#208 
 
Guidance for Industry 
 
Studies to Evaluate the Metabolism and Residue 
Kinetics of Veterinary Drugs in Food-Producing 
Animals:  Validation of Analytical Methods 
Used in Residue Depletion Studies 
 
VICH GL49(R) 
 
Submit comments on this guidance at any time.  Submit  electronic comments on the guidance 
to http://www.regulations.gov.   Submit written comments to the Division  of Dockets 
Management (HFA-305), Food and Drug Administration,  5630 Fishers Lane, Room 1061, 
Rockville,  MD  20852.  All written comments should be identified  with the Docket No. FDA-
2010-D-0165. 
 
For further information  regarding this document, contact Julia Oriani, Center for Veterinary 
Medicine (HFV-151), Food and Drug Administration,  7500 Standish Place, Rockville, MD 
20855, 240-402-0788, email:   julia.oriani@fda.hhs.gov. 
 
Additional  copies of this guidance document may be requested from the Policy and 
Regulations Staff (HFV-6), Center for Veterinary Medicine, Food and Drug Administration, 
7519 Standish Place, Rockville,  MD  20855, and may be viewed on the Internet at either 
http://www.fda.gov/AnimalVeterinary/default.htm  or http://www.regulations.gov. 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
March 2015 
 
    Contains Non-Binding Recommendations 
VICH GL 49 (R) (MRK)  METABOLISM AND RESIDUE KINETICS 
January 2015 
Revision at Step 9 
For implementation at Step 7 - Final 
 
 
 
 
 
 
STUDIES T O EVALUATE THE METABO LISM 
AND RESIDUE  KINETICS  OF VET ERINARY 
DRUGS IN  FOOD PRO DUCING ANIMALS: 
VALIDATION OF  ANAL YTICAL  METHOD S 
USED IN  RESIDUE  DEPLETION STUDIES 
 
 
 
Revision at step 9 
 
 
Adopted at Step 7 of the VICH Process by the VICH Steering Committee in January 2015 
for implementation by January 2016. 
 
 
 
This Guidance has been revised by the appropriate VICH Expert Working Group. At Step 7 of the 
Process the final draft is recommended for adoption to the regulatory bodies of the European 
Union, Japan and the USA. 
 
 
 
 
   
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Table of Contents 
 
1.INTRODUCTION............................................................................................................4 
1.1. Objective of the Guidance ..........................................................................................4 
1.2. Background ...............................................................................................................4 
2.GUIDANCE ....................................................................................................................5 
2.1 Purpose.......................................................................................................................5 
2.2.Scope .........................................................................................................................5 
3.PERFORMANCE CHARACTERISTICS .........................................................................5 
3.1. Linearity ....................................................................................................................6 
3.2. Accuracy ...................................................................................................................6 
3.3. Precision ....................................................................................................................7 
3.4. Limit of Detection ......................................................................................................8 
3.5. Limit of Quantitation..................................................................................................8 
3.6. Selectivity ..................................................................................................................8 
3.7. Stability in Matrix ......................................................................................................8 
3.8. Processed Sample Stability .........................................................................................9 
3.9. Robustness.................................................................................................................9 
4.GLOSSARY .................................................................................................................. 10 
Annex 1 ............................................................................................................................ 12 
Annex 2 ............................................................................................................................ 13 
Annex 3 ............................................................................................................................ 18 
 
   
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Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs 
in Food-Producing Animals:  Validation of Analytical Methods Used  in  
Residue Depletion Studies 
 
This guidance represents the Food and Drug Administrations (FDA or Agency) 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 this guidance using 
the contact information on the title page of this guidance. 
 
 
 
1.  INTRODUCTION 
1.1. Objective of the Guidance 
This guidance document is intended to provide a general description of the criteria that have 
been found by the European Union (EU), Japan, United States of America (USA), Australia, 
New Zealand and Canada to be suitable for the validation  of analytical methods used in 
veterinary drug residue depletion studies. 
 
1.2. Background 
During the veterinary drug development process, residue depletion studies are conducted to 
determine the concentration of the residue or residues present in the edible products (tissues, 
milk,  eggs or honey) of animals treated with veterinary drugs.  This information  is used in 
regulatory submissions  around the world.  Submission  of regulatory methods (i.e., post 
approval control methods) and the validation  requirements of the regulatory methods are 
usually well defined by various regulatory agencies worldwide and might even be defined by 
national or regional law.  However, the residue depletion studies are generally conducted 
before the regulatory methods have been completed.  Oftentimes the in-house validated 
residue methods provide the framework for the methods submitted for regulatory monitoring.   
Harmonization  of the validation  requirements for methodology  used during residue depletion 
studies and submitted to the regulatory agencies in support of the maximum  residue limits 
(MRLs) and withdrawal periods should be achievable.  It is the intent of this document to 
describe a validation  procedure that is acceptable to the regulatory bodies of the VICH 
regions for use in the residue depletion  studies.  This validated method could continue on to 
become the regulatory  method but that phase of the process will not be addressed in any 
detail in these guidances. 
 
