TABLE OF CONTENTS

Page No.

Introduction 4

Discussion 5

1. Specificity 6

2. Linearity 7

3. Range 7

4. Accuracy 8

5. Precision 8

5.1. Repeatability 8

5.2. Intermediate Precision 8

5.3. Reproducibility 9

5.4. Proof of Performance 9

6. Limit of Detection 9

6.1. Based on Visual Evaluation 9

6.2. Based on Signal-to-Noise 9

6.3. Based on the Standard Deviation of the Response and the Slope 10

6.3.1. Based on the Standard Deviation of the Blank 10

6.3.2. Based on the Calibration Curve 10

7. Limit of Quantitation 10

7.1. Based on Visual Evaluation 10

7.2. Based on Signal-to-Noise Approach 11

CONTAINS NON-BINDING RECOMMENDATIONS

3

7.3. Based on the Standard Deviation of the Response and the Slope 11

7.3.1. Based on the Standard Deviation of the Blank 11

7.3.2. Based on the Calibration Curve 11

8. Robustness / Ruggedness 11

9. System Suitability Testing 12

10. Recommended Data 12

Glossary 15

CONTAINS NON-BINDING RECOMMENDATIONS

4

Guidance for Industry

Validation of Analytical Procedures for Type C

Medicated Feeds1

INTRODUCTION

The purpose of this guidance is to provide recommendations on how to consider the

various validation characteristics for each analytical procedure used in medicated

feed assays. This guidance is written primarily for chromatographic methods;

however, the guidance does not limit the analytical technique to chromatographic

procedures, as other techniques may be appropriate. In some cases (for example,

demonstration of specificity), the overall capabilities of a number of analytical

procedures in combination may be investigated in order to ensure the quality of the

medicated feed.

Section 512(b) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. § 360b)

establishes the requirements for new animal drug approval. 21 C.F.R. § 514.1

specifies the information required to be submitted as part of the application and the

proper form for the submission. Section 514.1(b)(5)(vii) requires an applicant to

describe analytical procedures that should be capable of determining the active

component(s) within a reasonable degree of accuracy and of assuring the identity of

such components. Section 514.1(b)(5)(vii)(a) states that a description of practicable

1 This guidance has been prepared by the Office of New Animal Drug Evaluation in the Center for

Veterinary Medicine at the Food and Drug Administration.

This guidance represents the agency’s 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 statute(s) and regulation(s). If you want to

discuss an alternative approach, contact the FDA staff responsible for

implementing this guidance. If you cannot identify the appropriate FDA staff,

call the appropriate number listed on the title page of this guidance.

CONTAINS NON-BINDING RECOMMENDATIONS

5

methods of analysis of adequate sensitivity to determine the amount of the new

animal drug in the final dosage form should be included.

FDA’s guidance documents, including this guidance, do not establish legally

enforceable responsibilities. Instead, guidances describe the Agency’s 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.

DISCUSSION

The objective of the analytical procedure should be clearly understood since this

will govern the validation characteristics that are evaluated. Typical validation

characteristics that may be considered are listed below:

- Specificity

- Linearity

- Range

- Accuracy

- Precision

- Limit of Detection

- Limit of Quantitation

- Robustness

Each of these validation characteristics is defined in the attached Glossary.

Approaches other than those set forth in this guidance may be acceptable. It is the

responsibility of the applicant to choose the validation procedure and protocol most

suitable for the product. However, it is important to remember that the main

objective of validation of an analytical procedure is to demonstrate that the

procedure is suitable for its intended purpose.

It is recommended that a well-characterized reference standard, with documented

purity, be used throughout the validation study. The degree of purity necessary

depends on the intended use.

For the sake of clarity, this document considers the various validation

characteristics in distinct sections. The arrangement of these sections reflects the

process by which an analytical procedure may be developed and evaluated.

In practice, it is recommended to design the experimental work such that the

appropriate validation characteristics can be considered simultaneously to provide a

sound, overall knowledge of the capabilities of the analytical procedure.

CONTAINS NON-BINDING RECOMMENDATIONS

6

Appropriate validation characteristics may include: specificity, linearity, range,

accuracy, and precision.

1. SPECIFICITY

It is recommended that an investigation of specificity be conducted during the

validation of the medicated feed assay. The procedures used to demonstrate

specificity will depend on the intended objective of the analytical procedure.

