SECTION 100
Pesticide Analytical Manual Vol. I
100–1
Transmittal No. 98-1 (10/97)
Form FDA 2905a (6/92)
Table of Contents
page date
101: Regulatory Policy
101 A: Regulatory Authority 101-1 1/94
101 B: Regulatory Operations 101-2 1/94
101 C: Monitoring 101-3 1/94
References 101-3 1/94
102: Preparation of Analytical Samples
102 A: Introduction 102-1 1/94
102 B: Portion of Food Commodity to 102-1 1/94
be Analyzed
Raw Agricultural Commodities 102-1 1/94
Processed Foods 102-4 1/94
102 C: Compositing and Comminuting the 102-5 1/94
Laboratory Sample
Chapter 1
Regulatory Operations
Chapter 2
General Analytical
Operations and Information
Chapter 3
Multiclass
MRMs
Chapter 5
GLC
Chapter 4
Selective
MRMs
Chapter 6
HPLC
SECTION 100
Transmittal No. 98-1 (10/97)
Form FDA 2905a (6/92)
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Pesticide Analytical Manual Vol. I
page date
102 D: Handling Samples for Special Analyses 102-6 1/94
102 E: Retention of Reserve Portions 102-6 1/94
References 102-6 1/94
103: Method Application in Regulatory Analysis
103 A: Introduction 103-1 1/94
103 B: Choice of Method 103-1 1/94
PAM 103-1 1/94
Other “Official” Methods 103-2 1/94
Other Published Methods 103-2 1/94
103 C: Tentative Identification 103-3 1/94
103 D: Residue Quantitation 103-3 1/94
103 E: Confirmation of Identity 103-4 1/94
103 F: Documentation 103-6 1/94
Analytical Reports 103-6 1/94
Chromatograms 103-7 1/94
Chromatographic Data 103-7 1/94
References 103-8 1/94
104: Analytical Results
104 A: Introduction 104-1 1/94
104 B: Reporting 104-1 1/94
Portion of Commodity 104-1 1/94
Nature of the Residue 104-2 1/94
Residues Measured from Derivative 104-2 1/94
or Breakdown Product
Significant Figures 104-3 1/94
Trace 104-3 1/94
Confirmation 104-4 1/94
104 C: Determining Compliance with Regulations 104-4 1/94
General Rule for Multicomponent Residues 104-4 1/94
Special Situations 104-4 1/94
References 104-5 1/94
105: Analytical Limits of Quantitation
105 A: Definition 105-1 10/97
105 B: Calculation 105-1 10/97
105 C: Implementation 105-2 10/97
105 D: Factors Affecting Target Limits of Quantitation 105-2 10/97
SECTION 100
Pesticide Analytical Manual Vol. I
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Transmittal No. 98-1 (10/97)
Form FDA 2905a (6/92)
page date
Figures
102-a: Fraction of Cheese Units to Take for 102-5 1/94
Compositing
Tables
102-a: Portion of Raw Agricultural Commodity 102-2 1/94
to be Analyzed for Pesticide Residues
102-b: Portion of Processed Food to be Analyzed 102-4 9/96
for Pesticide Residues
103-a: Information Provided by Use of Element- 103-5 1/94
Selective Detectors
104-a: Portion of Commodity for Calculation 104-2 1/94
and Reporting of Residue Levels
105-a: Examples of Method Specifications 105-4 10/97
Used to Calculate Lqs
SECTION 100
Transmittal No. 98-1 (10/97)
Form FDA 2905a (6/92)
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Pesticide Analytical Manual Vol. I
Pesticide Analytical Manual Vol. I SECTION 101
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 101–1
101: REGULATORY POLICY
The expressed purpose of the Pesticide Analytical Manual is to publish analytical
methodology used by the FDA in enforcing pesticide tolerances. To understand
FDA’s application of methodology published in the manual, it is important to
understand pesticide tolerance regulations and related FDA regulatory operations.
Material in Chapter 1 reflects FDA regulatory policies that affect its application of
analytical methodology.
101 A: REGULATORY AUTHORITY
Information related to and the characteristics of pesticide tolerances include the
following:
A tolerance is the maximum concentration of a pesticide residue that is
legally permitted to remain in a food. The tolerance is not expected to be
exceeded if the pesticide’s registered use directions are followed.
The statutory authority for the Environmental Protection Agency’s
(EPA’s) establishment of tolerances is provided by sections 408 and 409
of the Federal Food, Drug, and Cosmetic Act.
Tolerances established by EPA are set forth in Title 40 of the Code of Federal
Regulations (CFR), Part 180 for raw agricultural commodities, Part 185 for
processed food, and Part 186 for animal feed. The concentration of toler-
ances listed in 40 CFR 180, 185, and 186 is expressed in terms of ppm (i.e.,
mg pesticide residue/kg food). In addition, certain pesticides are exempted
from the need for tolerances; such exemptions are listed in 40 CFR 180.1001.
A tolerance for a pesticide residue on a raw agricultural commodity, e.g.,
tomatoes, also applies to processed forms of that commodity, e.g., canned
tomatoes. In cases where processing may concentrate the residue, a food
additive regulation may be issued in 40 CFR Part 185 to establish a higher
tolerance on that processed commodity, e.g., tomato paste.
A tolerance regulation specifies the composition of pesticide residue for
which the limit applies; i.e., a tolerance can apply to the parent form of the
active ingredient only, parent compound plus one or more metabolites
and/or degradation products, one or more metabolites and/or degrada-
tion products only, or some chemical moiety that can be measured analyti-
cally for calculating the pesticide residue. A chemical entity not specified
by the tolerance regulation is not included in the residue for tolerance
enforcement purposes (Section 104).
A tolerance regulation also specifies each individual food (e.g., apples) or
food group (e.g., citrus fruit) to which the limit applies. No tolerance exists
for a residue on a commodity unless the commodity itself or the group to
which it belongs is specified.
In the examination of a food lot to determine whether it complies with
tolerances, it is necessary to provide a sample for analysis that is represen-
tative of the lot in order to determine the average pesticide residue content
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Transmittal No. 94-1 (1/94)
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of the lot. Tolerances apply to that sample or a representative portion of
that sample.
Unless otherwise specified in a tolerance regulation, each tolerance ap-
plies to the whole portion of a food commodity that moves in commerce.
In practice, however, some food commodities (mainly raw agricultural
commodities) require further definition as to the portion of commodity
to which a tolerance applies and which is to be analyzed.
In summary, a tolerance provides a means of ascertaining that a pesticide was
properly used. If a pesticide residue is found to exceed a tolerance or be present
in a food for which there is no tolerance, then the pesticide was not used in a
manner consistent with the tolerance. Under Sections 402 (a) (2) (B) or (C) of
the Federal Food, Drug, and Cosmetic Act, this constitutes a violation of the law;
i.e., the food commodity is adulterated because it contains an “unsafe” or illegal
pesticide residue.
101 B: REGULATORY OPERATIONS
To fulfill its responsibility to enforce regulations on pesticide residues in foods,
FDA maintains a comprehensive pesticide program, including the analysis of food
for enforcement of pesticide tolerances. Although the majority of samples ana-
lyzed contain no violative residues, sample handling must be consistent for all
analyzed samples, because it is impossible to know in advance which samples will
contain violative residues.
