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Guidance for Industry 
Mass Spectrometry for Confirmation 
of the Identity of Animal Drug Residues 
FINAL GUIDANCE 
This final guidance document is intended to provide specific guidance for the development, 
evaluation, and application of mass spectrometric methods for confirming the identity of 
animal drug residues. It elaborates the description of method specificity in CVM Guidance 
Document 3, General Principles for Evaluating the Safety of Compounds Used in 
Food-producing Animals, Part V, Guideline for Approval of a Method of Analysis for 
Residues, section B.1. 
Comments and suggestions regarding the document should be submitted to Division of 
Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 
1061, Rockville, MD  20852.  Submit electronic comments to http://www.regulations.gov.  
All comments should be identified with the Docket No.O1D-0224.   
 
For questions regarding this final document, contact David N. Heller, Center for Veterinary 
Medicine (HFV-510), Food and Drug Administration, 8401 Muirkirk Rd., Laurel, MD 20708, 
301-210-4579, e-mail: david.heller@fda.hhs.gov. 
Additional copies of this final guidance document may be requested from the 
Communications Staff (HFV-12), Center for Veterinary Medicine, Food and Drug 
Administration, 7519 Standish Place, Rockville, MD 20855 and may be viewed on the 
Internet at 
http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/default.htm  
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
May 1, 2003 
 
    CONTAINS NON-BINDING RECOMMENDATIONS 
FINAL GUIDANCE FOR INDUSTRY: 
MASS SPECTROMETRY FOR CONFIRMATION OF THE IDENTITY OF ANIMAL 
DRUG RESIDUES1 
This guidance represents the Food and Drug Administrations (FDAs) current thinking 
on this topic.  It does not create or confer any rights for or on any person and does not 
operate to bind FDA or the public.  You can use an alternative approach if the approach 
satisfies the requirements of the applicable statutes and regulations.  If you want to 
discuss an alternative approach, contact the FDA staff responsible for implementing the 
guidance.  If you cannot identify the appropriate FDA staff, call the appropriate number 
listed on the title page of this guidance. 
 
 
I. INTRODUCTION 
CVM develops, evaluates, and applies qualitative mass spectrometric methods for confirming 
the identity of animal drug residues.  Methods developed in support of a New Animal Drug 
Application (NADA methods) are designed for residues of an approved new animal drug used 
in the approved manner.  Methods may also be developed for unapproved new animal drugs 
or approved new animal drugs used in an unapproved manner (non-NADA methods).  This 
final guidance document describes the basic principles recommended by CVM for 
developing, evaluating and applying these methods. 
The purpose of this document is to facilitate and expedite coordination between CVM and its 
stakeholders so these activities may be carried out in a consistent and timely manner.  This 
final document does not commit CVM to accepting a specific method or data package prior to 
reviewing the relevant data. This final document is intended for technical professionals 
familiar with mass spectrometry.  Please contact CVM for further information on this 
document or any technical explanations that may be necessary.  For a historical perspective, 
please see the Bibliography.  For definitions of terms used in this document, please see the 
Glossary. 
This guidance document is applicable in the following areas: 
1.  Consultations on confirmatory methodology 
2.  Desk reviews of confirmatory procedures 
3.  Method trials or second-laboratory evaluations of confirmatory procedures 
4.  Development of confirmatory procedures 
                                                 
1 This guidance has been prepared by the Division of Residue Chemistry, Office of Research, Center for 
Veterinary Medicine (CVM) at the Food and Drug Administration. 
  1   CONTAINS NON-BINDING RECOMMENDATIONS 
5.  Desk reviews of data generated with confirmatory procedures 
It is CVMs view that methods should fit the purpose.  This document applies to work done 
for CVMs purposes, and does not necessarily apply to or invalidate work done for other 
purposes.  This guidance applies only if a reference standard is available. 
