AOAC Guidelines for Single Laboratory
Validation of Chemical Methods for Dietary
Supplements and Botanicals
CONTENTS
1.0 Introduction
1.1 Definitions
1.1.1 Validation
1.1.2 Method of analysis
1.1.3 Performance characteristics of a method of analysis
2.0 Single-Laboratory Validation Work
2.1 Preparation of the Laboratory Sample
2.2 Identification
2.3 Method of Analysis or Protocol
2.3.1 Optimization
2.3.2 Reference standard
2.3.3 Ruggedness Trial
2.3.4 Specific variables
a. Analyte addition
b. Reextraction of the extracted residue
c. Comparison with different solvents
d. Comparison with results from a different procedure
e. System Suitability Checks
3.0 Performance Characteristics
3.1 Applicability
3.2 Selectivity
3.3 Calibration
3.3.1 External Standard Method
3.3.2 Internal Standard Method
3.3.3 Standard Addition Method
3.4 Reliability Characteristics
3.4.1 Accuracy
3.4.2 Repeatability Precision (sr, RSDr)
3.4.3 Measurement Uncertainty
3.4.4 Reproducibility Precision (sR, RSDR)
3.4.5 Intermediate Precision
3.4.6 Limit of Determination
3.4.7 Reporting Low-level Values
3.4.8 Dichotomous Reporting
3.5 Controls
3.5.1 Control Charts
3.5.2 Injection Controls
3.5.3 Duplicate Controls
3.6 Confirmation of Analyte
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4.0 Report (as applicable)
Identity, source, purity of reference material
Scope, applicability, nonapplicability
Recovery, levels, percent (n = 5 at each concentration level)
Repeatability, RSDr (n = 5 at each concentration level)
Intermediate precision, RSDi (n = 5 at each concentration level)
Limit of determination
Because of the time and expense required for the determination of modern
analytes such as pesticide residues, industrial contaminants, veterinary drugs,
allergens, botanicals, dietary supplements, and alternative medicines in complex
matrices, there is considerable interest in obtaining acceptable methods of
analysis faster and cheaper. It has been suggested that accreditation of
laboratories, internal quality control, and external proficiency exercises can
improve laboratory performance to the point where interlaboratory validation is no
longer an absolute necessity. To this end AOACI has been exploring alternatives
to the full interlaboratory study design that requires the examination of a
minimum of 5 matrices by 8 laboratories (see www.AOAC.org under method
validation programs). These have included “minicollaborative” studies that
reduced the required number of matrices and laboratories, the “Peer-Verified
Methods Program” which merely required verification of the analytical parameters
by a second laboratory, “Performance Tested Methods” for test kits, the
developing e-CAM compiling program (www.AOAC.org/AOAC_e-CAM.pdf), and
the International Union of Pure and Applied Chemistry (IUPAC) sanctioned single
laboratory validation protocol (Pure & Appl. Chem. 74(5), 835-855(2002).
The IUPAC single-laboratory protocol necessarily deals in generalities and
specifically points out, “The total cost to the analytical community of validating a
specific method through a collaborative trial and then verifying its performance
attributes in the laboratories wishing to use it, is frequently less than when many
laboratories all independently undertake single-laboratory validation of the same
method.” The protocol also indicates that the degree of validation depends upon
the status of the method in the analytical structure. At one extreme is the initial
application of a well-established method in a laboratory that merely requires
verification of the capability of that laboratory to achieve the published
performance characteristics. The opposite extreme is the initial presentation of a
new method or the initial application of an established method to a new matrix or
application. Methods that are developed in response to a continued need for
compliance, surveillance, and enforcement of laws and contracts involving a
number of laboratories are expected to proceed to a multilaboratory validated
status.
This AOACI document is intended to present guidelines for the evaluation of the
initial use of a new or old method in a laboratory. It assumes that a proposed or
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available method is fairly well developed, optimized, and stabilized, that it has
been applied to some practical test samples with acceptable results, and that a
description of the method and its initial performance results are available in some
kind of document. The initiating or another laboratory must then decide if the
demonstrated performance appears to be satisfactory for the same or for another
purpose.
Although the output from method development is the input to method
validation, method developers cannot expect much input from method validators.
Although method validators may have had considerable experience in the
analysis of practical analytical samples, they are not expected to have the basic
knowledge to recommend improvement in methods, such as certain solvents as
useful for extraction of certain classes of analytes or column-solvent
combinations as useful for optimization of separations. Method developers are
expected to bring methods to the point where they satisfy validation
requirements.
By definition, single-laboratory validation does not provide any information
on what values would be expected on examination of identical test samples by
other laboratories. Therefore such methods probably would be used by
regulatory agencies only for monitoring purposes––to explore compliance with
laws and regulations unless the statutes under which they operate assign
correctness to their results. Ordinarily such methods would not be used to bring
a legal action or to settle a commercial dispute until their properties had been
further explored in an environment provided by an interlaboratory collaborative
study or a proficiency study utilizing that method. As stated in the FDA Center
for Drug Evaluation and Research (CDER) “Reviewer Guidance/Validation of
Chromatographic Methods” (November 1994), “Methods should be reproducible
when used by other analysts, on other equivalent equipment, on other days and
locations, and throughout the life of the drug product.”