Accordingly, the following procedures must be followed by FDA laboratories to
establish that a product contains illegal pesticide residue(s):
1) A laboratory sample of food must be collected from a consignment in
accordance with agency sample collection instructions [1]; this laboratory
sample is then considered representative of the food consignment.
2) The portion of food taken from the laboratory sample (whole product
or specified parts of product) must be in accordance with agency
instructions, and that portion must be appropriately composited and com-
minuted (Section 102). From that resulting test sample, a test portion is
taken for analysis; test portion size is dictated by requirements of the
analytical method. Residues found in the test portion are considered rep-
resentative of the average residue content of the original consignment.
(Note that terminology related to food products in this chapter, i.e., “con-
signment,” “laboratory sample,” “test sample,” “test portion,” reflects rec-
ommendations of IUPAC Analytical Chemistry Division, Commission on
Analytical Nomenclature [2]. Common usage, however, usually refers to
the test portion as “sample,” and this convention is used throughout most
of PAM I.)
3) The test portion must be analyzed by a published, official analytical method
or one that has been validated for the specific pesticide/commodity por-
tion, and findings of residues must be confirmed (Section 103). For FDA
monitoring purposes, analytical methods must be capable of accurately
Pesticide Analytical Manual Vol. I SECTION 101
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Form FDA 2905a (6/92) 101–3
measuring pesticide residues as defined by the tolerance regulation at not
only the tolerance limit but also the lower limit of quantitation (Section
105).
4) If the residue level found in the original analysis exceeds an established
tolerance, or if no tolerance exists for the residue in that commodity,
another analysis of a second test portion of the same composited test
sample must be conducted by a second analyst (normally a senior ana-
lyst); the second analysis is referred to as a “check analysis.”
5) If check analysis verifies that the residue violates a regulation, i.e., the
results of both original and check analyses exceed a tolerance and are
in close agreement or are in close agreement for pesticide residues for
which there is no tolerance, the analytical findings will support enforce-
ment action against the food consignment. If the check analysis result
is below a tolerance or if the results of the original and check analyses
are widely divergent, enforcement action cannot be supported. Addi-
tional analyses may be required to resolve widely divergent analytical
results.
101 C: MONITORING
The FDA pesticide program has two main objectives: (1) to enforce residue toler-
ances and (2) to determine incidence and level of pesticide residues in the food
supply. The section above defines operations established to enforce regulations.
Monitoring aspects of the programs can be accomplished simultaneously, because
levels of all residues found are calculated and recorded, whether or not they
support enforcement action. Section 104 provides information about reporting
residues for monitoring purposes, as well as determining compliance with regula-
tions.
References
[1] Investigations Operation Manual, Sample Schedule Chart 3, FDA, Rockville,
MD
[2] Horwitz, W. (1990) Pure Appl. Chem. 62, 1193-1208
Pesticide Analytical Manual Vol. ISECTION 101
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SECTION 102
102–1
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Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
102: PREPARATION OF ANALYTICAL SAMPLES
102 A: INTRODUCTION
This section contains directions for preparation of test samples of food from labo-
ratory samples collected for pesticide residue analysis. The following topics are
considered, but not all are pertinent to every sample situation: (1) instructions for
portion of commodity to be analyzed for pesticide residues, (2) directions for
compositing and comminuting food items, (3) procedures for samples that are to
undergo special analyses, and (4) requirements for retention of reserve portions
of test samples.
102 B: PORTION OF FOOD COMMODITY TO BE ANALYZED
As a general approach, the “portion of commodity” composited to create the test
sample consists of the entire food commodity (e.g., whole cantaloupe). For many
raw and processed foods, however, only specific portions of the food are included
in the composite for the test sample. To ensure uniformity and consistency in
tolerance enforcement and related monitoring, it is necessary to adhere to the
following instructions on the portion of commodity to be analyzed.
Raw Agricultural Commodities
EPA regulations [1] specify that a raw agricultural commodity examined for com-
pliance with a pesticide tolerance consist of the “whole raw agricultural commod-
ity.” The regulations contain some specific instructions on what constitutes the
whole raw agricultural commodity; e.g., “caps (hulls) shall be removed and dis-
carded from strawberries before examination for pesticide residues.” Such instruc-
tions are provided for only nine individual food commodities (e.g., bananas) and
crop group commodities (e.g., root vegetables).
Recognizing the limitation of these regulations, FDA developed directions for
additional commodities, taking into account practical considerations of sample
preparation (e.g., removal of stones from peaches to facilitate preparation of a
homogenate). Table 102-a is a compilation of EPA regulations and FDA direc-
tions. (An EPA rulemaking is expected to be initiated that would amend the above
existing regulation to incorporate FDA’s more complete instructions on the por-
tion of commodity to which a tolerance applies and that is to be analyzed.)
In some instances, a pesticide tolerance regulation specifies an exception to direc-
tions in Table 102-a. For example, the tolerance for mevinphos residues on mel-
ons states that compliance with the tolerance is to be “determined on the edible
portion with rind removed,” [2] even though the tolerances for most other pes-
ticides on melons apply to the whole commodity including the rind.
Follow these directions to prepare test samples of raw agricultural commodities:
? Use the entire raw agricultural commodity, as specified in Table 102-a.
? When a pesticide residue is found in a commodity for which the tolerance
applies to a portion different from that specified in Table 102-a, prepare a
new test sample in accordance with the pesticide’s tolerance regulation.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)102–2
SECTION 102 Pesticide Analytical Manual Vol. I
1
Members of food groups are listed in 40 CFR 180.34 (f)(9).
Table 102-a: Portion of Raw Agricultural Commodity to be
Analyzed for Pesticide Residues
Root and tuber vegetables group
1
Where separate tolerances are established for root
or tuber, analyze whole commodity after removing
adhering soil by lightly rinsing in running water.
Where a tolerance is established on a root veg-
etable including tops and/or with tops, and tops
and roots are marketed together, analyze tops and
roots separately. Neither the pesticide residue on
the roots nor the pesticide residue on the tops
shall exceed the tolerance level. For carrots,
parsnips, and rutabagas, remove and discard tops.
Bulb vegetables (green or dry) Whole commodity after removing and discarding
group roots. Remove adhering soil by lightly rinsing in
running water. In the case of dry bulb onions and
garlic, remove and discard stems and outer sheaths
(husk or parchment skin) that are easily removed.
Leafy vegetables (except Whole commodity after removing and discarding
Brassica vegetables) group obviously decomposed or withered leaves. In the
case of rhubarb, analyze only the stem without leaves.
Remove adhering soil from celery by lightly rinsing
in running water.
Brassica (cole) leafy vegetables Whole commodity after removing and discarding
group obviously decomposed or withered leaves, except
remove and discard all leaves from cauliflower and
headed broccoli and use sprouts only from brussels
sprouts.
Legume vegetables (succulent Whole commodity, including pods for succulent and
or dried) group without pods for dry.
Fruiting vegetables (except Whole commodity after removing and discarding
cucurbits) group stems and husks.
Cucurbit vegetables group Whole commodity after removing and discarding
stems.
Citrus fruits group Whole commodity.
Pome fruits group Whole commodity after removing and discarding
stems.
Stone fruits group Whole commodity after removing and discarding
stems and stones.
Small fruits and berries group Whole commodity after removing and discarding
caps and stems, except for currants, where the stems
are to be included.
SECTION 102
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Peanuts Whole peanut meat (kernel) after removing hulls.
Peanut hulls Whole commodity after removing peanut meat.
