quantitative LC/MS procedures: limited availability of LC/MS
instrumentation in government laboratories, and the significant lag time between a
sponsor developing an analytical procedure and the submission of the procedure to
the Agency as part of a proposed regulatory method. In the future, LC/MS will likely
become the dominant technique used for the quantitation of drug residues owing to
its inherent advantages, including simplified sample preparation procedures.

2.2

Confirmatory procedures

The ability to identify a drug residue positively and unambiguously is needed to
support any legal action that the FDA may take against the person responsible for an
illegal residue. Historically, mass spectrometry (MS) has been the method of choice
for confirmatory methods. MS provides structural information about the residue and
is well suited for working with sample concentrations in the parts per million or billion
range that are typically observed in residue analysis. Additionally, both the animal
drug industry and the FDA have extensive knowledge and expertise in the use of MS
for the confirmation of drug residues in animal products.

The confirmatory procedure should be developed for the same tissues for which the

determinative procedure was developed, preferably using the same extraction proce-
dure as used for the determinative portion of the method. Storage and stability data
are necessary for dried or liquid sample extracts if MS analyses of the confirmatory
samples are to be conducted in a laboratory other than the laboratory of sample prepa-
ration. Analytes present in sample extracts must be stable long enough for the samples
to be shipped to the MS laboratory and analyzed.

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Regulatory and scientific consideration for residue analytical methods

The FDA does not prohibit the use of other techniques that could provide unambigu-

ous structural information such as Fourier transform infrared (FTIR) spectrometry.
However, the requirement for relatively large amounts of sample for an analysis has
limited the use of such techniques.

Historically, the Agency has allowed the use of orthogonal (mutually exclusive)

chromatographic techniques for confirmatory procedures. However, confirmation of
residues by multiple chromatographic procedures has been rarely utilized. The use of
an independent chromatographic technique is an option of last resort. The FDA has
not approved the use of an orthogonal chromatographic technique unless the sponsor
demonstrates that the use of more specific techniques such as MS is not a viable option
for the residue in question. Since the advent of practical LC/MS systems in the 1980s,
no orthogonal chromatographic techniques for confirmation have been approved for
regulatory use.

3

Development of methods for regulatory use

A drug sponsor or a government laboratory developing a regulatory method should
design the method based on its intended use. The successful validation of a method
begins with considering the required elements for a regulatory method and incorpo-
rating them from the start of method development.

3.1

Practicability of methods

One of the primary requirements for methods is that it be practicable
[Section 512(b)(1)(G)]. A method that cannot be used in Federal laboratories has
no value in the protection of the food supply. Method developers should avoid the
use of rare or custom-made equipment, prohibitively expensive equipment, untested
technologies, or reagents that are not commercially available. For a determinative
procedure, an analysis should not exceed two working days, and methods should
have a minimum sample throughput of at least six samples per analyst-day.

3.2

Analyte selection

The sponsor of a NADA is responsible for generating the data needed to determine
the marker residue, tolerance, and target tissue. Typically, this requires a variety of
studies using a radioisotope-labeled drug to generate information on total drug residue
concentration, metabolites, and residue depletion. The tolerance for the marker residue
is based on concentrations of that residue using the proposed regulatory method
without correction for recovery. Because of the manner in which the tolerance is set,
the tolerance is a method-dependent value. Therefore, alternative methods developed
for a NADA drug with an approved tolerance should have a bridging study. The new
method should generate results for the marker residue in a known relationship to those
determined using the NADA method.

The process of development and validation of animal drug residue methods

81

For non-NADA methods developed for unapproved drugs or unapproved uses of

a drug, data required to set a tolerance may not be available. Although some data
may exist in the literature to aid in choosing an appropriate residue to monitor, basic
information is often lacking. Often, the parent molecule is chosen as the monitored
analyte. For the majority of compounds, at least some parent drug residue is assumed
to be present in the target tissue. Owing to a paucity of toxicological data for many
compounds, the limit of detection for the method is set as low as is practical. The
FDA will consider all available data on the metabolism and toxicity of the compound
in setting these values.