A variety of validation  guidances exist for analytical methodology  and many of the aspects of 
those validation  procedures are incorporated in this document (Guidance for Industry (GFI) 
#63, Validation  of Analytical Procedures: Definition  and Terminology  (VICH GL1) and 
GFI #64, Validation  of Analytical Procedures: Methodology  (VICH GL2), July 1999). 
However, there are aspects of residue validation  procedures that are addressed in this 
guidance document that are not addressed in previous documents.  The guidance provided 
here is intended to specifically  address the validation  of veterinary drug residue methods. 
 
FDAs guidance documents, including  this guidance, do not establish legally  enforceable 
responsibilities.  Instead, guidances describe the Agencys current thinking  on a topic and 
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should be viewed only as recommendations,  unless specific regulatory or statutory 
requirements are cited. The use of the word should in Agencys guidances means that 
something is suggested or recommended, but not required. 
 
2.  GUIDANCE 
2.1 Purpose 
The purpose of this document is to provide a general description of procedures that can be 
used for the validation  of the analytical methods developed for the analysis of tissue samples 
obtained in residue depletion studies. 
 
For purposes of this guidance, acceptable refers to the scientific evaluation  of the analytical 
method in terms of the described validation  criteria. 
 
2.2. Scope 
This guidance is only intended to apply to analytical procedures that have been developed for 
the evaluation of veterinary drug residue methods (assays developed to determine residues in 
marker residue depletion  studies). It is not intended to define the criteria needed for validation 
of regulatory monitoring  assay procedures. 
 
This document provides performance characteristics of the residue assays that if followed 
would generally be considered acceptable by the regulatory agencies in the VICH regions.  
The intent is that methods validated according to this guidance will provide residue data that 
would generally be considered acceptable by the regulatory agencies in determining 
appropriate withdrawal periods. 
 
While it is recognized that residue studies must be conducted under GLP using validated 
methodology,  the actual method validation  experiments do not fall within the scope of the 
GLP regulations.  However, raw data generated as a result of a method validation  should be 
archived as appropriate and be available for submission  to regulatory authorities upon request. 
 
3.  PERFORMANCE CHARACTERISTICS 
In general, there are specific performance characteristics of a method validation.   Those 
performance characteristics are defined as follows: 
 
  Linearity 
  Accuracy 
  Precision 
  Limit of Detection 
  Limit of Quantitation 
  Selectivity 
  Stability  in Matrix 
  Process Sample Stability 
  Robustness 
 
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Each of the characteristics will be described below as they apply to the validation  of methods 
intended for use in veterinary drug residue depletion studies. 
 
3.1. Linearity 
A calibration curve should be generated in which the linear relationship  is evaluated across 
the range of the expected matrix (tissue, milk,  egg or honey) concentrations.  Calibration 
standard curves can be generated in three formats depending upon the methodology:   
standards in solvent/buffer, standards fortified into control matrix extract and standards 
fortified into control matrix and processed through the extraction procedure.  Linearity should 
be described by a linear, polynomial  or other (as appropriate) regression plot of known 
concentration vs. response using a minimum  of 5 different concentrations. Acceptability  of 
weighting  factors should be determined by evaluation of the residuals across three runs to 
determine if the residuals are randomly distributed.   Evaluation  of the residuals should be 
carried out across at least three separate runs. 
 
The recommended acceptance criterion for a standard curve is dependent upon the format 
of the standard curve.  Calibration  standard curves generated by fortification  of control 
matrix and processed through the procedure are subject to the same acceptance criteria as 
the samples (see Section 3.3. Precision).  Calibration  standard curves generated by 
standards in solvent/buffer or by fortification  of control matrix extract would require more 
stringent acceptance criteria (Repeatability   15% at all concentrations except at or below 
LOQ where it can be  20%). 
Some assays (e.g. microbiological  assays) could require log transformations to achieve 
linearity  where other assays (e.g., ELISA, RIA) could require a more complicated 
mathematical function to establish the relationship  between concentration and response.  
Again, acceptability  of the function selected should be verified by evaluation  of the 
residuals generated when that function is used. 
 