Dates and olives Whole commodity after removing and discarding
stems and stones or pits.
Pineapples Whole commodity after removing and discarding
crowns (leaves at top of fruit).
Avocados and mangoes Whole commodity after removing and discarding
stones.
Bananas Whole commodity including peel after removing
and discarding crown tissue and stalk.
Miscellaneous raw fruits Whole commodity after removing and discarding
and vegetables not previously obviously decomposed or withered leaves, stems,
included stones or pits, shells or husks; if commodity has
adhering amounts of soil, remove by lightly rinsing
in running water.
Almond hulls Whole commodity after removing shell and
nutmeat.
Cereal grains group Whole commodity (grain) except for fresh corn
(including sweet corn). Include kernels plus
cob after removing and discarding husk.
Eggs Whole commodity after removing and discarding
shells.
Fish Edible portion of the commodity after removing
and discarding heads, tails, scales, fins, viscera,
bones (if inedible), and skin (if inedible).
Crab (hard shell) Edible portion of commodity after removing
and discarding shells, gills, and viscera.
Crab (soft shell) Edible portion of commodity after removing
and discarding gills.
Shrimp and crayfish Edible portion of commodity after removing
and discarding heads, shells, and inedible tails
of shrimp.
Lobster Edible portion of commodity including tomalley
(liver) after removing and discarding shells and
stomachs (hard sac near head).
Oyster, clam, and other shellfish Edible portion of commodity including the
liquor, after removing and discarding shells.
Rabbits and other game Edible portion of commodity after removing
and discarding bones.
Transmittal No. 96-1 (9/96)
Form FDA 2905a (6/92)102–4
SECTION 102 Pesticide Analytical Manual Vol. I
Table 102-b: Portion of Processed Food to be Analyzed
for Pesticide Residues
Processed food consisting of one ingredient Analyze the whole processed com-
and sold in a ready-to-eat form (e.g., canned modity including any liquid or
fruits packed in syrup or their own juice, other edible media in which the
canned vegetables packed in water or commodity is packed. Discard inedible
brine, or frozen fruits or vegetables, dried media, e.g., brine.
fruits, single-strength juices, catsup)
Processed food consisting primarily of one Analyze the whole processed com-
ingredient and sold in a form requiring modity after compensating for or
further preparation before it is ready to reconstituting to the commodity’s
eat (e.g., fruit juice concentrates, dehydrated normal moisture content.
vegetables, and powdered potatoes)
Processed food in a form not ready to eat, Analyze the whole processed com-
used as an ingredient or component of modity on an “as is” basis.
other food (e.g., flour, tomato concentrates
such as paste, and citrus oils)
Cheese Analyze the whole commodity
including natural cheese rind after
removing and discarding waxed or
oiled rinds.
Frozen seafood (e.g., fish or shrimp) Analyze the edible portion after
thawing; discard water.
Canned seafood Analyze the edible portion including
edible liquor and media, such as oil,
broth, or sauces in which commodity
is packed. Discard media that is not
edible.
Frog legs Analyze the edible portion of
commodity after removing and
discarding bones.
Processed Foods
In the absence of EPA regulations, FDA also developed the instructions listed in
Table 102-b on the portion of processed food to be analyzed for tolerance enforce-
ment purposes. These instructions, like the ones for raw agricultural commodities,
ensure uniformity and consistency in FDA analysis of processed food for pesticide
residues. The instructions take a practical approach for sample preparation of
processed food; e.g., fruit juice concentrates that are normally reconstituted before
consumption are also reconstituted prior to analysis for pesticide residues. There-
fore:
? Follow the directions in Table 102-b to prepare test samples of processed
foods.
SECTION 102
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Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
102 C: COMPOSITING AND COMMINUTING THE LABORATORY SAMPLE
Laboratory samples are comminuted or homogenized so that the relatively small
(25-100 g) test portion taken for analysis is representative of the entire sample.
Meaningful residue data can only be obtained when sample representation is
preserved. Specialized equipment is employed to provide as much homogeneity as
possible for the particular commodity.
The general procedure for comminuting the commodity is:
? Comminute the test sample, prepared according to Section 102 B direc-
tions.
? Use comminuting operations (grinding, chopping, etc.) that produce the
best possible homogenate for that commodity. Chopping procedures
adequate for fruits and vegetables are often inadequate for homogenizing
commodities such as dried hays and fish. See Section 203 for useful
information about equipment and procedures.
? After comminuting, handle the homogenate carefully to minimize loss of
residue by volatilization and concentration of residue through physical
separation of product.
Exceptions to the general rule occur in several situations related to commodity type
and special directions for the particular analysis. For example, removal of pits or caps
from large quantities of small units can be too time consuming; similarly, melting,
dicing, shredding, or blending a large unit of a food commodity such as butter or
cheese is impractical. The following procedures are to be used in these situations:
? When the commodity consists of small units (e.g., grains, cherries, nuts,
dried peas and beans), mix and reduce by quartering to approximately 4
lb or 4 qt. Prepare the portion of commodity from this smaller amount,
according to the appropriate directions in Table 102-a or 102-b. Chop or
grind that material to obtain >1 lb or >1 qt comminuted sample.
? When the laboratory sample consists of large units of commodities of
homogeneous nature (e.g., butter, cheese), prepare the test sample by
taking equal portions from each packaged unit. Select the appropriate
portion of commodity (for cheese, see Table 102-b) and comminute by
dicing, shredding, or blending.
? When the laboratory sample consists of large blocks, wedges, or wheels of
cheese, take fraction shown as shaded area in Figure 102-a. Select the
appropriate portion of commodity for cheese (Table 102-b) and commi-
nute by dicing, shredding, or blending.
Figure 102-a
Fraction of Cheese Units to Take for Compositing
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)102–6
SECTION 102 Pesticide Analytical Manual Vol. I
102 D: HANDLING SAMPLES FOR SPECIAL ANALYSES
Analyses for residues of ethylenebisdithiocarbamates (EBDCs) require special han-
dling of the laboratory sample. EBDCs decompose rapidly as soon as the crop surface
is broken and residues contact water, enzymes, and sugars [3]. Recoveries continue
to decrease with time of contact in aqueous crop solution. Samples for EBDC analysis
must either be analyzed immediately or frozen for storage.
Follow these directions for samples to be analyzed for EBDCs:
? Select representative units for EBDC analysis prior to chopping, grinding,
or blending the laboratory sample.
? If the individual units are small and free flowing (e.g., grains, beans, ber-
ries), mix well and take whole units for analysis.
? If the individual units are large, take wedges from each unit. Analyze
immediately or freeze immediately after cutting.
? If the commodity contains free juices (e.g., tomatoes, apples, oranges) and
also requires cutting in pieces to fit into the digestion apparatus, freeze
representative whole units before cutting. Dice frozen units without allow-
ing them to thaw; mix and take sample for analysis.
102 E: RETENTION OF RESERVE PORTIONS
The following directions apply to all test samples (i.e., comminuted material pre-
pared from appropriate portion of commodity):
? Select three 1 qt portions from the total sample homogenate; identify
them, respectively, as: “original analysis,” “check analysis,” and “reserve.”
? Take analytical test portions from the “original analysis” and “check analy-
sis,” as appropriate, and analyze. Store the “reserve” portion in a freezer
to provide to the claimant if requested.