3.3

Specificity

Specificity is the ability of the method to measure accurately the analyte response in the
presence of potential analyte interference that might be expected to be in a sample.
Specificity is determined by comparing the detector response of a sample extract
containing potential interference (e.g., drug metabolites, other animal health drugs,
synthetic intermediates of the target drug, degradation products of the target drug,
etc.) with the detector response of a solution containing only the analyte. In addition,
chromatographic procedures should be tested using sample extracts from control
animals from various regions of the country to determine if regional differences affect
the matrix components observed in sample extracts. For guidance, CVM recommends
that interferences of no greater than 10% of the response of the marker residue at
tolerance (for an analyte with an approved tolerance) be present in control matrix
chromatograms.

3.4

Ruggedness

One of the key aspects in developing a method for regulatory analysis is method
ruggedness. The more rugged a method, the less susceptible it is to failure or to
excessive variations due to differences in equipment, analyst technique, and other
differences that are typically present among laboratories. Several factors contribute
to poor method ruggedness: insufficient testing by the developer, excessive method
complexity, and a failure of the developer to identify and communicate critical
points.

Insufficient testing is one of the major causes of method failure. The amount of

data needed to publish a new procedure in a peer-reviewed journal and the procedural
detail supplied therein are often insufficient to allow a different user to validate a
method rapidly. The developer should evaluate if the method will work using chem-
icals, reagents, solid-phase extraction columns, analytical columns, and equipment
from various vendors. Separate lots of specific supplies within a vendor should be
evaluated to determine if lot-to-lot variation significantly impacts method perfor-
mance. Sufficient numbers of samples should be assayed to estimate the lifetime of
the analytical column and to determine the effects of long-term use on the equipment.

A complex method with many steps, compared with a simple straightforward pro-

cedure, is likely to have many more critical steps that need to be well defined with

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Regulatory and scientific consideration for residue analytical methods

proper control limits. Increased method complexity multiplies the work of the de-
veloper in identifying and defining critical steps, and also increases the probability
that control points for a critical step will not be identified properly. Although method
design is primarily driven by the required method detection limit and resources avail-
able to the developer, method developers should consider strategies to minimize the
complexity of a method in the early design stages.

The method developer should identify critical points in the method. Frequently,

the Youden test

2

may be used to determine if temperature, time, flow rate for solid-

phase extraction, weight, volume, and other variables in the method are critical. The
developer needs to identify if it is acceptable to take a break during a procedure, length
of the break, and steps that need to be completed quickly. Because of differences in
background and training between analysts, method developers should not assume
that other analysts will perform a technique in the same way as in the developer’s
laboratory. Often analysts will have different interpretations of simple terms such as
‘shake’, ‘slow’, ‘complete’, and ‘fast’.

3.5

Stability

The method developer should evaluate the stability of the analyte in the target tissue
after short- and long-term storage (days to months) and through multiple freeze and
thaw cycles. This assessment should also include an evaluation of analyte stability in
stock solutions at the appropriate concentrations and storage conditions. The devel-
oper should consider the stability of the analyte during the extraction procedure and
during the time a sample may be on an autosampler awaiting chromatographic anal-
ysis. Storage stability should be evaluated for fortified and incurred tissue residues
at an appropriate temperature (freezer or refrigerator) for a length of time in excess
of the time between sample collection and analysis of the last sample. Freeze–thaw
analysis should include at least three cycles. Fortified and incurred residue samples
should be frozen for at least 24 h and then thawed unassisted at room temperature. The
completely thawed samples should be refrozen for 12–24 h; the freeze–thaw cycle
should be repeated two more times and the samples analyzed. If the analyte is deter-
mined to be unstable, the number of cycles can be shortened or the freezer storage
temperature may be lowered to determine the appropriate stability.