Identification of the analyte may be made by means of retention time of the

standard.

For chromatographic procedures, it is recommended that representative

chromatograms be used to demonstrate specificity, and individual feed components

and drug products be appropriately labeled. The chromatographic profile using

peak shape and tailing criteria may be used to indicate either co-eluting peaks or

sample matrix effects. The peak parameters should be in agreement between the

standard and analyte peaks. In addition, to ensure that the peaks are single

components, a diode array detector may be used to obtain peak purity information

for the analyte peaks in a variety of feed matrices. Similar considerations may be

given to other separation techniques.

For the assay, it is recommended that there be a demonstration of a lack of

interference by feed ingredients or other drug products that may be in the feed.

This may be done by demonstrating that the responses of a blank placebo made

from the feed ingredients and/or drug products, either separately or in combination,

are either different from the absorbance (for Ultraviolet (UV) methods) or retention

time (for Gas Chromatography (GC) and High Performance Liquid

Chromatography (HPLC) methods) of the analyte of interest or not significant (i.e.,

that the signal measured as a percent concentration is not greater than 10%). It is

recommended that additional information be provided showing that common feed

ingredients do not interfere with the detection system. If potential interference is

observed, it is recommended that the ingredient be evaluated by the complete

method. Some examples of interfering ingredients are clay agents (for flowability)

and pellet binding, molasses, grass meals (e.g., alfalfa), high mineral content, corn

cob meal, cottonseed by product meal, meat and bone meal, and fish meal. Many

methods developed to give good recovery in a simple corn-soy feed do not work

well when the analyte is added to high mineral feeds, and are not recommended.

Typically, 2-3 feed mixtures, based on the species that will be medicated,

geographical location where the feed may be prepared, and life cycle of the species

(e.g., starter, finisher), should be tested. For drug products, it is recommended that

CONTAINS NON-BINDING RECOMMENDATIONS

7

applicants consider the most common products that may typically be present within

the feed.

Applicable literature references demonstrating non-interference may be supplied in

lieu of actual testing.

2. LINEARITY

It is recommended that a linear relationship be evaluated across the range (see

section 3) of the analytical procedure. It may be demonstrated directly on the drug

substance by separate weighings (two separate weighings preferred) and/or dilution

of a standard stock solution, using the proposed procedure.

It is recommended that linearity be evaluated by visual inspection of a plot of

signals as a function of analyte concentration or content. If there is a linear

relationship, it is recommended that test results be evaluated by appropriate

statistical methods, for example, by calculation of a regression line by the method

of least squares. Data from the regression line itself may be helpful to provide

mathematical estimates of the degree of linearity. It is recommended that the

correlation coefficient (R) be at least 0.995. The regression line intercept should

not differ from zero if a single point calibration technique is used. This may be

demonstrated if the confidence limits of the intercept include zero or if the intercept

value is a small percentage of the target level. If the intercept is significantly

different from zero, then a single point calibration technique is not recommended.

For the establishment of linearity, a minimum of 5 concentrations, covering the

intended dosing range with one concentration 50% of the lowest dose, is

recommended. It is recommended that the sponsor contact CVM if other

approaches are used.

3. RANGE

The specified range should be derived from linearity studies and depends on the

intended application of the procedure. It may be established by confirming that the

analytical procedure provides an acceptable degree of linearity, accuracy, and

precision when applied to samples containing amounts of analyte within or at the

extremes of the specified range of the analytical procedure.

For the assay of a drug in a medicated feed, the range should be from 50 to 150

percent of the labeled concentration.

CONTAINS NON-BINDING RECOMMENDATIONS

8

4. ACCURACY

It is recommended that accuracy be established across the specified range of the

analytical procedure used for medicated feed assays.

It is recommended that two (2) typical feed matrices with known quantities of the

drug added be analyzed.

It is recommended that accuracy be assessed using a minimum of 15 - 20

determinations over the concentration levels covering the specified range for each

feed matrix tested (e.g. 3 - 4 concentrations (depending on the dose range) / 5

replicates each of the total analytical procedure). Recovery from fortified blank

matrix samples should be between 80 - 110%.

5. PRECISION

Validation of tests for assay of medicated feeds should include an investigation of

precision.