? In addition to their sample homogenate, remaining fractions of large
samples (e.g., blocks of cheese) must be retained in a manner that pre-
vents decomposition of product and/or residue. This requires that all
products be frozen until findings of original analysis have been verified.
The amount of commodity retained is governed by the extent of analysis
required on the sample. However, in no case should portions be <1 qt
each (or for products of high density, <1 lb) for original, check, and
reserve.
References
[1] 40 CFR 180.1(j)
[2] 40 CFR 180.157
[3] Cullen, T.E. (1964) Anal. Chem. 36, 221-224
Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 103–1
SECTION 103
103: METHOD APPLICATION IN REGULATORY ANALYSIS
103 A: INTRODUCTION
The Pesticide Analytical Manual (PAM) is published to provide analytical method-
ology for determination of pesticide residues in foods. Method application in a
tolerance enforcement program imposes certain requirements and restrictions,
and this section provides information about FDA operations concerning choice of
method, identification, quantitation, and confirmation of residues(s), and docu-
mentation of analysis. Procedures established to support enforcement also apply
to analyses that result only in monitoring data (i.e., nonviolative samples), to the
degree required in a program that fulfills both needs. (When PAM methods are
used by other organizations for different purposes, e.g., environmental monitoring
without regulatory consequences, application requirements may vary from those
expressed here.)
103 B: CHOICE OF METHOD
To support enforcement action against a commodity, original, check, and any
additional analyses must be performed using official methods or methods shown
by the analyst to support the validity of the result. The following minimum evi-
dence is required to demonstrate that a method is valid for a particular analyte in
a particular commodity:
1) Reagent blank analysis performed using reagents only (no commodity)
shows no detector responses that could be mistaken for the analyte.
2) Historical or concurrent analyses of a residue-free lot of the same or similar
commodity show no interfering detector responses.
3) Recovery of the analyte, added to a residue-free sample at or near the level
of residue in the violative sample, is in the range 80-110%. If a residue-free
lot is not readily available, recovery determination may be performed on
another portion of the sample of interest by fortifying it with at least twice
the level of residue found (e.g., add 2 ppm if original analysis is 1 ppm).
Validation tests may be performed by either the original or check analyst and must be
carried out concurrently with analysis of the sample of interest. The method used to
determine any illegal residue found in a commodity for the first time by a laboratory
must be validated in this way.
Analytical methods may be taken from the following sources, in decreasing order of
preference:
PAM
Analytical methods from the PAM are suitable for regulatory analysis. As specified by
regulation in the CFR, PAM contains the methods FDA uses for determining
compliance with pesticide tolerances [1]. Volume I methods are capable of determin-
ing more than one residue (multiresidue methods, MRMs) and are most often used
for routine analysis.
SECTION 103
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PAM I MRMs are presented as choices of several extraction, cleanup, and determi-
native modules. At the beginning of each method section in Chapters 3 and 4,
method combinations that have undergone interlaboratory validation are listed.
Absence of a particular combination of modules from this list does not prevent its
use in regulatory analysis, but the analyst must provide suitable supporting evi-
dence, as described above, that the combination is indeed valid for the particular
commodity and residue combination.
PAM II methods are designed to determine residues of a single pesticide (single
residue methods, SRMs). SRMs are published in PAM II for residues of all pesti-
cides subject to EPA tolerances. These methods, usually from the pesticide manu-
facturer that petitioned for the tolerances, may be used by FDA to target residues
not determined by MRMs. SRMs are also useful for check analyses when a residue
has been determined first by an MRM, especially when a residue determined by an
MRM is known to represent only part of the expected residue of a particular
pesticide. For example, it may be known that the MRM does not completely re-
cover the residue, or that the tolerance definition of the pesticide residue includes
metabolites not detectable by the MRM. In these cases, MRMs may detect the
presence of a residue but further analysis must be performed using a PAM II SRM
to determine the total residue.
Other “Official” Methods
AOAC International maintains a system of interlaboratory testing through which
methods passing the requirements are designated “AOAC Official”; many PAM I
methods have undergone this process and are AOAC Official for certain residue/
commodity combinations. Other AOAC official methods are not included in PAM
but are acceptable for use in FDA regulatory analyses; AOAC methods have also
been designated in the CFR as methods suitable for use [2]. In some cases, the
AOAC method is preferable; e.g., AOAC method 977.19 [3], official for
hexachlorobenzene and mirex in adipose tissue, is preferred to PAM I method
304, through which the two chemicals involved are incompletely recovered.
Other Published Methods
When investigational evidence suggests that a commodity may contain a residue for
which no PAM I or other official method is acceptable, published methods from the
scientific literature must be sought. Use of such a method must be supported by
documented evidence of its applicability to the residue and commodity in question,
in the hands of the analyst who performed the regulatory analysis. The requirements
described above are the minimum acceptable as supporting evidence.
Liquid-liquid partitioning and column chromatography cleanup steps that vary
from established versions only in the use of proportionately smaller amounts of
reagents are usually considered equivalent to the original; such methods are often
referred to as “scaled-down” or “miniaturized.” Miniaturized procedures that are
published, validated, and used routinely in FDA are included as method modules
in PAM I, e.g., the 4 g Florisil column of Section 302 C1, developed as an alterna-
tive to the 20 g column. Certain other miniaturized versions of PAM I method
modules have not undergone validation, but analyses performed in this way are
considered adequate to support enforcement action and to assume equivalent
coverage of residues for monitoring purposes. Extraction steps that involve smaller
sample weight and extractant volume have not yet been studied sufficiently to
Pesticide Analytical Manual Vol. I
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SECTION 103
endorse, however; it is recommended that miniaturization be applied only to steps
subsequent to filtration of the original extract.
103 C: TENTATIVE IDENTIFICATION
Application of methods in this manual results in tentative identification of resi-
dues based on the analyte’s behavior matching that of a reference standard. “Be-
havior” of the analyte refers to its recovery through methods, including the eluate(s)
in which it elutes from cleanup columns, its GLC or HPLC retention time, and the
response it elicits from selective detectors or detection systems.
PAM I tables list test results of chemical behavior through various analytical proce-
dures. The tables are provided to supply analytical chemists with information
useful in residue identification. Typically, a sample is analyzed by a particular
method and the extract examined by one or more determinative steps. When a
GLC or HPLC response is recorded, the analyst measures the retention time of the
response, calculates a relative retention time, and refers to the appropriate PAM I
table (e.g., Appendix I, PESTDATA; Table 403-a) to find chemicals that elute at
that approximate time. Detector response data and molecular formulas included
in PESTDATA offer further clues about which residue is likely to have caused
response by the GLC element-selective detector used. Other details of chemical
behavior through methods provide the analyst with means to eliminate certain
candidate chemicals from consideration and strengthen the case for others; e.g., if
the method included a column chromatographic cleanup step and more than one
eluate was used, only those chemicals known to elute in the pertinent eluate
should be considered further.
In most cases, this preliminary evaluation provides the analyst with a limited number
of potential candidates for residue identification. A reference standard solution of
each likely chemical is then chromatographed on the appropriate GLC or HPLC
system for direct comparison with the residue. In no circumstance is a table of data
alone adequate for residue identification.
The expertise of the residue chemist is most critical during the determinative step of
the analysis. The chemist’s knowledge of pesticide usage and the chemistry and
metabolism of pesticides is invaluable to the correct interpretation of evidence.