3.6

System suitability

System suitability defines the critical performance characteristics that a method must
meet to show that it is being used correctly and can generate acceptable data. Re-
tention times, linearity of calibration curves, peak shape, recovery of analyte from
fortified samples, and background in control samples are some of the factors ad-
dressed by system suitability. Setting system suitability criteria is often a balancing act.
Criteria set either too stringently or not stringently enough can lead to method failure.
Overly stringent criteria can create a situation where even a well-qualified analyst is
unable to meet the suitability criteria. Overly lax criteria can make poor workmanship
acceptable, and allow flawed results to be considered valid. For example, failure to

The process of development and validation of animal drug residue methods

83

define the minimum resolution between the analyte and interfering peaks can lead to
the analyst accepting results from an analysis in which the peaks had partially merged.
The developer needs to determine the performance criteria for the method and base
the system suitability on these criteria.

4

Method criteria

Prior to submitting a method for trial, the sponsor should develop data demonstrating
the performance of the method. At a minimum, the following sample sets for the
target tissue should be evaluated for a determinative procedure:

r

five control samples

r

five control samples fortified with the marker residue at half the tolerance concen-
tration

r

five control samples fortified with the marker residue at the tolerance concentration

r

five control samples fortified with the marker residue at twice the tolerance con-
centration

r

10 incurred tissues (two concentrations with five at each) containing residues be-
tween half and twice the tolerance concentration generated by treating animals
with the drug.

For confirmatory procedures, the fortified sample sets at half and twice the tolerance
are not required.

4.1

Standards

Standards should be analyzed contemporaneously for both determinative and con-
firmatory procedures. The method developer needs to describe fully the preparation
of all the standards and the calibration procedure to be used, such as calibration
prior to sample analysis, interspersed standards, or bracketing standards (confirmatory
only).

The use of standards prepared in control matrices is typically not allowed for deter-

minative procedures because control tissues are not routinely available to regulatory
laboratories. When a matrix effect alters the spectrum or chromatography of an ana-
lyte relative to the pure standard, so that confirmatory criteria cannot be met, a control
extract containing standard may be substituted for pure standard. Justification, with
CVM concurrence, should be provided for confirmatory methods that use fortified
control extracts.

4.2

Precision

The precision of an analytical method is a measure of the variability of repetitive
measurements. Contributions from numerous sources affect precision, but the ma-
jor components are within-laboratory (repeatability) and between-laboratory (repro-
ducibility) variations. Precision is expressed as the relative standard deviation (or CV)

84

Regulatory and scientific consideration for residue analytical methods

because it is relatively constant over the concentration range of interest. For determi-
native procedures, CVM guidance allows a within-laboratory CV of

≤10% when the

tolerance is

≥100 µg kg

−1

. When the tolerance is

<100 µg kg

−1

, the within-laboratory

CV should be

≤20%. To be meaningful, precision should be determined using the

exact sample and standard preparation procedures that will be followed in the final
method.

4.3

Accuracy

Accuracy (systematic error or bias) expresses the closeness of the measured value to
the true or actual value. Accuracy is usually expressed as the percentage recovery of
added analyte. Acceptable average analyte recovery for determinative procedures is
80–110% for a tolerance of

≥100 µg kg

−1

and 60–110% is acceptable for a tolerance

of

<100 µg kg

−1

. Correction factors are not allowed. Methods utilizing internal stan-

dards may have lower analyte absolute recovery values. Internal standard suitability
needs to be verified by showing that the extraction efficiencies and response factors of
the internal standard are similar to those of the analyte over the entire concentration
range. The analyst should be aware that in residue analysis the recovery of the fortified
marker residue from the control matrix might not be similar to the recovery from an
incurred marker residue.

4.4

Other considerations

Adequate sensitivity should be demonstrated and estimates of the limit of detection
(LOD) and the limit of quantitation (LOQ) should be provided. The slope of the
calibration line may indicate the ability of the method to distinguish the true analyte
concentration. The LOD of a method is the lowest analyte concentration that produces
a reproducible response detectable above the noise level of the system. The LOQ is the
lowest level of analyte that can be accurately and precisely measured. For a regulatory
method, quantitation is limited by the lowest calibration standard. The techniques for
these estimations should be described.

The FDA requests that the method exhibit sufficient sensitivity to measure ac-

curately the residue of interest after fortification of the control matrix at half the
tolerance concentration. Minimally, the detector response at the tolerance should be
at least 10 times the average background response.