3.2. Accuracy 
Accuracy refers to the closeness of agreement between the true value of the analyte 
concentration and the mean result that is obtained by applying  the experimental procedure.  
Accuracy is closely related to systematic error (analytical method bias) and analyte recovery 
(measured as percent recovery).  Recommended accuracy for residue methods will vary 
depending upon the concentration of the analyte.  The accuracy should meet the range listed 
below: 
   
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Table 1.  Acceptable Range for Accuracy for Analyte Concentrations 
 
Analyte Concentration*  Acceptable Range for Accuracy 
< 1 g/kg  -50 % to +20 % 
 1 g/kg < 10 g/kg  -40 % to +20 % 
 10 g/kg < 100 g/kg  -30 % to +10 % 
 100 g/kg  -20 % to +10 % 
* g/kg =ng/g = ppb 
 
3.3. Precision 
Precision of a method is the closeness of agreement between independent test results obtained 
from  homogenous test material under stipulated conditions  of use.  Analytical variability 
between different  laboratories  is defined as reproducibility, and  variability from repeated 
analyses within a laboratory is repeatability.  Single-laboratory validation precision should 
include a within-run (repeatability)  and between-run component. 
 
The within- and between-run precision of the analytical method can be determined as part of 
the validation procedure.  There is generally not a need to determine reproducibility (between-
laboratory precision) in order to conduct a residue depletion study, because the laboratory that 
is developing the method is often the same laboratory assaying the samples from the residue 
study.  Instead of establishing reproducibility of the assay, a within-run precision, can be 
determined.  Within- and between-run precision should be determined by the evaluation of a 
minimum of three replicates at three different concentrations representative of the intended 
validation  range (which should include the LOQ) across three days of analysis. 
 
For the purposes of the residue method validation, acceptable variability is dependent upon 
the concentration of the analyte.  The precision should meet the range listed below: 
 
Table 2.  Acceptable Precision (%CV) for Analyte Concentrations 
 
Analyte Concentration  Acceptable within-run  precision  Acceptable between-
(Repeatability),  %CV  run precision %CV* 
< 1 g/kg  30 %  45% 
 1 g/kg < 10 g/kg  25 %  32% 
 10 g/kg < 100 g/kg  15%  23% 
 100 g/kg  10 %  16% 
    * as determined by the Horwitz equation CV = 2(1-0.5 log C) where C = concentration 
    expressed as a decimal fraction (e.g. 1 g/kg is entered as 10-9). 
 
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3.4. Limit of Detection 
The limit  of detection (LOD) is the smallest measured concentration of an analyte from which 
it is possible to deduce the presence of the analyte in the test sample with acceptable certainty.  
There are several scientifically  valid ways to determine LOD and any of these could be used 
as long as a scientific justification  is provided for their use.  See Annex 1 and Annex 2 for 
examples of acceptable methods for determining  LOD and Annex 3 for a suggested protocol 
for determining  accuracy, precision,  LOD, LOQ and selectivity in a single study. 
3.5. Limit of Quantitation 
The LOQ is the smallest measured content of an analyte above which the determination  can 
be made with the specified degree of accuracy and precision.  As with the LOD, there are 
several scientifically  valid ways to determine LOQ and any of these could be used as long as 
scientific justification  is provided.  See Annex 1 and Annex 2 for examples of acceptable 
methods for determining  LOQ and Annex 3 for a suggested protocol for determining 
accuracy, precision, LOD, LOQ and selectivity in a single study. 
 
3.6. Selectivity 
Selectivity  is the ability  of a method to distinguish  between the analyte being measured and 
other substances which might be present in the sample being analyzed.  For the methods used 
in residue depletion studies, selectivity  is primarily  defined relative to endogenous substances 
in the samples being measured.  Because the residue depletion  studies are well controlled, 
exogenously  administered components (i.e., other veterinary drugs or vaccines) could either 
be known or not be allowed during the study. If it is the intent to submit the validated method 
as a regulatory method, it might be prudent for the investigator  to test known products used in 
the animals being tested for possible interference. 
 
A good measure of the selectivity  of an assay is the determination  of the response of control 
samples (see section 3.5 above).  That response should be no more than 20% of the response 
at the LOQ.  See Annex 3 for a suggested protocol for determining  accuracy, precision, LOD, 
LOQ and selectivity in a single study. 
 