Familiarity with commonly encountered artifacts from commodities, reagents, and
environmental contaminants helps avoid incorrect conclusions.
103 D: RESIDUE QUANTITATION
Quantitation of residues is considered appropriate when:
1) The level of residue in a commodity is quantitated according to standard
practices of GLC (Section 504) and HPLC (Section 606). In addition:
a) Peak sizes of sample and standard match within ±25%.
b) Time between sample and standard injection is ≤1 hr; chromato-
graphic sequence ends with a standard.
c) Replicate injections of standard and sample have been made when
determining difficult-to-chromatograph residues.
SECTION 103
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2) Directions from Section 104 have been followed for summing levels of
related residues for purposes of determining compliance with regulations.
3) Determination occurred at conditions that provided a limit of quantitation
as directed in Section 105.
103 E: CONFIRMATION OF IDENTITY
Because analytical processes are subject to possible error in interpretation or mea-
surement, confirmatory evidence must be developed to increase confidence in the
tentative residue identification (Section 103 C). Attempting to define minimum
confirmation requirements that are adequate for every situation is impractical.
Instead, a philosophy of confirmatory analyses and discussion of certain minimum
expectations are presented.
The extent of confirmatory effort will be influenced by the significance of the
sample, nature and level of the residue, sample history, purpose of the analysis,
and practical considerations such as time, cost, number of other samples, etc. The
choice of confirmatory procedures depends on the tentative identity of the pesti-
cide, amount of residue available for testing, sample type, and availability of instru-
mentation required for confirmatory tests.
The logic of most confirmatory schemes relies upon presumptive evidence; i.e., if
the behavior of an unknown in a particular analytical technique is the same as that
of a reference standard, it is presumed that they are the same chemical. Any
analytical technique that measures a single property of an analyte may err in
distinguishing between two chemicals that behave the same; e.g., two different
chemicals may have the same GLC retention time. To avoid this potential error,
either the analytical technique must inherently measure more than one property
of the analyte or more than one analytical technique must be employed, each of
which measures a different property. Following this logic, FDA laboratories con-
firm most residues by one of two approaches:
1) FDA requires that mass spectrometry (MS) be used to confirm the identity
of any residue found for the first time. Modern laboratories usually have
access to compact, highly automated mass spectrometers, configured as
either mass-selective or ion trap detectors for GLC. Such instruments are
capable, in some instances, of simultaneously detecting, quantitating, and
confirming the residue, especially when reference standards are available.
Errors may still occur, however, if GLC-MS is operated in the single ion
monitor mode, which is not adequate to distinguish between two chemi-
cals that elute from the column at the same time and are detected at the
same m/z. For identification to be confirmed, GLC-MS must be operated
by monitoring at least three ions [4].
Full spectrum MS on a high resolution instrument may be required for
full structural elucidation of previously unidentified chemicals. Such analy-
ses usually require research MS instruments, operated by specialists, that
offer several different modes of ionization that may be needed for unam-
biguous identification.
2) Residues previously reported may be confirmed by less rigorous techniques,
such as additional chromatographic analysis (GLC or HPLC), with different
Pesticide Analytical Manual Vol. I
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SECTION 103
columns, mobile phases, and/or detectors. Confirmation of identity re-
quires an accumulation of corroborating evidence sufficient to prove that
the residue and reference standard must in fact be identical because they
behave the same way in different tests. Such evidence is provided by
measurement of a different chemical or physical property in each test used.
Selective MRMs (Chapter 4) are designed to be applicable to residues of a single
chemical type; the steps of the method provide some built-in confirmation
because only residues with that chemistry are recovered and determined. Other
available confirmatory analyses are referenced as part of these method descrip-
tions.
The GLC determinative steps included with multiclass MRMs (Section 302 DG1-
DG23) offer a series of alternatives that can be used as confirmatory analyses. To
the degree that a detector is selective to a single element or group, its use inher-
ently provides some confirmatory evidence during the original tentative identifica-
tion. Additional chromatography using other element-selective detectors is ideal
for confirming a residue that contains appropriate elements. For example, many
chemicals contain both nitrogen and phosphorus, others both phosphorus
and sulfur. Complementary evidence from phosphorus and sulfur mode flame
photometric (FPD-P and FPD-S), N/P, and electrolytic conductivity nitrogen mode
(ElCD-N) detectors, as shown in Table 103-a, can provide excellent confirmatory
information.
Table 103-a: Information Provided by Use of Element-Selective
Detectors
Detector Response Conclusion
Example
1
N/P
1
Measurable Either N or P in molecule
and
FPD-P
2
None N in molecule
Example
2
N/P None No N or P in molecule
and
FPD-P
2
Measurable Large amount of S in molecule
(verify with FPD-S
3
)
Example
3
N/P Measurable Either N or P in molecule
and
FPD-P Measurable P in molecule; N also possible
(verify N with E1CD-N
4
)
1
responds to both N and P
2
responds to P; large amount of S can also cause response
3
responds to S; presence of P can also cause response
4
responds to N only
SECTION 103
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No detector is completely element-specific, however, and the analyst must be aware
of the potential for false-positive responses. Replacement of the relatively nonse-
lective electron capture detector with element-selective detectors encouraged de-
velopment of methods with minimal cleanup. These methods are popular because
they reduce analytical time and solvent volumes, and because they permit determi-
nation of residues that are removed by traditional column chromatographic cleanup
steps (Section 301). However, extracts from such methods contain relatively large
amounts of co-extractives, to which even selective detectors may respond. Columns
of dissimilar polarity should be used with the various element-selective detectors to
strengthen confirmation.
Special precautions are necessary when determining residues in which nitrogen is
the only element to which element-selective detectors respond. Because many
nonpesticidal organonitrogen chemicals occur naturally in foods, chromatograms
from nitrogen-selective detectors often display a pattern of responses related to
the commodity being analyzed. Additional columns and/or cleanup steps should
be employed to confirm identification of residues found with nitrogen detectors.
Other techniques are available for confirmation if chromatography with element-
selective detectors is not applicable or available. Chemical or photolytic derivatization
and thin layer chromatography are among those most frequently employed.
103 F: DOCUMENTATION
FDA regulatory operations require that analytical results be adequate to support
enforcement actions, if necessary, in a court of law. The analytical package accompa-
nying a recommendation for enforcement action must demonstrate that the require-
ments described above for sampling, sample handling, and analysis have been met.
Descriptions of analytical operations must be documented in a format readily
understandable to another residue chemist and explainable to nonscientists. To that
end, FDA established the following minimum requirements for documenting analy-
ses that support recommendations for enforcement action against food or feeds
because of the presence of violative pesticide residues.
Analytical Reports
Analytical reports for both original and check analyses must provide complete
information on the sample and on sample handling steps used, as well as the following
analytical information:
1) Method reference or memorandum of analysis, including description of any
modifications made to the referenced method
2) Measured weights and volumes used for sample weight calculations
3) Volume of final sample solution or eluate
4) Details of residue determination record
a) Sample solution/eluate identity (e.g., 6% ethyl ether/petroleum ether)
b) Sample weight per unit volume
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c) Volume and sample weight equivalent injected
d) Retention time or distance and retention time relative to the marker
compound appropriate to the system
e) Peak size; area or height for sample(s) and standard(s)
5) Calculation of results; may also appear on chromatograms
6) Column and detector used for each injection
Chromatograms
Each chromatogram must be labeled with identity of solution, volume and weight
injected, date, analyst’s initials, and time of injection. Time is not required if a
continuous chromatogram is submitted. The following chromatograms must be
submitted:
1) Marker compound. For each column-detector combination used, a chro-
matogram of the reference standard used as a marker compound for
that combination; it is preferable for additional reference standards also
to be included in a mixed standard solution appropriate to the determi-
native step.