1.0 Introduction
The primary purpose of validating a method of analysis is to show that the
method is fit for its intended purpose. Some purposes are:
1. Determine how much of a valuable, necessary, or characteristic
ingredient is present in a product.
2. Determine if a product meets specifications.
3. Determine if a product meets regulatory requirements.
4. Survey an environment to determine the presence and amount of a
component, contaminant, or a nutrient.
5. Identify a product and/or its components.
The purposes usually answer the questions, “What is this product?” in the sense
of its common or usual name, chemical identity, or components, and “How much
of something [an analyte] is in this product [matrix]?”
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At least at the initial stages of a problem, only a single or at most a very
few laboratories require validation of a method of analysis. These circumstances
include situations similar to the following:
1. Methods for research.
2. Only a few test samples are anticipated.
3. For quality control of a manufacturing process of a single item by a
single producer,
4. Checking the reliability of a method imported from another source.
5. Rechecking the reliability of a previously used method after a
period of disuse.
6. Situations where there is a lack of interest by other laboratories in
participating in an interlaboratory validation exercise.
7. Multi-analyte, multi-matrix methods where a conventional
interlaboratory validation exercise is impractical.
For the present purpose we assume:
1. We know or can assume the chemical identity of the material we
are dealing with.
2. We have a specimen of the material that can be used as a
reference to compare the signal produced by the analyte isolated from the
product we are examining with the same signal produced by a known amount of
the reference analyte (traceable to a stated reference).
If either or both of these requirement are not met, much useful information
can still be obtained, but our information will be “floating” in the same sense as a
ship at sea does not know where it is without landmarks to determine its position.
If the identity of an analyte must be determined, not merely verified, a whole new
dimension is added to the problem. This involves bringing in a laboratory or an
individual with skill in determining chemical structure, a highly specialized,
expensive, and time-consuming exercise.
It is often found during the initial experience with application or validation
of a method that deficiencies appear, unexpected interferences emerge,
reagents and equipment are no longer available, instruments must be modified,
and other unanticipated problems require returning the method to a development
phase. Frequently a method that functions satisfactorily in one laboratory fails to
operate in the same manner in another. Often there is no clear-cut differentiation
between development and validation and the two procedures constitute an
iterative process. For that reason some aspects of method development that
provide an insight into method performance, such as ruggedness, are included in
this document.
In some cases it is impossible to set specific requirements because of
unknown factors or incomplete knowledge. In such cases it is best to accept
whatever information is generated during development and validation and rely
upon the “improvements” that are usually forthcoming to asymptotically approach
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performance parameters developed for other analytes in the same or in a similar
class.
1.1 Definitions
1.1.1 Validation is the process of demonstrating or confirming the performance
characteristics of a method of analysis.
This process of validation is separate from the question of acceptability or
the magnitude of the limits of the characteristics examined, which are
determined by the purpose of the application. Validation applies to a
specific operator, laboratory, and equipment utilizing the method over a
reasonable concentration range and period of time.
Typically the validation of a chemical method of analysis results in the
specification of various aspects of reliability and applicability. Validation is
a time-consuming process and should be performed only after the method
has been optimized and stabilized because subsequent changes will
require revalidation. The stability of the validation must also be verified by
periodic examination of a stable reference material.
1.1.2 The method of analysis is the detailed set of directions, from the
preparation of the test sample to the reporting of the results that must be followed
exactly for the results to be accepted for the stated purpose.
The term “method of analysis” is sometimes assigned to the technique,
e.g., liquid chromatography or atomic absorption spectrometry, in which
case the set of specific directions is referred to as the “protocol.”
1.1.3 The performance characteristics of a method of analysis are the functional
qualities and the statistical measures of the degree of reliability exhibited by the
method under specified operating conditions.
The functional qualities are the selectivity (specificity), as the ability to
distinguish the analyte from other substances; applicability, as the
matrices and concentration range of acceptable operation; and degree of
reliability, usually expressed in terms of bias as recovery, and variability as
the standard deviation or equivalent terms (relative standard deviation and
variance).
Measurements are never exact and the “performance characteristics of a
method of analysis” usually reflect the degree to which replicate
measurements made under the same or different conditions can be
expected or required to approach the “true” or assigned values of the
items or parameters being measured. For analytical chemistry, the item
being measured is usually the concentration, with a statement of its
uncertainty, and sometimes the identity of an analyte.
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2.0 Single-Laboratory Validation Work
2.1 Preparation of the Laboratory Sample
Product and laboratory sampling are frequently overlooked aspects of analytical
work because very often product sampling is not under the control of the
laboratory but the sample is supplied by the customer. In this case, the customer
assumes the responsibility of extrapolating from the analytical result to the
original lot. If the laboratory is requested to sample the lot, then it must
determine the purpose of the analysis and provide for random or directed
sampling accordingly.