3.7. Stability in Matrix 
Samples (tissue, milk,  eggs or honey) collected from residue depletion studies are generally 
frozen and stored until assayed.  It is important to determine how long these samples can be 
stored under the proposed storage conditions  without excessive degradation prior to analysis.  
As part of the validation  procedure or as a separate study, a stability  study needs to be 
conducted to determine the appropriate storage conditions  (e.g., 4C, -20C, or -70C) and 
length of time the samples can be stored prior to analysis. 
 
Samples should be fortified with known quantities of analyte and stored under the appropriate 
conditions.   Samples should be periodically  assayed at specified intervals (e.g. initially,  1 
week, 1 month, 3 months).  If the samples are frozen, freeze/thaw studies should be conducted 
(3 freeze/thaw cycles  one cycle per day at a minimum).   Alternatively,  incurred samples can 
be used with initial  assays conducted to determine the starting concentrations.  The 
recommended protocol for assessing stability  in matrix is the analysis of two different 
concentrations in triplicate near the high and low end of the validation  range.  Stability  in 
matrix is considered acceptable if the mean concentration obtained at the specified stability 
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time point agrees with the initial  assay results or freshly fortified control sample assay results 
within the accuracy acceptance criteria established in Section 3.2. 
 
3.8. Processed Sample Stability 
Often, the samples are processed one day and assayed on a second day or because of an 
instrument failure are stored additional  days, e.g. over a weekend.  The stability  of the analyte 
in the process sample extract might be examined as necessary to determine stability  under 
processed sample storage conditions.   Examples of storage conditions  would be 4 to 24 hours 
at room temperature and 48 hours at 4C.  Other storage conditions might be investigated 
consistent with the method requirements. The recommended protocol for assessing processed 
sample stability  is the analysis of two different concentrations in triplicate near the high and 
low end of the validation  range.  Processed sample stability  is considered adequate if the 
mean concentration obtained at the specified stability  time point agrees with the initial  assay 
results or with freshly fortified and processed control sample assay results within the accuracy 
acceptance criteria established in Section 3.2. 
 
3.9. Robustness 
Evaluation  of the robustness of regulatory methods is of major importance.  Evaluation  of 
robustness for residue methodology  is less of a concern for residue methods as these are 
usually conducted within  a single laboratory using the same instrument.  However, robustness 
should still be evaluated particularly  for areas of the method that could undergo changes or 
modifications  over time.  These might include reagent lots, incubation  temperatures, 
extraction solvent composition  and volume,  extraction time and number of extractions, solid 
phase extraction (SPE) cartridge brand and lots, analytical column brand and lots and HPLC 
elution solvent composition.   During the development,  validation  or use of the assay, method 
sensitivity  to any or all of these conditions  can become apparent and variations in the ones 
most likely  to affect the method performance should be evaluated. 
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4.  GLOSSARY 
 
Accuracy  The accuracy of an analytical procedure expresses the closeness of agreement 
between the true value of the analyte concentration and the mean result that is obtained by 
applying  the analytical procedure.  This is generally expressed as % recovery or % bias. 
 
Control sample  Tissue, milk,  egg or honey from an animal that has not been treated with 
the veterinary drug under investigation. 
 
Between-run Precision  Between-run precision expresses within-laboratory  between-run 
variations. 
 
Incurred sample  Tissue, milk,  egg or honey from an animal treated with the veterinary 
drug under investigation  that has a residue concentration of the analyte of interest. 
 
Limit of Detection  The limit  of detection of an individual  analytical  procedure is the lowest 
amount of analyte in a sample that can be detected with acceptable certainty but not 
quantitated as an exact value. 
 
Limit of Quantitation  The limit  of quantitation  of an individual  analytical procedure is the 
lowest amount of analyte in a sample that can be quantitatively  determined with acceptable 
precision and accuracy. 
 
Linearity  The linearity  of an analytical procedure is its ability  (within  a given range) to 
obtain test results that are directly proportional  to the concentration (amount) of analyte in the 
sample. 
 
Marker residue  The residue whose concentration is in a known relationship  to the 
concentration of total residue in an edible tissue. 
 
Matrix  The matrix is basic edible animal products (tissue, egg, milk  or honey) that contains 
or could contain the residue of interest. 
 
Precision  The precision of an analytical procedure expresses the closeness of agreement 
between a series of measurements obtained from multiple  sampling  of the same homogenous 
sample under prescribed conditions.   The precision of an analytical procedure is usually 
expressed as the variance, standard deviation  or coefficient of variation of a series of 
measurements. 
 
Processed Sample  A processed sample is a sample that has been extracted or otherwise 
processed to remove the analyte from much of the original  sample matrix. 
 