5.1. Repeatability

For fortified medicated feed samples, it is recommended that repeatability be

assessed using a minimum of 15 determinations covering the specified range for

the procedure (e.g., 3-4 concentrations / 5 replicates each).

For drugs incorporated into medicated feeds at greater than 10 ppm, the withinlaboratory

variation coefficient should be less than 5.0%. For drugs

incorporated into medicated feeds at less than 10 ppm, the within-laboratory

variation coefficient should be less than 7.5%.

5.2. Intermediate Precision

The extent to which intermediate precision should be established depends on the

circumstances under which the procedure is intended to be used. It is

recommended that the applicant establish the effects of random events on the

precision of the analytical procedure. It is recommended that variations to be

studied include days, analysts, equipment, etc. It is not recommended to study

these effects individually. Instead, the use of a statistical experimental design

(matrix) is encouraged (see Statistical Manual of the AOAC by W.J. Youden

and E.H. Steiner, 1975, page 33 for more information on statistical design of

CONTAINS NON-BINDING RECOMMENDATIONS

9

experiments). The performance of the method by a second independent

laboratory is encouraged.

5.3. Reproducibility

It is recommended that reproducibility be assessed by means of an interlaboratory

trial. It is recommended that reproducibility be considered in the

case of standardization of an analytical procedure.

5.4. Proof of Performance

It is recommended that proof of performance of the assay be demonstrated by

testing two (2) batches of the proposed medicated feed manufactured in mixing

equipment of the appropriate size and under conditions representative of typical

commercial processing. When feasible, batches should be manufactured using

different configurations of mixers.

It is recommended that a minimum of 10 determinations covering the specified

range for the procedure be made (e.g., 2 concentrations (high and low) / 5

replicates each). If the feed is pelletized, it is recommended that the mash and

the pelletized feed be tested separately. Results should be reported in both

concentration and percent label claim.

6. LIMIT OF DETECTION

There are several approaches for determining the limit of detection (LOD),

depending on whether the procedure is non-instrumental or instrumental.

Approaches other than those listed below may be used.

6.1. Based on Visual Evaluation

Visual evaluation may be used for non-instrumental or instrumental methods.

The detection limit may be determined by the analysis of samples with known

concentrations of analyte and by establishing the minimum level at which the

analyte can be reliably detected.

6.2. Based on Signal-to-Noise

It is recommended that this approach be applied only to analytical procedures

that exhibit baseline noise.

CONTAINS NON-BINDING RECOMMENDATIONS

10

Determination of the signal-to-noise ratio may be performed by comparing

measured signals from samples with known low concentrations of analyte with

those of blank samples, and establishing the minimum concentration at which

the analyte can be reliably detected. A signal-to-noise ratio of between 3 or 2:1

is generally recommended for estimating the detection limit.

6.3. Based on the Standard Deviation of the Response and the Slope

It is recommended that the LOD be expressed as:

LOD = 3.3 δ / S

where δ = the standard deviation of the responses and S = the slope of the

calibration curve. The slope S may be estimated from the calibration curve of

the analyte. The estimate of δ may be carried out in a variety of ways, for

example:

6.3.1. Based on the Standard Deviation of the Blank

It is recommended that the measurement of the magnitude of analytical

background response be performed by analyzing an appropriate number of

blank samples and calculating the standard deviation of these responses.

6.3.2. Based on the Calibration Curve

It is recommended that a specific calibration curve be studied using samples

containing an analyte in the range of the LOD. The residual standard deviation

of a regression line or the standard deviation of y-intercepts of regression lines

may be used as the standard deviation.

7. LIMIT OF QUANTITATION

Several approaches for determining the limit of quantitation (LOQ) are possible,

depending on whether the procedure is non-instrumental or instrumental.

Approaches other than those listed below may be used.

7.1. Based on Visual Evaluation

Visual evaluation may be used for non-instrumental or instrumental methods.

The LOQ may be determined by the analysis of samples with known

concentrations of analyte, and by establishing the minimum level at which the

analyte can be quantified with acceptable accuracy and precision.

CONTAINS NON-BINDING RECOMMENDATIONS

11

7.2. Based on Signal-to-Noise Approach

It is recommended that this approach be applied only to analytical procedures

that exhibit baseline noise. Determination of the signal-to-noise ratio may be

performed by comparing measured signals from samples with known low

concentrations of analyte with those of blank samples, and establishing the

minimum concentration at which the analyte can be reliably quantified. A

signal-to-noise ratio of 10:1 is recommended.