This guidance document should be used to help in the development of new methods, the 
review of methods submitted to CVM, and in the laboratory trial of methods submitted to 
CVM.  The document should also help in making decisions about appropriate methodology in 
various regulatory situations and ensuring consistency in work done for CVMs purposes.  
This document sets guidance standards and performance specifications as targets.  CVM 
recommends that methods meet or exceed these standards. 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the Agencys current thinking on a topic and 
should be viewed only as recommendations, unless specific regulatory or statutory 
requirements are cited.  The use of the word should in Agency guidances means that 
something is suggested or recommended, but not required.   
II. GUIDANCE 
Where CVM can predict that use of a new animal drug in food animals will likely result in the 
presence of drug residues in edible tissue of the treated animal, a full CVM Confirmatory 
Procedure should be developed and validated.  For cases when a full procedure is unavailable 
and time does not permit a procedure to be fully validated, an Ad Hoc Confirmatory Package 
may be assembled. (See Glossary for these terms.)  The following sections list the specific 
elements that should be addressed in each case. 
CVM Confirmatory Procedures  
CVM Confirmatory Procedures are developed and validated in advance of their application.  
These methods should address each of the following points:   
I.  Validation package from originating laboratory 
A.  Replicate samples 
1.  Five Controls (may be subsamples from one source, but see part 1.F. below.) 
2.  Five Fortified Controls at tolerance/safe level 
3.  Residue-incurred: 
a.  Internal (within-laboratory) validation:  Five per analyte (may be subsamples 
from one source) 
b.  Interlaboratory study (e.g., trial of NADA method):  Ten total (five at each of 
two levels) 
  2   CONTAINS NON-BINDING RECOMMENDATIONS 
B.  Demonstration of zero false positive rate.   
C.  Demonstration of  10% false negative rate at or above the tolerance or safe level is 
recommended (based on fortified and incurred samples).  If this criterion cannot be 
met during method development, contact CVM. 
D.  Demonstration that suitable data can be acquired on more than one day. This helps to 
ensure that the method is rugged and under control.  
E.  Demonstration of non-interference by other animal drugs.  It is CVMs view that 
interference testing should be conducted in proving the specificity of a confirmatory 
procedure.  The selection of drugs for interference testing should be reasonable as 
opposed to exhaustive.  For example, a reasonable selection includes drugs approved 
in the same species, and may include drugs from the same class of compounds, drugs 
of similar molecular weight, or known metabolites and degradants. Some compounds  
may be reasonably excluded based on obvious chemical differences.  For clarification, 
please contact CVM.    
F.  Demonstration of non-interference by matrix components in control samples from 
more than one source (i.e., an individual animal).  Note:  although data to address Part 
I.A.1. may be acquired with subsamples from one source, more than one source should 
be tested. 
  II.  Method Description (Standard Operating Procedure, SOP) 
A.  Scope of applicability 
B.  Method principles, including technique for mass spectral data acquisition. 
C.  Stepwise, unambiguous description of all reagents, apparatus, and steps. 
D.  Structure and full spectrum of marker residue. 
E.  Spectral data based on at least three structurally-specific ions that completely define 
the parent molecule (may or may not include the parent ion), or more if non-specific 
ions are included.  Use of water loss and isotopic ions are discouraged, but will be 
evaluated on a case-by-case basis.  
F.  Proposed fragment ion structures, consistent with fragmentation pattern. 
G.  Justification for specificity of selected ions or scan range.  
H.  System Suitability parameters. 
I.  Confirmation criteria specified in advance (see Section III of this guidance). 
J.  Operational criteria for repeat injection of same sample. 
K.  Estimate of concentration limits for confirmation in matrix.  
  3   CONTAINS NON-BINDING RECOMMENDATIONS 
L.  Quality Control section (see Section IV of this guidance). 
  III.  Confirmation criteria 
These criteria are an expansion and updating of criteria that CVM has applied in the past.  
The new, expanded criteria are in response to the use of newer mass spectral techniques 
for regulatory confirmation.  In CVMs judgment, all methods approved prior to issuing 
this document meet these criteria.  Criteria should be specified in the SOP in advance. 