Repeatability  Repeatability  expresses the precision under the same operating conditions 
over a short interval of time. 
 
Reproducibility  Reproducibility  expresses the precision between laboratories. 
 
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Residue  Veterinary drug (parent) and/or its metabolite. 
 
Robustness  The robustness of an analytical procedure is a measure of its capacity to remain 
unaffected by small variations in method parameters and provides an indication  of its 
reliability  during normal usage. 
 
Selectivity  Selectivity is the ability  to assess the analyte in the presence of components 
(endogenous materials, degradation products, other veterinary drugs) that might be expected 
to be present. 
 
Within-run Precision  Within-run precision expresses within-laboratory within-run 
variations. 
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Annex 1 
Examples of Methods for Determining LOD and LOQ 
 
One commonly  used approach is referred to as the IUPAC definition.1    In that procedure the 
LOD is estimated as mean of 20 control sample (from at least 6 separate sources) assay results 
plus 3 times the standard deviation of the mean. The LOQ then becomes the mean of the same 
results plus 6 or 10 times the standard deviation of the mean.  Testing of the accuracy and 
precision at the estimated LOQ will provide the final evidence for determination  of the LOQ.  
If the %CV for the repeatability  measurement at that concentration is less than or equal to the 
accuracy and precision acceptance criteria (Section 3.2 and 3.3), then the estimated LOQ is 
acceptable. 
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Annex 2 
U.S. Environmental Protection Agency Method for Determining LOD and LOQ 
 
The procedure described below is a slight modification  of a procedure used by USDAs 
Interregional Project No. 4 program which is published,  in 40 CFR Part 136, Appendix B).2  
This modified  procedure can be found in Appendix 1 of the U.S. Environmental  Protection 
Agencies document entitled Assigning  Values to Non-detected/Non-quantified  Pesticide 
Residues in Human Health Food Exposure Assessments3.  The procedure is provided below 
with minor modifications  making it more representative of a tissue marker residue assay 
procedure example. 
 
In this procedure, the estimation of the LOD and LOQ of a specific method for a specific 
analyte in a specific matrix can be done in the following  two steps. 
 
  The first step is to produce a preliminary  estimate of the LOD and LOQ and to verify 
that a linear relationship  between concentration and instrument response exists.  These 
preliminary  estimates correspond to what some term the IDL (Instrument Detection 
Limit) and IQL (Instrument Quantitation  Limit),  respectively.  The matrix of interest 
will be fortified (spiked) at the estimate LOQ in the next step for the actual estimation 
of LOD and LOQ of the method. 
 
  The second step is to use the initial  estimate of the LOD and LOQ determined in Step 
1 to estimate the method detection limit  and the method quantitation  limit  in the 
matrix of interest. 
 
An illustrative  example follows: 
 
Step 1.  The analyst derives a standard curve for the method of interest.  In this particular 
instance, the analyst prepares the standard solution  in buffer or water with the following 
concentrations of the analyte of interest:  0.005, 0.010, 0.020, 0.050 and 0.100 g/mL. For 
each concentration in the sample solution,  the following  instrument responses (measure peak 
height) are recorded: 
 
Table 1.  Instrument Responses for Concentrations of Sample Solutions 
 
Concentration (g/mL)  Instrument Response (peak height) 
0.100  206,493 
0.050  125,162 
0.020  58,748 
0.010  32,668 
0.005  17,552 
 
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In order to verify that a linear response is seen throughout the tested range, the instrument 
response is plotted as a function of injected concentration.  The results (and associated 
statistics) are shown in Figure 1.  Note from these results that the instrument response appears 
to be adequately linear throughout  the range of tested concentrations (0.005 to 0.100 g/mL), 
and that the R2 value from the Summary of Fit box in Figure 1 as the Root Mean Square 
Error) is 8986.8.  The equation which describes this relationship  (provided in the Parameter 
Estimates box of Figure 1) is as follows: 
 
Y = 15,120 + 1,973,098  * (Concentration) 
 
Where Y is the instrument response (peak height) 
 
The estimated LOD and LOQ are calculated as follows (assuming these values are set to 3 
and 10 standard deviations above the blank response, respectively): 
 
1.  The Peak Height at the LOD (Y ) is calculated at 3 times the standard deviation 
LOD
while the Peak Height at the LOQ (Y ) is calculated at 10 times the standard 
LOQ
deviation 
 