2) Sample and standard chromatograms used for quantitation
3) Any other chromatograms (for both sample and standard) associated with
additional tests to confirm identity of the residue
Chromatographic Data
The following information must be included on the chromatograms. (If a data
collection system is used, much of the required information can be automatically
entered into the chromatographic report.)
1) Brand name and model of chromatograph
2) Column: type, size, liquid phase, plus solid support and percentage load-
ing for GLC packed column; coating identification, film thickness, length
and internal diameter for GLC wide bore or capillary column; packing
identification, length, and internal diameter for HPLC column
3) Temperatures: column, detector, injector, transfer lines, furnaces, etc.
4) Description of injector and/or inlet system
5) Gas flow rates and identities for carrier, fuel, purge and make-up gases
(GLC); mobile phase, gradient if applicable, flow rate (HPLC)
SECTION 103
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6) Detector type, including design, mode of operation, and specifics related to
operation of the particular detector, such as ion exchange resin, electrolyte,
and electrolyte flow rate of the electrolytic conductivity detector
7) Range settings of electrometer, integrator, linearizer, etc., along with perti-
nent detector voltages
8) Parameters for signal measurement: recorder span and speed, integrator
settings, etc. (recommended chart speed is 1 cm/min or 0.5"/min)
References
[1] 40 CFR 180.101(c)
[2] 21 CFR 2.19
[3] Official Methods of Analysis (1990)15th ed., Association of Official Analytical
Chemists, Inc., Arlington, VA
[4] Sphon, J.A. (1978) J. Assoc. Off. Chem. 61, 1247-1252
Pesticide Analytical Manual Vol. I SECTION 104
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104: ANALYTICAL RESULTS
104 A: INTRODUCTION
Three separate but related responsibilities must be managed within any regulatory
program of analyses for pesticide residues: (1) determination of residue identity
and calculation of level, (2) reporting residue identity and level, and (3) judging
whether the presence of that level of residue is in compliance with regulations or
whether it warrants enforcement action.
The first responsibility, residue identification and calculation, is a scientific en-
deavor treated in the remaining chapters of this manual; regulatory requirements
for these operations were discussed in Section 103. Regardless of the purpose of
the analysis, the basic instructions remain the same. Appropriate application of
Chapters 3 or 4 methods, combined with the precepts of accurate quantitation
described in Sections 504 and 606, will permit the chemist to ascertain the pres-
ence or absence of particular residues and to calculate the quantity of each resi-
due present in the sample.
Beyond the scientific endeavor, the use to which analytical results are put is within
the province of the organization sponsoring the work. In this section, current FDA
procedures for reporting residues and determining compliance with prevailing
regulations are noted. Operations are often dictated by the needs of the dual-
purpose FDA pesticide program: to monitor incidence and levels of pesticide
residues in foods and to enforce regulations concerning permissibility of these
residues.
104 B: REPORTING
In this context, reporting the presence and levels of pesticides refers to laboratory
entry of information into a computerized data base. FDA has monitored residue
trends for more than 30 years by reporting results of all analyses, including those
in which no residues were found, into a data system. Pertinent information de-
duced from accumulated data includes the decline in residue levels of persistent
chlorinated hydrocarbons in the years following their ban and the identity of
terminal residues in different commodity types. One goal of the residue reporting
system is to retain all pertinent information obtained during the analysis; of spe-
cial interest is the precise identity of the residue. Accumulated reports of residue
analyses are used to answer many questions about the prevalence of residues in the
food supply; most prominently, the data are used to prepare an annual report of
residue findings [1].
The following reporting practices have evolved over the years to produce the most
meaningful possible data for agency interpretation:
Portion of Commodity
The exact portion of food that is analyzed for pesticide residues is dictated by the
purpose of the analysis. Section 102 reflects agency procedures for the portions of
particular commodities used in FDA regulatory monitoring. Once analyzed, resi-
due levels are calculated and reported based on the exact portion of food taken
for analysis as indicated in Table 104-a.
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Pesticide Analytical Manual Vol. ISECTION 104
Table 104-a: Portion of Commodity for Calculation and Reporting of
Residue Levels
Commodity Report Results On:
Raw agricultural commodities Whole commodity as prepared for analysis
Milk products
1
Whole product
Juice concentrates or powders Reconstituted basis
Concentrated/dehydrated products Whole product, “as is”
consumed “as is” or used as
ingredients
Dehydrated vegetables intended for Calculated equivalent weight of original
use after reconstitution product before dehydration
Other processed foods Whole product
1
Includes whole, low fat, skim, and other milk products. Note that reporting residues in whole milk
on the whole basis (effective 10/1/91) does not change the way of determining compliance with
regulations for residues whose tolerances in milk are on the fat basis.
Nature of the Residue
Whenever possible, pesticide residues in foods are identified and calculated as
individual chemicals (i.e., parent compound, metabolites, and degradation prod-
ucts). Although many pesticides are formulated and marketed as technical mix-
tures of related chemicals, their residues are calculated separately whenever chro-
matographic conditions and availability of separate reference standards permit.
Residues are reported as the individual isomer or congener that is identified and
calculated during the determination. If the residue can be identified and its level
calculated only by comparison to a mixed or technical reference standard, the
residue may be reported as such in the data system.
Residues of polychlorinated biphenyls (PCBs) are calculated by comparison to
commercial mixtures known as Aroclors (Section 504 D). Levels are reported in
terms of the particular Aroclor(s) used as reference standard.
Residues Measured from Derivative or Breakdown Product
Some analytical methods convert the residue(s) to a derivative or breakdown
product so that common determinative steps can be used. In these cases, some
convention must be established for reporting the residues. Specific instances of
this situation are:
Acids and Phenols. Residues of acids and phenols are converted to respective
methyl esters/ethers by Section 402 method, to produce chemicals that can be
measured by GLC. Reference standard solutions are prepared from standards of
the ester/ether, if available, or from standards of the acid/phenol carried through
the procedure. Levels of residue are calculated and reported as the acid or
phenol.
Pesticide Analytical Manual Vol. I SECTION 104
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Benomyl, Thiophanate-Methyl, and Carbendazim (MBC). Residues of these ben-
zimidazole pesticides and related residues are determined by Section 404. MBC,
the most common residue, may result from use of benomyl, which converts rapidly
to MBC; thiophanate-methyl, which converts slowly; or carbendazim, a fungicide
that is the same chemical as MBC. Residues are reported according to assumptions
made about the source of MBC found.
In absence of evidence to the contrary, a residue of MBC is assumed to result from
use of benomyl. MBC residues are quantitated by comparison to a reference stan-
dard of MBC and the level converted to the equivalent benomyl level, which is
reported.
However, if MBC and thiophanate-methyl are both found in the sample, or if
investigatory evidence indicates the commodity was treated with thiophanate-me-
thyl, the quantitated level of MBC is converted to equivalent thiophanate-methyl
and reported as such.