Linearity verifies that sample solutions are in a concentration range in which the de-

tector response is linearly proportional to analyte concentration. Current FDA guide-
lines call for establishing linearity. For regulatory methods, this is generally performed
by preparing standard solutions at four or five concentrations, from 30 to 200% of
the tolerance.

Linearity is often assessed by examining the correlation coefficient (r) [or the

coefficient of determination (r

2

)] of the least-squares regression line of the detector

response versus analyte concentration. A value of r

= 0.995 (r

2

= 0.99) is generally

considered evidence of acceptable fit of the data to the regression line. Although
the use of r or r

2

is a practical way of evaluating linearity, these parameters, by

The process of development and validation of animal drug residue methods

85

themselves, may be misleading and should not be used without a visual examination
of the response versus concentration plot. A determination of standardized residuals
may be a better estimate of system linearity.

4.5

Confirmatory procedure criteria

For the confirmatory procedure, it is recommended that the sponsor develop spectral
data based on at least three structurally specific ions that completely define the marker
residue molecule. These ions may or may not include the molecular ion. The use of
water loss and isotopic ions is usually unacceptable and CVM concurrence should be
sought when water loss ions or isotopic ions are selected for the confirmatory analysis.
The proposed fragment ion structures should be consistent with the fragmentation
pattern, and justification for specificity of selected ions or scan range should be
included. All confirmation criteria should be specified in the standard operating
procedure.

Selected ion chromatographic peak(s) should exceed a signal-to-noise ratio (S/N)

threshold of 3 : 1. The technique used for estimating S/N should be included. Criteria
for retention time (t

R

) matching should be specified. The t

R

criteria should not exceed

2% for gas chromatography/mass spectrometry (GC/MS) or 5% for LC/MS, relative
to the retention time of the standard. The relative abundance for three structurally spe-
cific ions should match the relative abundance of the reference standard within 10%
(arithmetic difference, not relative difference). For example, at 50% relative abun-
dance, the corresponding window would be 40–60%, not 45–55%. These guidelines
apply to selected ion monitoring. Currently, the FDA is in the process of updating
the guidance on the use of MS for the confirmation of drug residues to address issues
regarding confirmation using MS/MS techniques and the matching of full-scan and
partial-scan spectra.

3

To be acceptable, the confirmatory procedure should confirm

the presence of the analyte in all fortified and incurred samples (no false negatives)
at or above the tolerance, and fail to confirm the presence of the drug in all control
samples (no false positives).

5

Standard operating procedures (SOPs)

5.1

Determinative procedure

The format for analytical methods proposed as the regulatory method should be clear
and should contain all necessary information needed successfully to perform the
laboratory steps and calculate the results. The following is a recommended format
for a determinative procedure:

A. Title. A descriptive title should be provided.
B. Scope. The analytes measured and the applicable matrices should be included.

The reason why the method is being submitted for regulatory evaluation should
be explained. The advantages of the method over existing methodology should be
included.

86

Regulatory and scientific consideration for residue analytical methods

C. Principles. The physical and chemical principles of the method should be described.

The structure of the new animal drug should be provided.

D. Reagents. All the reagents (including grade) used in the procedure and their prepa-

ration should be listed in this section. If the method uses reagents that may be
limited in supply or availability, the specific source of these reagents and ordering
information should be provided. Any critical sources or types of reagents should
be identified.

E. Equipment. The equipment required should be included along with the manufac-

turer and model information. All equipment should be commercially available. If
equivalent equipment is available, it should be listed or criteria provided to judge
the acceptability of equivalent equipment.

F. Procedure. The procedure section should unambiguously describe the step-

wise preparation of samples, standards, and blanks. Instrumental variables should
be described. Weight and volume measurements should include the acceptable
range. The procedure should also include methods for any calculations. Proce-
dures should include, but are not limited to, the following recommended elements:

1. Standard preparation

a. Extraction
b. Cleanup
c. Dilution

d. Other

2. Controls and fortification sample preparation

a. Blank reagents
b. Control matrix preparation
c. Fortification procedure for control matrix

3. Sample preparation

a. Extraction
b. Cleanup
c. Dilution

d. Other

4. Instrument operating variables

a. Instrumental configuration
b. Monitored response
c. Specific operating conditions

5. Procedure for instrumental analysis of samples, controls, and standards.
6. Calculations: all dilution factors and calculation parameters should be clearly

explained. An example calibration curve should be provided.