Y  = 15120 + 3 * (8987) = 42,081 
LOD
 
Y  = 15120 + 10 * (8987) = 104,990 
LOQ
 
2.  These values (peak height at LOD and peak height and LOQ) are then used to 
calculate the concentrations associated with these peak heights as follows: 
 
Y = 15,120 + 1,973,098  * (Concentration) 
Rearranging, 
Concentration = (Y  15,120) / 1,973,098 
Therefore, 
 
LOD = Y  - 15,120 / 1,973,098  = (42,081 - 15,120) / 1,973,098  = 0.014 g/mL 
LOD
 
LOQ = Y  - 15,120 / 1,973,098  = (104,990 - 15,120) / 1,973,098  = 0.046 g/mL 
LOQ
 
Thus, the initial  estimated LOD and LOQ are 0.014 and 0.046 g/mL, respectively 
which correspond to the IDL and IQL. 
These estimated LODs (or IDLs) and LOQs (or IQLs) are expressed in terms of the 
solution  concentration and not in terms of the matrix concentration.  At this stage, the 
solution  concentration (g/mL solution)  should be converted to the effective 
concentration in the matrix (e.g., g/g of matrix). 
   
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Figure 1.  Statistical Results 
 
 
 
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Step 2.  With the initial  estimate of LOD (or IDL) and LOQ (or IQL) obtained and linearity 
verified, Step 2 involves estimating  the LOQ and LOD in spiked matrix samples.  This 
procedure uses the estimated instrumental LOQ and the procedure detailed in 40 CFR Part 
136, Appendix B4 to provide a better estimate of LOQ and verifies that method recoveries are 
acceptable. 
 
The method calls for the analysis of 7 or more untreated control samples spiked at the 
estimated LOQ.  The standard deviation  of these samples is measured and the LOD and LOQ 
are determined as follows: 
 
LOD = t  * S 
0.99
 
LOQ = 3 * LOD 
 
where   t = one-tailed t-statistic at the 99% confidence level for n-1 replicates 
    S = Standard Deviation of n sample spikes at the estimated LOQ 
 
The following  is a set of t-values for use in the above equation: 
 
Table 2.  T-values for use in LOD Equation. 
 
# of  Degrees of  t # of Replicates  Degrees of  t
0.99  0.99 
Replicates  Freedom (n-1)  (n)  Freedom (n-1) 
(n) 
3  2  6.965  13  12  2.681 
4  3  4.541  14  13  2.650 
5  4  3.747  15  14  2.624 
6  5  3.365  16  15  2.602 
7  6  3.143  17  16  2.583 
8  7  2.998  18  17  2.567 
9  8  2.896  19  18  2.552 
10  9  2.821  20  19  2.539 
11  10  2.764  21  20  2.528 
12  11  2.718  22  21  2.518 
 
   
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In this example, the analyst prepared 7 untreated control samples spiked at the above 
estimated LOQ of 0.05 g/g.  The following  results were obtained: 
 
Table 3.  Percent Recover of Untreated Control Samples Spiked at 0.05 g/g. 
 
Concentration detected  % Recovery 
(g/g) 
0.0397  79.4 
0.0403  80.6 
0.0400  80.0 
0.0360  72.0 
0.0498  99.6 
0.0379  75.8 
0.0388  77.6 
 
Average Concentration:  0.0404 g/g 
 
Standard Deviation:  0.0044 g/g 
 
Average Recovery:  80.7% 
 
Given that recoveries are adequate at the LOQ (average = 80.7%, range = 72.0% to 99.6%), 
the LOD and LOQ for the method are estimated as follows: 
 
LOD = t * S (for 7-1 = 6 degrees of freedom) 
0.99 
= 3.143 * 0.0044 g/g 
= 0.0138 g/g 
 
LOQ = 3 * LOD 
= 3 * 0.0138 g/g 
= 0.0414 g/g 
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Annex 3 
Protocol for Residue Method Validation 
 
Selectivity,  LOD and LOQ are all interrelated and are affected by endogenous interferences 
that might be present in the matrix being assayed.  LOD is often difficult  to determine 
particularly  in LC/MS assays where control samples actually provide zero response at the 
retention time of the analyte.  Without a response, it is impossible  to calculate a standard 
deviation  and therefore impossible  to determine the LOD based on the mean plus 3 times the 
SD of the mean.  Even if a mean plus 3 times the SD of the mean can be determined, it is 
often related to the instrument limit  of detection rather than the method limit  of detection. The 
following  protocol is designed to determine specificity,  LOD, LOQ, precision and accuracy in 
one study. 
 