EBDCs. Tolerances for EBDCs are established in terms of parts per million
(ppm) zineb, one of the EBDCs. Residues of EBDCs are determined by methods
that convert these chemicals to carbon disulfide, which is measured and calculated
as zineb. This analytical approach precludes identification of which EBDC was
present. By convention, FDA laboratories report levels found as “EBDC (identity
unknown),” unless investigatory evidence suggests which of the EBDCs was used
on the product.
Because registrations for zineb have been cancelled, supplies of the chemical may
not be available for use as a reference standard. If necessary, another EBDC ana-
lytical standard may be used and appropriate molecular weight conversion made
to permit reporting residues in terms of ppm zineb.
Significant Figures
The level of each residue that appears at or above its limit of quantitation for the
method is calculated (Section 105 discusses limit of quantitation). Residues are
calculated and reported in ppm. Unless the quality of the chromatography or
other factors necessitates fewer significant figures, levels are reported as follows:
≥100 ppm to nearest ppm
10 to 99.9 ppm to nearest 0.1 ppm
1 to 9.99 ppm to nearest 0.01 ppm
0.010 to 0.999 ppm to nearest 0.001 ppm
Trace
Residues that are detectable by the method but present at less than the limit of
quantitation are reported as “Trace.”
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Pesticide Analytical Manual Vol. ISECTION 104
Confirmation
Identities of residues are confirmed before reporting, according to the principles
discussed in Section 103. Confirmation of nonviolative levels of frequently found
residues do not require confirmation in every sample; frequency of confirmation is at
the discretion of the laboratory.
104 C: DETERMINING COMPLIANCE WITH REGULATIONS
For monitoring purposes, all residues of the same pesticide are calculated and
reported as individual chemicals. For tolerance enforcement purposes, however,
the residue definition as stated in the particular regulation applies; in some situ-
ations, not all residues are included in the total residue for determining compli-
ance with the tolerance. FDA’s Compliance Policy Guide [2] provides criteria that
must be met to initiate an enforcement action for violative pesticide residues
found in a food commodity. Directions included in this section assume that
quantitation has been accurately performed, according to directions in Sections
504 and 606, and that individual residues have been reported into the data system.
General Rule for Multicomponent Residues
Residues that consist of more than one isomer of a technical pesticide, or that
consist of parent and degradation products, are added together to determine
compliance with existing tolerances, insofar as the degradation products are in-
cluded in the tolerance expressions of 40 CFR Parts 180, 185, or 186 [3].
Special Situations
BHC. Determining compliance of residues of BHC is complicated by the fact that
γ-BHC, also known as lindane, is marketed as a separate pesticide and is also an
isomeric component of technical BHC, which may have up to six different iso-
mers. At one time separate tolerances for BHC and for lindane were established,
and the possibility existed that both might be used on the same commodity.
Currently, U.S. tolerances for BHC have been revoked, but residues are still found
in domestic and imported commodities; U.S. tolerances for lindane remain for
several commodities.
When residues of BHC isomers are found in a commodity, the quantity of each
isomer that is present is calculated against an individual reference standard,
according to the general principle; quantities of α, β, and δ isomers are then
added together. If the amount of γ-BHC is <1/3 the total of α+β+δ, the total of
the four isomers is considered to be a residue of BHC. If the γ-isomer is >1/3
the total of α+β+δ, the amount in excess of 1/3 (α+β+δ) is considered to be
lindane and the remainder of the γ plus α, β, and δ is considered BHC. Appro-
priate regulatory action is decided based on these calculations.
Chlordane, Heptachlor, Heptachlor Epoxide. Two factors complicate the residue
situation for chlordane: (1) chlordane is a multicomponent mixture whose termi-
nal residue pattern varies considerably, and (2) one component of technical chlo-
rdane is heptachlor, which was marketed as a separate pesticide. At the time when
both chlordane and heptachlor were registered for food use, the possibility existed
that they might be used on the same product. U.S. tolerances for both chlordane
Pesticide Analytical Manual Vol. I SECTION 104
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and heptachlor are now revoked, and most residues now occur in fish as a result
of lingering environmental contamination, but the procedures developed during
the previous period still apply if needed.
Section 504 outlines the prescribed method for quantitating chlordane residues,
either against a technical standard or against individual reference standards, de-
pending on the residue pattern. It also specifies that peaks at the retention times
of heptachlor and heptachlor epoxide can be included in quantitation of chlor-
dane against the technical standard, if those peaks are relatively small.
If chlordane residues are calculated as individual terminal residues, they are added
together to determine total chlordane. If measured against a technical chlordane
reference standard, the calculated value is considered total chlordane. In either
case, heptachlor and heptachlor epoxide peaks are included as part of total chlor-
dane if they are relatively small and in reasonable proportion to the rest of the
residue. If heptachlor and/or heptachlor epoxide are much out of proportion, as
shown in Figure 504-d, they are considered as separate residues. Appropriate regu-
latory action is decided based on these calculations.
PCBs. Regulations related to PCB residues establish tolerances for “PCBs,” so
compliance is based on total PCBs calculated, regardless of which Aroclor(s) was
used as reference standard.
Residues of More Than One Pesticide. In certain cases, pesticides “that cause
related pharmacological effects [are] regarded, in the absence of evidence to the
contrary, as having an additive deleterious action” [3]. Special rules for adding
together residues in these categories may apply.
Residues Calculated From a Derivative. As described above under reporting,
some residues can be quantitated only by methods that form a derivative prior to
the determinative step. Compliance with regulations in these cases depends on the
precise statement of the regulation and on investigatory evidence related to the
particular sample. For regulatory purposes, the residue level must be calculated in
terms of the chemical(s) specified in the tolerance; if necessary, a conversion is
made.
References
[1] Food and Drug Administration (1993) J. AOAC Int. 76, 127A-148A, and previous
annual reports
[2] Compliance Policy Guide, Section 7141.02, Food and Drug Administration,
Rockville, MD
[3] 40 CFR 180.3
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Pesticide Analytical Manual Vol. ISECTION 104
Pesticide Analytical Manual Vol. I SECTION 105
105–1
Transmittal No. 98-1 (10/97)
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105: ANALYTICAL LIMITS OF QUANTITATION
105 A: DEFINITION
FDA defines limit of quantitation (Lq) as the lowest level of residue that can be
quantitated by a given method and whose identity can be confirmed in regulatory
laboratories operating under routine conditions. Levels less than the Lq are defined
as trace.
When MRMs are used, a separate Lq applies to each residue determined by the
method because each represents a different analytical situation.
The following factors must be specified in order to define the analytical situation; only
then can an Lq be calculated:
1) Analytical method used
2) Sample (matrix) type
3) Sample weight equivalent introduced to the determinative step
4) Sensitivity of the determinative step to the analyte; sensitivity is dependent
on the following instrumental conditions:
a) Determinative technique (In MRMs, the determinative step is usually
GLC or HPLC; operational parameters must be defined as part of the
method description.)
b) Range of analyte weight that produces a linear detector response
c) Overall condition of the system
d) Amplification and/or attenuation of the detector signal
e) Characteristics of the signal processing or recording device
f) Chromatographic elution characteristics of the analyte
105 B: CALCULATION
FDA Lqs for each method are arrived at by (1) specifying a sample weight equivalent
to be examined by the determinative step (the amount chosen must be compatible
with long-term instrument stability); (2) establishing a recommended determinative
step sensitivity that is stable, reproducible, and achievable by all laboratories; and
(3) establishing a response equivalent to 10% of full scale deflection (FSD) on the
signal-processing device as the minimum considered quantifiable and confirmable.