The laboratory is responsible for handling the sample in the laboratory to
assure proper preparation with respect to composition and homogeneity and to
assure a suitable analytical sample. The laboratory sample is the material
received by the laboratory and it usually must be reduced in bulk and fineness to
an analytical sample from which the test portions are removed for analysis.
Excellent instructions for this purpose will be found in the “Guidelines for
Preparing Laboratory Samples” prepared by the American Association of Feed
Control Officials, Laboratory Methods and Service Committee, Sample
Preparation Working Group (2000) (Ms Sharon Senesac, AFFCO Assistant
Secretary-Treasurer, POBox 478, Oxford IN 47971) that cover the preparation of
particularly difficult mineral and biological material. The improper or incomplete
preparation of the analytical sample is an often overlooked reason for the
nonreproducibility of analytical results.
If a laboratory prepares test samples for the purpose of validating a
method, it should take precautions that the analyst who will be doing the
validation is not aware of the composition of the test samples. Analysts have a
bias, conscious or unconscious, of permitting knowledge of the identity or
composition of a test sample to influence the result (J. AOAC Int. (2000) 83, 399-
406).
2.2 Identification
Identification is the characterization of the substance being analyzed, including
its chemical, mineral, or biological classification, as applicable. In many
investigations the identity of the analyte is assumed and the correctness of the
assumption is merely confirmed. With some products of natural origin, complete
identification and characterization is not possible. In these cases identification
often may be fixed by chemical, chromatographic, or spectrophotometric
fingerprinting –– producing a reproducible pattern of reactions or characteristic
output signals (peaks) with respect to position and intensity.
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For botanical products provide:
the common or usual name of the item;
the synonyms by which it is known;
the botanical classification (variety, species, genus, family);
the active or characteristic ingredient(s) (name and Chemical Abstracts
Registry number or Merck Index number) and its chemical class. If the
activity is ascribable to a mixture, provide the spectral or chromatographic
fingerprint and the identity of the identifiable signals.
2.3 Method of Analysis or Protocol
The protocol or method of analysis is the set of permanent instructions for the
conduct of the method of analysis. The method of analysis that is finally used
should be the same as the one that was studied and revised as a result of
research, optimization, and ruggedness trials and edited to conform with
principles and practices for the production of Official Methods of Analysis of
AOAC International (OMA). At this point the text is regarded as fixed.
Substantive changes (those other than typographical and editorial) can only be
made by formal public announcement and approval.
This text should be in ISO-compatible format where the major heads
follow in a logical progression (e.g., Title, Applicability (Scope), Equipment,
Reagents, Text, Calculations, with the addition of any special sections required
by the technique, e.g., chromatography, spectroscopy). Conventions with
respect to reagents and laboratory operations should follow those given in the
section “Definitions and Explanatory Terms” which explains that “water is distilled
water,” reagents are of a purity and strength defined by the American Chemical
Society (note that these may differ from standards set in other parts of the world),
alcohol is the 95% aqueous mixture, and similar frequently used working
definitions.
AOAC-approved methods may be considered as “well-recognized test
methods” as used by ISO 17025. This document requires that those method
properties, which may be major sources of uncertainties of measurements, be
identified and controlled. In AOAC methods the following operations or
conditions, which may be major contributors to uncertainties, should be
understood to be within the following limits, unless otherwise specified more
strictly or more loosely:
Weights within ±10% (but use actual weight for calculations)
Volumes volumetric flasks, graduates, and transfer pipets (stated
capacity with negligible uncertainty)
Burets (stated capacity except in titrations)
Graduated pipets ― use volumes >10% of capacity
Temperatures set to within ±2°
pH within ±0.05 unit
Time within ±5%
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If the operational settings are within these specifications, together with any others
derived from the supporting studies, the standard deviation obtained from these
supporting studies in the same units as the reported result with the proper
number of significant figures, usually 2 or 3, may be used as the standard
measurement uncertainty.
2.3.1 Optimization
Prior to determining the performance parameters, the method should be
optimized so that it is fairly certain that the properties of the “final method” are
being tested. Validation is not a substitute for method development or for
method optimization. If, however, some of the validation requirements have
already been performed during the development phase, there is no need to
repeat them for the validation phase. A helpful introduction is the AOACI
publication “Use of Statistics to Develop and Evaluate Analytical Methods” by
Grant T Wernimont. This volume has only three major chapters: the
measurement process, intralaboratory studies, and interlaboratory studies. No
simpler explanation in understandable chemical terms exists of the analysis of
variance than that given in pages 28-31. It supplements, explaining in greater
detail, the concepts exemplified in the popular “Statistical Manual of AOAC” by
W. J. Youden. Other useful references are Appendix D and E of Official Methods
of Analysis of AOAC International, 17th Edition (2000).
2.3.2 Reference standard
All chemical measurements require a reference point. Classical gravimetric
methods depend on standard weights and measures, which are eventually
traceable to internationally recognized (SI) units. But modern analytical
chemistry depends on other physical properties in addition to mass and length,
usually optical or electrical, and their magnitude is based upon an instrumental
comparison to a corresponding physical signal produced from a known mass or
concentration of the “pure” analyte. If the analyte is a mixtur
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