A.  Comparison standard. 
Comparison standard(s) should be analyzed contemporaneously.  Preparation and 
analysis sequence of the comparison standard(s) should be fully described.  Examples: 
first single injection prior to or after samples; average of all standards injected the day 
of analysis; average of two closest bracketing standards.  If a matrix effect alters the 
spectrum or chromatography of a pure standard so that normal confirmation criteria 
cannot be met, a control extract containing standard may be substituted for pure 
standard.  Confirmatory procedures that call for spiked control extracts for comparison 
should be justified.  The tissue used as a control should be analyzed without a standard 
added to demonstrate the absence of interferences. 
B.  Chromatography/Mass Spectrometry. 
Any of the following chromatograms may be used:  total ion chromatogram (TIC); 
reconstructed ion chromatogram (RIC); all single ion chromatograms (from scan, 
Selected Ion Monitoring (SIM) or Selected Reaction Monitoring (SRM)).  Flow 
injection analysis is discouraged, but will be evaluated on a case-by-case basis.  
1.  The chromatographic peak(s) should exceed a signal-to-noise (s/n) threshold of 
3:1.  The technique used for estimating s/n should be described.   
2.  An acceptability range for retention time matching should be specified in the SOP.  
The acceptability range should not exceed 2% for GC/MS or 5% for LC/MS, 
relative to the retention time of standard. 
C.  Mass spectral matching. 
Refer to Section II.E. structurally-specific ions.  Confirmation criteria vary depending 
on the technique used for mass spectral data acquisition.  
1.  MS1 full scan 
The mass spectrum should include at least three structurally-specific ions.  The 
spectrum obtained from a suspect compound should visually match the spectrum 
obtained from a contemporaneous standard.  Since full scan data may include 
hundreds of significant data points for comparison, strict numerical criteria need 
not be applied. [An acceptability range of  20% on relative abundance of major 
ions is a useful rule of thumb, but is not required.  See Part III.C.2.a.]  Library-
  4   CONTAINS NON-BINDING RECOMMENDATIONS 
search algorithms should not be used to confirm identity.  The following elements 
should apply when MS1 full scan data are used: 
a.  All structurally-specific ions identified in Section II.E. are present above a 
specified level.  The method developer should describe an appropriate 
minimum level based either on relative abundance or signal-to-noise. 
b.  There is general correspondence between relative abundances or ranked 
abundances obtained for sample and standard. 
c.  Prominent ions other than from the target analyte can be explained (e.g. 
present in controls, blanks, etc.). 
d.  If background subtraction is used, this should be specified in the SOP.  The 
range used as background should always be indicated on the chromatogram.   
2.  MS1 Selected Ion Monitoring (SIM). 
a.  Relative abundances for three structurally-specific ions should match the 
comparison standard within 10%. This acceptability range is calculated by 
addition and subtraction. For example, at 50% relative abundance, the 
acceptability range would be 40-60%, not 45-55%. 
b.  Relative abundances for four or more unique, structurally-specific ions should 
match the comparison standard within 15%. 
c.  Relative abundances for more than three ions, which include ions due to 
isotopes or loss of water, should match the comparison standard within  10%. 
3.  MS1 scan acquisition, SIM treatment. 
If scan data is acquired, the data may be treated as for SIM acquisition (Section 
III.C.2.). 
4.  MS1 partial scan 
Criteria are the same as for full scan (Section III.C.1. above).  All structurally-
specific ions should appear in the scan range.   
5.  MSn full scan 
The spectrum obtained from a suspect compound should visually match the 
spectrum obtained from a contemporaneous standard.  Since full scan data may 
include hundreds of significant data points for comparison, strict numerical criteria 
need not be applied.   
a.  All structurally-specific ions identified in Section II.E. should be present above 
a relative abundance specified in the SOP. 