1.  Collect drug free matrix from 6 separate sources (animals) and screen for any possible 
analyte contamination. 
2.  Fortify (spike) 1 each of a minimum  of 3 samples (each source randomly selected such 
that each source is represented at least once at each concentration) of the 6 control 
samples at 0, at the estimated LOD (determined during assay development),  at 3 times 
the estimated LOD (estimated LOQ), and 3 other concentrations that will encompass 
the expected concentration range (Table 1).  Repeat the fortification  process for Day 2 
and Day 3 using a second and third set of 3 each (each source randomly selected such 
that each is represented at least once at each concentration) of the 6 control samples. 
 
Table 1.  Example of Minimum Study Design to Allow Determination of LOD, LOQ, 
Accuracy and Precision (Six Sources/Animals: A, B, C, D, E, and F) Within One Study 
Fortification  Concentration  Animal/Source  ID 
Day/Run 1  Day/Run 2  Day/Run 3 
0 (Control)  B, F, D  A, C, C  B, E, F 
eLOD*  B, C, E  D, F, F  A, B, E 
eLOQ (3 X eLOD)*  C, C, E  A, B, E  D, F, D 
Lower part of Validation  Range  A, B, E  A, C, D  B, E, F 
Middle of Validation  Range  B, C, E  C, E, F  A, D, F 
Upper Part of Validation  Range  A, B, B  D, F, F  A, C, E 
* eLOD (estimated LOD) is generally determined from preliminary  studies conducted 
during method development.  eLOQ (estimated LOQ) is determined as 3 times eLOD. 
 each source randomly  selected such that each source is represented at least once at each 
concentration across the 3 validation  runs. 
 
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3.  Assay the 18 samples each day and evaluate the results against a calibration standard 
curve. 
4.  Plot the results of concentration found against concentration added across all three 
days of assays.  This will normalize the data results across days and allow all the data 
from the 3 runs to be used in the determination  of the LOD and LOQ. 
5.  Establish a decision  limit  by calculating prediction  intervals around the weighted 
regression line with the upper confidence interval line based upon the probability  
(false positive) and the lower confidence interval line based upon the probability  
(false negative)4.  The decision limit (Y ) then becomes the point at which the upper 
C
confidence  limit  crosses  the  Y-axis  and  can  be  converted  to  concentration  by 
estimating from the regression line to the x-axis (L ).  This is the critical point where 
C
50% of the responses are real.  The L  or LOD can be determined by estimating 
D
concentration from the lower confidence limit  that reduces the false negative rate to 
what level is assigned to .  Typically,  both  and  are set equal to 5%. 
6.  Establish a determination  limit (Y )  by  multiplying  the detection  limit (Y ) by 3 
Q C
(commonly accepted ratio between LOD and LOQ is 3).  The LOQ (L ) can then be 
Q
determined by estimating where the line Y  crosses the lower confidence limit  that 
Q
reduces the false negative rate for the determination of LOQ to what level is assigned 
to  (typically  5%). 
7.  Inter-day precision can be determined by calculating the %CV at each concentration 
evaluated. Accuracy can be determined by comparison of the results obtained to the 
fortification levels.  Acceptance criteria for accuracy and precision are provided in 
Sections 3.2 and 3.3, respectively. 
 
Figure 1.  Plot of Concentration Found As a Function of Concentration Added 
 
1
nd YQ
u
o
F 
n 
o
ntrati 3 x YC 
e
c
n
o
C
YC
0
0 LC LD LQ 1
Concentration Added
 
 
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This approach takes into consideration the interrelationship  between specificity,  LOD and 
LOQ. By determining  LOD and LOQ using 6 different sources of matrix, the variability  due to 
the matrix as well as the variability  of the assay is taken into account. Since specificity  for 
residue methods is dependent upon the possible interference of matrix components this 
approach also addresses specificity and insures that specificity is acceptable at the LOD and 
LOQ determined.  This approach is consistent with the determination  of the detection limit  and 
quantitation  limit  specified in VICH GL2 (Validation  Methodology)  Guidance. 
 
Data Set Example: 
 
A validation  procedure based on the above methodology  was conducted on an LC-MS/MS milk 
assay procedure. 
 
Control bovine milk obtained from six different animals were each fortified with the analyte at  
0, 4.2, 14.0, 35, 140 and 400 ng/mL giving  a total of 36 samples.  Milk samples from 3 of the 6 
animals (insuring  that each of the 6 animals were run at least once) were randomly chosen at 
each of the fortification  levels to be run on each of the 3 days of assay for a total of 18 samples 
per day. 
 