7. System suitability information. Minimum requirements for instrument accept-

ability and any critical operating parameters should be identified.

G. Quality control information. All critical points, with recommended control

procedures and performance criteria, should be identified. If applicable, stopping
point(s) should be indicated. Performance specifications for instruments and stan-
dard materials should be included. Recommended actions to be taken if perfor-
mance does not meet the acceptance criteria need to be provided. Sample handling

The process of development and validation of animal drug residue methods

87

instructions and information on the stability of the analyte in the biological matrix
and final extracted samples should be included. Any additional items that the user
will need to duplicate the performance of the method set by the developer should
be included.

H. Safety considerations. The Occupational Safety and Health Administration

(OSHA), US Department of Labor, standard entitled ‘Occupational Exposure to
Hazardous Chemicals in Laboratories’ (29 CFR 1910.1450) makes it necessary
to address safety issues in the SOP. The standard requires laboratories that use
hazardous chemicals to maintain employee exposures at or below the permissi-
ble exposure limits specified for these chemicals in 29 CFR Part 1910, Subpart
Z. Hazards associated with any specific chemicals used in a method must be ad-
dressed so that the user has the information needed to follow the Chemical Hygiene
Plan for their laboratory. The method developer should limit the use of hazardous
chemicals where feasible. The use of toxic and/or carcinogenic reagents should
be avoided or eliminated as much as possible. Additionally, the cost of disposal
is increasing and could impact the practicality of a method. Material Safety Data
Sheets for the analyte(s) and any unusual or hazardous reagents should be provided
for the user.

5.2

Confirmatory procedure

The confirmatory procedure should follow the same format as the determinative
procedure, but also include the following additional information:

1. Method principles should include the technique used for mass spectral data acqui-

sition.

2. Structure and full spectrum of the marker residue should be included.
3. Spectral data based on at least three structurally specific ions that completely

define the parent molecule (may or may not include the molecular ion), or more if
nonspecific ions are included. Use of water loss and isotopic ions is discouraged.

4. Proposed fragment ion structures, consistent with fragmentation pattern.
5. Justification for specificity of selected ions or scan range.
6. Instrument operational settings. This includes settings such as zone pressures,

temperatures, voltages, and flow variables. If alternative instruments may be used,
their operational variables should be listed.

7. Confirmation criteria specified in advance.
8. Operational criteria for repeat injection of the same sample.
9. Estimate of concentration limits for confirmation in matrix.

5.3

Other considerations

At least one negative control and one positive control should be run each day. The
positive control should meet recovery or confirmation criteria. The negative con-
trol should have no interferences greater than those specified in the determinative

88

Regulatory and scientific consideration for residue analytical methods

procedure and must fail confirmation criteria for the day’s analyses to be valid. As
part of method development, sufficient blanks or negative controls should be analyzed
after standards or positive samples to ensure that carryover does not cause a false-
positive result. If necessary, the indicated number of blanks to be inserted between
samples should be added to the SOP.

The method should define all criteria used to determine if an analysis is valid

and the data are acceptable as part of the SOP. The analyst may not substitute or
modify criteria used to determine the acceptance of data after an analysis has been
completed.

6

The method trial

6.1

Second analyst/laboratory check

Prior to a method trial, the FDA strongly recommends that a second analyst or inde-
pendent laboratory perform the method. The independent analyst is asked follow the
method SOP as written. This analyst should not have been involved in developing the
method or be familiar with it in any way. The purpose of the independent analysis is
to determine if a qualified chemist can perform the method described without input
other than that provided in the written instructions. This ‘trial run’ will typically iden-
tify problems with the SOP that are not apparent to the method developer. Although
not required by the FDA, the independent assessment can identify potential problems
with the method SOP prior to the lengthy and costly method trial. A ‘trial run’ offers
the method developer an opportunity to correct problems and to increase the proba-
bility that subsequent method trials will be successful. Finally, the method developer
should realize that the variability achieved in his/her laboratory is often less than that
realized by less experienced analysts. If a method cannot achieve a suitable degree of
repeatability in the developer’s laboratory, it should not be expected to do any better
in other laboratories.