  5   CONTAINS NON-BINDING RECOMMENDATIONS 
b.  There should be general correspondence between relative abundances or 
ranked abundances obtained for sample and standard. 
c.  If a structurally-specific precursor ion completely dissociates to product ions 
after MSn, the appearance of at least two additional structurally-specific 
product ions in the MSn+1 spectrum should be sufficient.   
d.  Prominent ions other than from the target analyte can be explained (e.g. 
present in controls, blanks, etc.). 
e.  If background subtraction is used, the range used as background should be 
specified. 
6.  MSn partial scan 
Criteria are the same as for full scan (Section III.C.5. above).  All structurally-
specific ions should appear in the scan range.   
7.  MSn Selected Reaction Monitoring (SRM) 
a.  If a precursor ion selected by MSn is completely dissociated, and only two 
structurally-specific product ions are monitored in MSn+1, the relative 
abundance ratio should match the comparison standard within  10%. 
b.  If three or more structurally-specific ions are monitored, the relative abundance 
ratios should match the comparison standard within  20%.  
8.  MSn scan acquisition, SRM treatment 
If MSn scan data is acquired, the data may be treated as for SRM acquisition 
(Section III.C.7.). 
D. New technology.  
New Technology.  The availability of new instrumentation has led to new options as listed 
above, but also to new concerns in confirmatory analysis.  If the number of ions observed, 
their structural significance, or their mass measurement accuracy are issues, please contact 
CVM for further information.  Such issues will be handled on a case-by-case basis.  
 
  IV. Quality Control 
A.  System suitability should be established before valid data can be obtained. 
B.  At least one control and one fortified control sample should be run each day.  The 
fortified control should meet criteria and the control should fail criteria for the days 
analyses to be valid. 
  6   CONTAINS NON-BINDING RECOMMENDATIONS 
C.  Sufficient blanks or controls should be analyzed after standards or fortified control 
samples to ensure that carryover does not cause a false positive outcome.   
D.  Operational criteria for repeat analysis of same sample: If a sample is analyzed but it 
can be shown that system suitability was not adequate during that analysis, the sample 
may be reanalyzed after taking steps to improve system performance and reestablish 
suitability. 
E.  This document provides options for method developers, so that methods may be fit for 
their purpose.  However, once a procedure is developed and the SOP prepared, a 
single set of confirmation criteria should be specified and used.  Analysts should not 
substitute other criteria after analyses have been carried out.   
Ad Hoc Confirmatory Packages  
Ad Hoc Confirmatory Packages should meet or exceed the following minimal data 
recommendations: 
Ad hoc data packages arise when new procedures are applied in response to unanticipated 
situations, when full confirmatory procedures are unavailable, and when time does not permit 
a procedure to be fully validated.  CVMs confidence in ad hoc data packages is based on 
good quality assurance, good training, and high expertise in the laboratory.  The following 
analyses are the minimum recommended for an ad hoc data package, although additional 
supporting data is strongly encouraged (see above).  All recommendations for structural 
specificity (Section II.B-F.), confirmation criteria and recommendations for treatment of data 
(Section III.), and quality control (Section IV.) still apply. 
I.  At least two control samples should be analyzed.  No control sample should meet criteria 
(i.e., give a false positive outcome).  A surrogate control is from a similar matrix known to 
be free of the suspect compound, but is used to simulate the same matrix.  A survey 
control is from the same type of matrix, but is of unknown origin, and has been analyzed 
repetitively and found to fail confirmation in every case. 
II.  Control samples fortified with the suspect compound should meet confirmation criteria.  
At least two control samples should be fortified at the suspect compounds tolerance or 
safe level.  If the suspect compound has no tolerance or safe level, at least four fortified 
control samples should be prepared: two above and two near the suspect compounds 
estimated level.  
III. Sufficient replicate injections of the standard should be made to establish system 
suitability. 