Based  on  these  three  days  of  analyses  which  consisted  of  54  assays  total  the  following 
determinations were done:  repeatability (within-day precision), between-day precision, LOD 
and LOQ.  The raw data and the results of the statistical analyses are listed below: 
   
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Table 2.  Concentration of Analyte in Control Milk Fortified at 0, 4.2, 14.0, 
35.0, 140 and 400 ng/mL Across Three Days of Analysis 
Conc.  Run 1  Run 2  Run 3 
Added,  Animal  Conc.  Animal  Conc.  Animal  Conc. 
ng/mL  ID  Found,  ID  Found,  ID  Found, 
ng/mL  ng/mL  ng/mL 
0  B  0.494  A  0.233  B  0.154 
F  0.654  C  0.012  E  0.120 
D  0.588  C  0.117  F  0.313 
4.2  B  4.38  D  4.97  A  3.80 
C  4.13  F  3.85  B  4.12 
E  4.33  F  4.41  E  3.67 
14.0  C  13.2  A  11.1  D  11.8 
C  13.5  B  12.0  F  10.5 
E  11.9  E  12.8  D  11.7 
35.0  A  31.5  A  51.0  B  27.3 
B  32.7  C  33.2  E  29.4 
E  34.4  D  32.9  F  25.5 
140  B  131  C  137  A  118 
C  147  E  124  D  106 
E  127  F  131  F  118 
400  A  396  D  396  A  335 
B  394  F  390  C  316 
B  384  F  373  E  344 
 
The statistical evaluation  of the above data was conducted as follows:   The percentage 
recovery was calculated for each sample using the concentration obtained and the fortification 
concentrations prior to analysis.  A model which included  the fixed effect of treatment 
(fortification  level) and the random effects of run (day), run by treatment interaction and 
residual was used to obtain the least squares means and estimates of variation. To obtain the 
data in the table, the method calibration  curve was calculated by weighted (1/variance) 
regression. Other weighting  factors (e.g. 1/x, 1/x2) may be used, as appropriate, in calculating 
the regression equation of the calibration  curve (see Zorn reference). 
In order to assess within-day variability,  the residual variance was used in calculating  the CV 
for each treatment and across treatments.  The CVs were calculated by dividing  the square 
root of the residual variance by the mean and multiplying  by 100. 
In order to assess across-day variability,  the sum of the residual variance, the variance due to 
run, sample within run and run by treatment was used as the estimate of variance when 
calculating  CVs for each treatment and overall treatments. 
The results of the analysis were as follows: 
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Table 3.  Within- and Between-Run Assay Precision and Accuracy Determination* 
Theoretical  n  Mean*  95%  Precision, %CV 
Concentration,  Recovery, %  Confidence  Within- Between-
ng/mL  Interval  Run  Run 
4.2  9  99.6  87.9  111.4  7.8  10.2 
14.0  9  86.1  75.0  97.2  7.1  7.5 
35.0  9  94.6  77.3  111.9  19.3  22.6 
140  9  90.4  79.5  101.3  5.8  9.2 
400  9  92.4  82.1  102.8  3.0  8.2 
 
*  The reported data in the above table were derived using statistical software capable 
of mixed-model analysis (e.g. SAS) 
 
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A graphical representation of the determination  of LOD and LOQ is provided below. 
 
Figure 2.  Plot of the Determination of LOD and LOQ with Concentration Found As a 
Function of Concentration Added. 
 
 
LOD = 1.6 ng/mL 
LOQ = 3.7 ng/mL 
This is a straightforward way to accurately determine precision, accuracy, LOD and LOQ 
within one study across three days of validation. 
                                              
1 Codex Alimentarius Procedural Manual, 15th Ed., Twenty-eight Session of the Codex Alimentarius 
Commission, Rome, 2005, p 81. 
2 U.S. Code of Federal Regulations, Title 40: Protection of Environment , Part 136  Guidelines Establishing 
Test Procedures for the Analysis of Pollutants, Appendix B to Part 136  Definition and Procedure for the 
Determination of the Method Detection Limit  Revision 1.11. 
3 U.S. Environmental Protection Agency, Office of Pesticide Programs, March 23, 2000, Assigning Values to 
Non-detected/Non-quantified Pesticide Residue in Human Health Food Exposure Assessments  Appendix 1, A-
1 through A-8. 
4 Zorn ME, Gibbons RD, Sonzogni WC.  Weighted Least-Squares Approach to Calculating Limits of Detection 
and Quantification by Modeling Variability as a Function of Concentration, Anal Chem 1997,  69, 3069-3075. 
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