7.3. Based on the Standard Deviation of the Response and the Slope

The LOQ may be expressed as:

LOQ = 10 δ / S

where δ = the standard deviation of the responses and S = the slope of the

calibration curve. The slope S may be estimated from the calibration curve of

the analyte. The estimate of δ may be carried out in a variety of ways, for

example:

7.3.1. Based on the Standard Deviation of the Blank

Measurement of the magnitude of analytical background response may be

performed by analyzing an appropriate number of blank samples and calculating

the standard deviation of these responses.

7.3.2. Based on the Calibration Curve

It is recommended that a specific calibration curve be studied using samples

containing an analyte in the range of the LOQ. The residual standard deviation

of a regression line or the standard deviation of y-intercepts of regression lines

may be used as the standard deviation.

8. ROBUSTNESS / RUGGEDNESS

It is recommended that the evaluation of robustness be considered during the

development phase and demonstrated during the analytical validation phase.

Robustness depends on the type of procedure under study. It should show the

reliability of an analysis with respect to deliberate variations in method parameters.

CONTAINS NON-BINDING RECOMMENDATIONS

12

If measurements are susceptible to variations in analytical conditions, it is

recommended that the analytical conditions be suitably controlled or a

precautionary statement be included in the procedure. One consequence of the

evaluation of robustness should be that a series of system suitability parameters

(e.g., resolution test) are established to ensure that the validity of the analytical

procedure is maintained whenever used.

Examples of typical variations are:

-stability of analytical solutions and feed extracts; and

-extraction time.

(Note: it is recommended that results of the stability studies of the

analytical solutions and feed extracts be included in the procedure)

In the case of high performance liquid chromatography (HPLC), examples of

typical variations are:

-influence of variations of pH in a mobile phase;

-influence of variations in mobile phase composition;

-different columns (different lots and/or suppliers); and

-temperature-flow rate.

In the case of gas chromatography (GC), examples of typical variations are:

-different columns (different lots and/or suppliers); and

-temperature-flow rate.

9. SYSTEM SUITABILITY TESTING

System suitability testing is an integral part of many analytical procedures. The

tests are based on the concept that the equipment, electronics, analytical operations,

and samples to be analyzed constitute an integral system that can be evaluated as

such. System suitability test parameters to be established for a particular procedure

depend on the type of procedure being validated and may include, for example, data

acceptability testing for feed controls. It is recommended that system suitability

tests and criteria for the HPLC or GC detection system be evaluated. Performance

specifications for critical reagents and steps, such as solid phase extraction, should

be included when appropriate. If specific tests and criteria are used, then the

recommended actions taken if performance does not meet the criteria should be

determined. Additional information is available in Pharmacopoeias.

10. RECOMMENDED DATA

CONTAINS NON-BINDING RECOMMENDATIONS

13

It is recommended that data collected during validation and formulae used for

calculating validation characteristics be submitted for each feed type and discussed

as outlined below:

Specificity:

It is recommended that representative sample sets of chromatograms be

provided so that recalculation can be performed, including:

Baseline / mobile phase

Extraction solvent

Feed ingredient placebo that cause interference

Other drug product placebo that causes interference

Standards

Samples (high and low concentration, different feed mixtures)

Retention times and a comparison of relative retention times should be

provided.

Tabular listing of feed mixture ingredients and other drug products tested

should be provided.

Linearity & Range:

It is recommended that the correlation coefficient, y-intercept, slope of the

regression line, and residual sum of squares be submitted. A plot of the data

should be included. In addition, an analysis of the deviation of the actual data

points from the regression line may also be helpful for evaluating linearity.

Accuracy:

It is recommended that accuracy be reported as percent recovery by the assay of

known added amount of analyte in the sample or as the difference between the

mean and the accepted true value together with the confidence intervals.

Tabular listing of feed mixture ingredients used should be provided.

For each feed matrix studied, it is recommended that the complete set of data

including weighings, sample and standard preparation, chromatography,

calculations, and results be provided. A representative set of chromatograms

should be provided and, for the concentration(s) in between, a table of relevant

parameters should be provided. All individual area or height measurements for

controls, standards, and samples and all other information such as sample

weights, standard concentrations, and dilutions should also be provided.