IV. A blank or control sample should be analyzed after a fortified sample or standard to 
demonstrate that carryover does not cause a false positive outcome.  Otherwise, blanks 
should be analyzed after each sample until the blank analysis appears free of the suspect 
compound.  
 
  7   CONTAINS NON-BINDING RECOMMENDATIONS 
Exact Mass Measurements 
The following recommendations apply to the use of exact mass measurements in 
confirmatory analyses.  Until specific standards for exact mass measurements in animal 
drug residue analysis are generally accepted, their use will be evaluated on a case-by-case 
basis.  Multiple structurally-specific ions should be used to confirm the presence of a 
compound of a specified structure. 
* The mass spectrometer design and operating conditions used to assign an exact mass 
(e.g., analyzer geometry, resolution, lock masses, mass range) should be described.   
* Mass resolution and peak purity should be demonstrated to be sufficient to provide only 
one predominant component per mass peak in the range of the peak of interest. 
* Suitability should be demonstrated by applying the method to a standard of known 
composition to show that mass accuracy is acceptable.  Accuracy should be reported as 
parts-per-million (ppm) = [measured  calculated mass] x 106  / calculated mass. 
* The possibility of interferences within the mass measurement accuracy of the instrument 
should be evaluated.  Candidate compositions should be calculated using a range of 
reasonable elements, including but not limited to C, H, N, and O.  The candidate 
compositions should be shown, along with the ranges of each element used to generate 
them.  At low mass (well below m/z 500) < 5 ppm difference may be sufficient to 
confirm a unique elemental composition.  However, as mass increases, the chance of 
interference increases dramatically.  At roughly m/z 500 and above, many elemental 
compositions can occur within a 5-10 ppm window around the theoretical mass.   
* If multiple candidates occur within the mass measurement accuracy of the instrument, 
the alternatives should be evaluated for their reasonableness.  Parameters to consider 
could include the number of heteroatoms, the isotopic distribution pattern, or results of 
other chemical analyses.  
* If all alternative compositions cannot be excluded by virtue of the instruments mass 
accuracy or the unreasonableness of the alternatives, then exact mass measurements do 
not improve the analysis relative to nominal mass measurements. 
  8   CONTAINS NON-BINDING RECOMMENDATIONS 
BIBLIOGRAPHY 
1.  J.A. Sphon, J. Assoc. Off. Anal. Chem. 61 (1978) 1247-52.  
2.  T. Cairns, E.G. Siegmund, and J.J. Stamp, Mass Spectrom. Reviews 8 (1989) 93-117. 
3.  T. Cairns, E.G. Siegmund, and J.J. Stamp, Mass Spectrom. Reviews 8 (1989) 127-145. 
4.  J.B. Schilling, S.P. Cepa, S.D. Menacherry, L.T. Bavda, B.M. Heard, and B.L. Stockwell, 
Anal. Chem. 68 (1996) 1905-1909. 
5.  W.G. de Ruig, R.W. Stephany, and G. Dijkstra, J. Assoc. Off. Anal. Chem.  72 (1989) 487. 
6.  Baldwin, R., et al, J. Am. Soc. Mass  Spectrom. 8 (1997) 1180-1190 . 
7.  Bethem, R.A.; Boyd, R.K., J. Am. Soc. Mass  Spectrom. 9 (1998) 643-648. 
8.  Gross, M.L, Giordani, A.B., Price, P.C., J. Am. Soc. Mass Spectrom. 5 (1994) 57. 
  9   CONTAINS NON-BINDING RECOMMENDATIONS 
GLOSSARY 
Ad Hoc Confirmatory Package - A data package accompanied by a conclusion that is 
supported by the data.  Ad hoc data packages may be acceptable when a CVM Confirmatory 
Procedure is unavailable, and time does not permit a procedure to be fully validated.  
Examples of the need for such procedures include unanticipated misuse of an approved drug; 
unanticipated use of an unapproved drug, suspected presence of drug in unexpected tissue 
matrix, or sabotage of food products.  