FDA methods applied according to these guidelines are capable of analyzing for most
residues at levels well below established tolerances.
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Pesticide Analytical Manual Vol. ISECTION 105
Determinative step sensitivity is established by reference to a “marker compound”; i.e.,
the instrumental parameters are adjusted to cause a specified response to a specified
quantity of the marker compound. This approach makes it possible for different
laboratories to achieve approximately the same Lq even though the instrument
settings may be different for each. Lq for the marker compound can then be
calculated with the formula below for any particular method. Lqs for all other
compounds recovered through the method will vary according to the determinative
step sensitivities for each.
With these guidelines established, Lq for a method is calculated thus:
ng 50% FSD = ng analyte injected x ng marker specified x marker peak height
ng marker injected analyte peak height
ng 10% FSD = ng 50% FSD/5
Lq = (ng 10% FSD)/(mg sample injected)
Round the Lq result following the guidance for significant figures and reporting
analytical results in Section 104, page 104-3. For general purposes, results at or below
0.010 ppm are deemed to have an Lq of 0.010 ppm.
105 C: IMPLEMENTATION
Guidelines for applying analytical methods are required to provide consistency
among laboratories performing regulatory analyses. Otherwise, variations in the
amount of sample equivalent injected and/or the sensitivity of the determinative
step can cause different Lqs in different laboratories. Lqs that result from fol-
lowing FDA guidelines are adequate for the enforcement of tolerances and, in
most cases, are sufficient to determine residues below the tolerance level so that
data on incidence and levels of residues in foods and feeds can be collected.
The following rules are established to maintain consistent Lqs among FDA labo-
ratories:
? Establish the sensitivity recommended in each determinative step method
module (e.g., Section 302 DG1-DG12, Section 401 DL1). Note that the
requirement for GC determinations to be based on columns of 100%
methyl siloxane is in effect as of FY'98 (October 1, 1997); prior to that
time, other DG modules may have been used to calculate Lq.
? Inject a volume of extract containing the equivalent sample weight recom-
mended for each method (e.g., Section 302, Determination).
? If one of the recommended specifications above cannot be achieved, or
if changing one is advisable for any reason, adjust the other parameter to
maintain the targeted limit of quantitation. Section 105 D describes fac-
tors that may cause problems in specific situations.
Table 105-a lists examples of Lqs that can be calculated from the recommended
sample weight equivalent and determinative step sensitivity for particular PAM I
methods. The list is not exhaustive but does illustrate the way in which the Lq for
any method in PAM I can be calculated.
Pesticide Analytical Manual Vol. I SECTION 105
105–3
Transmittal No. 98-1 (10/97)
Form FDA 2905a (6/92)
105 D: FACTORS AFFECTING TARGET LIMITS OF QUANTITATION
The following factors, individually or in combination, may reduce the certainty of
quantitation and/or identification of a residue in any specific analytical situation.
They may also cause the Lq to differ from the recommended limit defined by the
formula above and by Table 105-a. Measures taken to compensate for one factor may
trigger the influence of another.
1) Determinative step sensitivity to any particular residue. A distinct Lq
applies to each residue determinable by a particular MRM, because the
sensitivity of the determinative step to each compound may be different.
2) Limited detector sensitivity. Not all individual detectors are capable of
reaching the sensitivity specified; in such cases, the Lq will be higher
than targeted.
3) Greater detector sensitivity. Directions here recommend sensitivity at
which detectors should be operated, even though some are capable of
greater sensitivity. However, operation at conditions that produce rec-
ommended sensitivity may sometimes be precluded by other disadvan-
tages in detector performance. For example, many models of
63
Ni elec-
tron capture detectors are not linear at conditions that produce sensi-
tivity of 50% FSD to 1.5 ng chlorpyrifos, as is recommended for other
detectors; most are linear, however, at conditions that produce 50% FSD
to 0.15 ng chlorpyrifos. The rules in Section 105 C specify that, in this
situation, the laboratory should operate at the greater sensitivity in or-
der to work in a linear range, then proportionately reduce the weight
of sample equivalent injected in order to maintain Lqs consistent with
those achieved by other laboratories.
4) Other improvements that affect determinative step. Wide bore capillary
GLC columns (Section 502 C) permit analytes to elute in a tighter band
than was possible with packed column chromatography. When detector
response is measured in terms of peak height, use of capillary columns
results in an apparent improvement of response. Injection of a smaller
amount of equivalent sample, as directed in Section 105 C, is appropri-
ate and, at the same time, beneficial to the longevity of the column.
5) Excessive interferences from sample co-extractives. Interferences from
sample co-extractives raise the Lq of a method by masking the detector
response to the residue or by preventing injection of the specified sample
equivalent without undesirable damage to the system. Additional proce-
dures to clean up the sample extract prior to determination may im-
prove the Lq by removing these interferences.
105–4
Transmittal No. 98-1 (10/97)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. ISECTION 105
1
Parenthetical codes indicate the detector used in the GLC determinative step.
2
Ng marker compound that causes detector response of 50% FSD; where residues targeted
by the method are different from the marker compound, weight of example target that
caused 50% FSD is also listed.
3
Calculated by formula in Section 105 B; note that sensitivity is divided by 5 to produce ng
causing 10% FSD.
OR
Table 105-a: Examples of Method Specifications Used to
Calculate Lqs
Recommended Recommended
PAM I Method
1
Mg Injected Sensitivity
2
Lq (marker compound)
3
302 E1+DG2 (FPD-P) 20 mg 1.5 ng chlorpyrifos 0.015 ppm chlorpyrifos
302 E3+C1+DG3 20 mg 1.5 ng chlorpyrifos 0.015 ppm chlorpyrifos
(ElCD-X)
302+E1+C3+DL1 116 mg 10 ng carbofuran 0.017 ppm carbofuran
303 E1+C1+DG1 (EC) 20 mg 1.5 ng chlorpyrifos 0.015 ppm chlorpyrifos
2 mg 0.15 ng chlorpyrifos 0.015 ppm chlorpyrifos
304 E4+C2+DG1 (EC) 10 mg (cheese 1.5 ng chlorpyrifos 0.03 ppm chlorpyrifos,
with 30% fat) whole product basis
401 E1+C1+DL1 200 mg 10 ng carbofuran 0.01 ppm carbofuran
402 E1+C1+DG3 5 mg Eluate 1 1.5 ng chlorpyrifos 0.008 ppm PCP
(fatty foods) (0.2 ng PCP methyl ether
methyl ether)
10 mg Eluate 2 1.5 ng chlorpyrifos 0.01 ppm 2,4,5-T
(0.5 ng 2,4,5-T methyl ester
methyl ester)
402 E2+C1+DG3 10 mg Eluate 1 1.5 ng chlorpyrifos 0.004 ppm PCP
(nonfatty foods) (0.2 ng PCP methyl ether
methyl ether)
20 mg Eluate 2 1.5 ng chlorpyrifos 0.005 ppm 2,4,5-T
(0.5 ng 2,4,5-T methyl ester
methyl ester)
403 E1+C1+DL3 800 mg 40 ng diuron 0.01 ppm diuron
404 E1+DL5 125 mg 62.5 ng MBC 0.1 ppm MBC
404 E1+DL7 125 mg 6.25 ng 0.01 ppm thiabendazole
thiabendazole
(fluorescence
detector)