CONTAINS NON-BINDING RECOMMENDATIONS

14

Precision:

It is recommended that the standard deviation, relative standard deviation

(coefficient of variation), and confidence interval be reported for each type of

precision investigated.

It is recommended that the complete set of data including weighings, sample

and standard preparation, chromatography, calculations, and results be

provided. A representative set of data should be provided and, for the

concentration(s) in between, a table of relevant parameters should be provided

for each type of precision investigated.

Limit of Detection:

It is recommended that the limit of detection and the method used for

determining the detection limit be presented. If the LOD is determined based

on visual evaluation or based on signal-to-noise ratio, the presentation of the

relevant chromatograms may be considered acceptable for justification.

In cases where an estimated value for the LOD is obtained by calculation or

extrapolation, this estimate may subsequently be validated by the independent

analysis of a suitable number of samples known to be near, or prepared at, the

LOD.

Limit of Quantitation:

It is recommended that the limit of quantitation and the method used for

determining the LOQ be presented. The limit should be subsequently

confirmed by the analysis of a suitable number of samples known to be near, or

prepared at, the LOQ.

Robustness/Ruggedness:

It is recommended that tabular representation including conditions tested,

retention times, tailing factors, effects on resolution, and potency be presented.

CONTAINS NON-BINDING RECOMMENDATIONS

15

GLOSSARY

1. ANALYTICAL PROCEDURE

The analytical procedure refers to the way an analysis is performed. It describes in

detail the steps that should be followed to perform each analytical test. This may

include, but is not limited to, the sample, the reference standard and the reagents

preparations, use of the apparatus, generation of the calibration curve, and use of

the formulae for the calculation.

2. SPECIFICITY

Specificity is the ability to assess unequivocally the analyte in the presence of

components that may be expected to be present. Typically, these might include

impurities, degradation products, matrix, other approved drugs, etc.

Lack of specificity of an individual analytical procedure may be compensated for

by other supporting analytical procedure(s).

This definition includes the following:

Identification: to ensure the identity of an analyte.

Assay (content or potency): to provide an exact result which allows an

accurate statement on the content or potency of the analyte in a sample.

3. 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.

4. RANGE

The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations)

for which it has been demonstrated that the analytical procedure has a suitable level

of precision, accuracy, and linearity.

5. ACCURACY

The accuracy of an analytical procedure refers to the closeness of agreement

between the value that is accepted either as a conventional true value or an accepted

reference value, and the value found.

This is sometimes termed trueness.

CONTAINS NON-BINDING RECOMMENDATIONS

16

6. PRECISION

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple

sampling of the same homogenous sample under the prescribed conditions.

Precision may be considered at three levels: repeatability, intermediate precision,

and reproducibility.

Precision is investigated using homogenous, authentic samples. However, if it is

not possible to obtain a homogenous sample, it may be investigated using

artificially prepared samples or a sample solution (although extraction variability

will not be measured).

The precision of an analytical procedure is usually expressed as the variance,

standard deviation, or coefficient of variation of a series of measurements.

6.1. Repeatability: Repeatability expresses the precision under the same

operating conditions over a short interval of time. Repeatability is also termed

intra-assay precision.

6.2. Intermediate precision: Intermediate precision expresses within-laboratories

variations: different days, different analysts, different equipment, etc.

6.3. Reproducibility: Reproducibility expresses the precision between

laboratories (collaborative or transfer studies, usually applied to standardization

of methodology).

7. 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, but not necessarily quantitated as an exact

value.

8. 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 suitable precision

and accuracy. The quantitation limit is a parameter of quantitative assays for low

levels of compounds in sample matrices and is used particularly for the

determination of impurities and/or degradation products.

CONTAINS NON-BINDING RECOMMENDATIONS

17

9. ROBUSTNESS / RUGGEDNESS

The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters. Robustness

provides an indication of its reliability during normal usage.

10. SYSTEM SUITABILITY

A procedure run prior to the individual analytical analysis to demonstrate that the

instrument, column, mobile phase, etc., parameters are within defined criteria.

Adequate system suitability is demonstrated before proceeding with the analysis.

## No comments:

Post a Comment