Comparison Standard - The reference standard which is analyzed contemporaneously with 
unknown samples.  The mass spectrum and retention time from the sample are evaluated 
against the corresponding data from the standard.  
Confirmation - Unambiguous identification of a compounds presence by comparison to a 
reference standard (mass spectrometric).   
Control Sample - A control sample is the same tissue type as the target tissue, but is known 
to be free of target analyte.  A fortified control sample is a control sample to which target 
analyte has been added. 
CVM - FDA Center for Veterinary Medicine 
CVM Confirmatory Procedure - A procedure that CVM considers valid for regulatory 
analyses. This procedure should include a stepwise description of the method for evaluation 
by CVM.  Such procedures can be developed in advance of their application because their 
need can be anticipated.  Examples of the need for such procedures include the approval 
process for new animal drugs and preparation for surveys of suspected drug misuse.  Such 
procedures are reviewed by CVM prior to a laboratory evaluation of the procedure at CVM.  
The evaluation consists of a sample set corresponding to Section I.A-D (see above). 
Exact Mass Measurement - A mass assignment to more than one decimal place. 
False Negative Rate - The percent of samples known to contain a marker residue which do 
not meet the confirmation criteria.   
False Positive Rate - The percent of samples known not to contain a marker residue which do 
meet the confirmation criteria. 
Full Spectrum - A mass range encompassing all diagnostic data or the full width of 
instrumental capability.  For example, full spectrum may include both the molecular ion and 
low molecular weight fragment ions. 
Limit of Confirmation (lower) - The methods limit of confirmation may be defined as the 
concentration where the weakest diagnostic ion no longer appears at an acceptable signal-to-
noise level or where the false negative rate becomes excessive.  The estimate of limit of 
confirmation is recommended for two reasons:  to show that the method is sufficiently 
sensitive for its purpose, and to predict transferability of the method to other laboratories. 
  10   CONTAINS NON-BINDING RECOMMENDATIONS 
Marker Residue - The residue selected for assay whose concentration is in a known 
relationship to the concentration of the residue of concern in the last tissue to deplete to its 
permitted concentration. 
MSn - Two or more stages of mass separation conducted sequentially. 
Residue - Any compound present in edible tissues of the target animal which results from the 
use of the compound, including the compound, its metabolites, and any other substances in or 
on food because of the compounds use.  
Safe Level  A conservative estimate of a drug residue level in edible animal tissue derived 
from food safety data or other scientific information.  Concentrations of residue in tissue 
below the safe level will not raise human food safety concerns.  A safe level is not a safe 
concentration or a tolerance and does not indicate that an approval exists for the drug in that 
species or category of animal from which the food is derived. 
Standard Operating Procedure (SOP) - A stepwise written procedure for carrying out an 
analytical method.  
Structurally-Specific - Characterizing a compounds molecular weight and/or unique 
substructure.  For a molecule to be completely defined, the spectral data should be unique to 
that compound and none other. 
Suitable Data - Data acquired when system suitability has been met. 
System Suitability - The fitness of analytical instruments for the purpose at hand, based on 
manufacturer specifications, instrumental Standard Operating Procedure, or specific 
requirements of the analytical method. Suitability may be established through verification of 
relevant instrumental parameters such as calibration, pressure, flows, temperature, multiplier 
gain, etc., or through verification of method-specific parameters such as signal-to-noise level 
for a known amount injected, peak shape, test spectra, etc. 
Target Analyte - The chemical entity that a particular method is designed to detect. 
Target Tissue - The edible tissue selected to monitor for residues in the target animals, 
including where appropriate, milk or eggs. 
Tolerance (Rm) - The concentration of the marker residue in the target tissue when the 
residue of concern is equal to the permitted concentration in the last tissue to deplete to its 
permitted concentration. 
Validation - Demonstration that a method performs as claimed through a defined 
experimental protocol and data evaluation.  A claim that a mass spectrometric method is valid 
for confirmatory analyses should be supported with experimental results. 
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