6.2

FDA review

The FDA reviews a method prior to trial to ensure that the data submitted by the
sponsor support the conclusion that the method is suitable for trial. The sponsor
should include the following: (1) a complete stepwise, unambiguous description of
the method including reagents, apparatus, sampling procedures, preparation of stan-
dards and analytical samples, storage conditions, and identification of critical steps
and/or stopping points; (2) system suitability criteria to verify and maintain method
performance; (3) a typical calibration curve; (4) individual and summary results de-
rived from control, fortified, and incurred residues in the matrix showing that the
method meets the specificity, precision, and recovery requirements; (5) raw data
and intermediate results including relevant worksheets, calculations, chromatograms,
statistical analyses, mass spectrograms, selected ion monitoring data, etc., from the
analyses of control, control fortified, and incurred target tissues.

The process of development and validation of animal drug residue methods

89

6.3

Inter-laboratory method trial

The method trial process for NADA methods is different to the process for non-NADA
methods. However, the validation protocol followed by the participating laboratories
and the requirements for acceptance of the method are the same. The trial process also
differs for determinative procedures and confirmatory procedures. Determinative pro-
cedures are evaluated using the multiple laboratory process, whereas the confirmatory
method needs to be evaluated only in a single government laboratory.

The evaluation of all NADA analytical methods was previously conducted exclu-

sively by the CVM. Since 1995, the CVM has offered sponsors of NADA residue
methods the option of conducting the method trial through a Sponsor Monitored
Method Trial (SMMT) process. The SMMT is conducted according to CVM speci-
fications with CVM oversight. The resultant performance data must be reviewed and
judged acceptable by CVM before the method is approved.

In the SMMT process, draft protocols are reviewed, and guidance provided to the

sponsor to help ensure that the format and specifications are adequate. The protocol
should be approved by CVM prior to the initiation of the method trial. Once the
protocol and method description are acceptable to CVM, the methods are sent to
the participating laboratories for review, and a method demonstration is scheduled.
The method demonstration, attended by all participating laboratory analysts, involves
review of the study protocol and method SOP and a laboratory demonstration of the
method. Ideally, all revisions are completed by the end of the demonstration and the
study protocol is signed.

At a minimum, the method will be tested in one FDA laboratory and two con-

tract laboratories selected by the sponsor. If the method is for a new animal drug in
tissue regulated by the United States Department of Agriculture (USDA) as part of
the meat inspection program, a Food Safety and Inspection Service (FSIS)/USDA
laboratory will be included if sufficient resources are available. The method trial
will be conducted using control and incurred target tissues that are supplied by
the sponsor. The sponsor may, on request, supply new or unusual reagents or
standards.

Each of the three laboratories analyzes the same sample sets that the developer was

required to analyze during method development:

r

five control samples

r

five control samples fortified with the marker residue at half the tolerance concen-
tration

r

five control samples fortified with the marker residue at the tolerance concentration

r

five control samples fortified with the marker residue at twice the tolerance con-
centration

r

10 incurred tissues (two concentrations with five at each) containing residues be-
tween half and twice the tolerance concentration generated by treating animals
with the drug.

If a separate confirmatory procedure is necessary, the analysis will be conducted in an
FDA laboratory. The sponsor may have one or more of the contract laboratories test

90

Regulatory and scientific consideration for residue analytical methods

the confirmatory procedure, but the confirmatory procedure must pass in the Federal
laboratory for acceptance. Each of the laboratories analyzes the following samples:

r

five control samples

r

five control samples fortified with the marker residue at the tolerance concentration

r

10 incurred tissues (two concentrations with five at each) containing residues be-
tween half and twice the tolerance concentration generated by treating animals
with the drug.

During the conduct of the method trial, participating laboratories are instructed to

use the method as written. Because the goal of the trial is to evaluate the method
as written in the SOP, one of the major challenges is to ensure that the participants
do not try to improve or modify the method. Minor modifications to accommodate
available equipment are allowed in the contract laboratories with the concurrence of
the method sponsor. Any deviations from the method by the contact laboratories are
to be reported to the sponsor’s Study Director for the trial, the CVM method trial
coordinator, and are to be listed in the study final report. Modifications or deviations
conducted by the government laboratory(ies) will require the concurrence of the
CVM method trial coordinator, the FDA reviewer overseeing the conduct of the trial.
The trial is conducted in three phases. At the completion of each phase, the Study
Director for the trial, or the CVM trial coordinator, reviews the results and gives
the analysts in the participating laboratories the clearance to proceed to the next
phase.

In the first phase, the performance of the instrumentation used for the method is

demonstrated. Based on the analysis of standards, results from the participating lab-
oratory should meet the system suitability requirements of the method. Successful
completion of this phase will qualify the analyst, his or her equipment, and the lab-
oratory for the trial. Failure in the first phase does not usually cause a method to
fail the trial. However, it can slow the process. When a procedure fails during the
first phase of a trial, the sponsor may need to write a cautionary note in the SOP dis-
cussing recommended or inadequate types of instruments. To correct the problem, the
participating laboratory analyst can substitute equipment that gives adequate perfor-
mance; alternatively, the sponsor must find a different laboratory to participate in the
trial.

In the second phase, analysts in participating laboratories prepare and analyze a

minimum of two control samples and two samples fortified at the proposed toler-
ance concentration. This phase allows analysts to become familiar with the method
before the analysis of samples that will be part of the method validation. Results
from the second phase should demonstrate that the control samples are without
interference and that the analysts in the participating laboratories can achieve ac-
ceptable recovery of analyte from the samples. It is not uncommon for an analyst
to have to repeat the second phase several times before adequate results are ob-
tained. Failure at this phase of the trial can cause a method to fail the trial. Often the
problems are related to a poorly written SOP that does not adequately describe the
procedure.

The third and last phase of the trial is the analysis of the validation samples. All

data collected are reported. No results are discarded unless a determinate error can
be identified. Any request to repeat the assay of a sample should be approved by

The process of development and validation of animal drug residue methods

91

the sponsor’s Study Director and/or the CVM method trial coordinator. The analyst
completes the analysis of the sample set described earlier in this section.

Following the completion of the trial, each participating contract laboratory pro-

vides a report of their results to the method trial Study Director. The government
laboratory(ies) provide their results to the CVM method trial coordinator. The spon-
sor compiles the final results from participating laboratories into a summary report.
A final version of the SOP is also provided that includes any revisions made be-
cause of observations made during the trial. The summary report, electronic and
hard copies of all laboratory results, work sheets, and reports from each of the
participating laboratories are sent to CVM for final review and acceptance. This
should include electronic copies of all information necessary to verify all of the
results.

6.4

Confirmatory procedure method trial

Confirmatory procedures are evaluated differently from determinative procedures
because of the different intended uses of the procedure. The primary differences are
the testing laboratories and evaluation of the resulting data. Because a confirmatory
procedure is needed for legal action, the procedure will be evaluated based on the
results obtained in a government laboratory.

Another difference between determinative and confirmatory method trial proce-

dures is the way in which sample extracts are prepared for analysis. Most current
methods submitted for review use the same sample extraction technique for both the
determinative and confirmatory procedures. In those cases where the same extraction
technique is used, the sponsor may provide the prepared extract to the FDA laboratory
for analysis. Any problems with the extraction procedure will have been corrected
during the determinative method trial.

The final difference is that the FDA analyst alone makes the recommendation based

on the data for the acceptance of the confirmatory procedure. The conclusion of the
analyst stating the suitability of the procedure for confirming the presence of the
marker residue is sent directly to the CVM method trial coordinator in the Office of
New Animal Drug Evaluation (ONADE) and not back to the sponsor as with the
determinative procedure.

6.5

Non-NADA method trial

The FDA coordinates the method trial process for non-NADA methods. The sample
requirements are the same as for the NADA trials. Non-Federal laboratories such
as contract laboratories and State laboratories can participate in the process. For a
single-residue method, the minimum numbers of samples and laboratories are the
same as for NADA method trials.

Non-NADA methods may be designed to detect multiple residues and they may be

designed for use in multiple species. In order to validate these multi-residue methods,
modifications to the validation protocol relative to single analyte methods are made.
Additional laboratories will participate in the method trial, but the number of samples

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Regulatory and scientific consideration for residue analytical methods

analyzed at each facility will be decreased. For example, five laboratories may assay
only three fortified samples at each concentration. Incurred residue samples can be
blended so that multiple residues will be present in the same sample. Two levels of
all incurred residues may not be included. However, each laboratory analyzes more
‘blinded’ samples than it would in a single-residue method. Because analysts are
blinded to sample contents and because samples may contain one or more analytes,
data generated from the evaluation of blinded samples for a multi-residue trial clearly
demonstrate the suitability of the method for regulatory use.

6.6

Evaluation of data and recommendation for use

Guidelines for acceptability of NADA and non-NADA methods are the same. For
the determinative procedure, the criteria described in ‘Method Criteria’ for accuracy
and precision are used to evaluate data generated at participating laboratories. There
are no criteria for accuracy in the analysis of the incurred residue samples; however,
the overall data set is reviewed to see if there is general agreement between results
obtained by contract laboratories and relative to the levels reported in the sponsor’s
laboratory.

On occasion, results from one of the participating laboratories will fail to meet

established acceptability criteria. In those cases, acceptance or rejection of the method
is determined by the CVM based on overall method performance. For example, a
method that has borderline but acceptable performance for both precision and accuracy
at two of three participating laboratories and fails badly at a third laboratory would
probably fail. A method that was a borderline failure in one laboratory but easily
passed in the other laboratories could be accepted.

For confirmatory methods, the confirmatory procedure criteria described previously

should be met. All negative control samples should fail to meet the confirmation
standard established in the procedure. All samples fortified at or above the tolerance
and all incurred residue samples at or above the tolerance should meet the confirmation
standard (to confirm) described in the SOP. It has been argued that it is not necessary
for incurred samples containing the marker residue at a concentration below the
tolerance to meet established confirmatory criteria. However, failure to confirm the
marker residue in these samples may indicate a lack of robustness of the procedure.
Any procedure that had this problem would be closely examined to ensure that the
method would meet the needs of the Agency.

7

Conclusion

The method trial process is long and involved. The primary purpose of the process
is to ensure that the FDA and the FSIS have the tools needed to both monitor the
Nation’s food supply. An acceptable method allows regulators to take regulatory
and/or criminal action against those who illegally use drug products in food animals.
Method trials are designed to ensure that the method is sufficiently defined so that
it can be successfully used in a government laboratory on short notice by an analyst
who may have little or no experience with the procedure. Usually, the analyst in the

The process of development and validation of animal drug residue methods

93

sponsor’s laboratory will have extensive experience with the method, having tested
hundreds of samples as part of the studies done to support the NADA. Even for
non-NADA methods, the method developer will have become an expert in the proce-
dure during the development process. A successful method trial tests and enhances
a method established in an expert laboratory and establishes a SOP for the method.
The goal of the process is to provide government data to support the conclusion that
the government will have a practicable method to enforce the Food Drug & Cosmetic
Act for the animal drug residues determined and confirmed by the method.

References

1. Guideline for Approval No. 3: General Principles for Evaluating the Safety of Compounds Used

in Food-producing Animals. Revised July 1994. US Department of Health and Human Services,
Public Health Service, Food and Drug Administration, Center for Veterinary Medicine, Rockville,
MD (1994).

2. W.J. Youden and E.H. Steiner, ‘Statistical Manual of the AOAC’, AOAC International,

Gaithersburg, MD p. 33 (1975).

3. Guidance for Industry No. 118: Mass Spectrometry for Confirmation of the Identity of Animal

Drug Residues DRAFT GUIDANCE June 6, 2001, US Department of Health and Human Services,
Public Health Service, Food and Drug Administration, Center for Veterinary Medicine, Rockville,
MD (2001).