Method validation and quality

control procedures

Department of Food Chemistry and Analysis, ICT Prague

Vladimír Kocourek
Prague, 2012
 WHAT IS VALIDATION?
► Validation is a process, within which the method is demonstrated
to be suitable for its purpose. It documents methods performance !

► During validation process, methods Performance characteristics

are estimated.

► Validation documents, that the methods performance

characteristics are capable of producing results in line with the
needs of the analytical problem.
Is it possible to detect pesticide residues at regulation levels using the method ?
Is it possible to correctly quantify the amount of residues in apple/orange/… ?
…

► Validation procedure (protocol) is related to a particular

analyte and matrix

Applicability
Analytical method for decision making

Defining Selection of
the task method

Sample
handling

Analytical
procedure
measuring

Calibration a Data Interpretation,

Calculation processing Presentation
 VALIDATION
VALIDATION PARAMETERS

► PRECISION
ACCURACY
► TRUENESS

►RANGE & LINEARITY

► LIMIT OF DETECTION & LIMIT OF QUANTIFICATION

► SPECIFITY & SELECTIVITY

► RUGGEDNESS
 TRUENESS AND PRECISION = ACCURACY
ERRORS OF MEASUREMENT

What is included in result value (X)?

X = μ + ε + Σδ
RELATED TO RELATED TO
TRUENESS PRECISION

X mean result of repeated measurement

µ true value – real content of analyte in sample
ε systematic error – always the same value and sign (+/-)
Σδ sum of random errors – variable value and sign (+/-)

Error should not be confused with a mistake !!!

 TRUENESS AND PRECISION = ACCURACY
ERRORS OF MEASUREMENT

What is included in result value (X)?

X = μ + ε + Σδ
True value is an idealized concept and „true value“ cannot be
known exactly!
Hence the REFERENCE VALUE represents a true value in routine
practice
Reference value usually provided with reference to:
► Certified reference material
► Reference measurement procedure
► Known amount of analyte added into the sample (spike)
 TRUENESS
TRUENESS is closeness of agreement between the mean of
an N number of replicate measured values and a
REFERENCE (TRUE) value.

Trueness is inversely related to systematic error:

The lower the systematic error, the higher the trueness…

Estimate of a systematic error: bias

In analytical chemistry: RECOVERY

Correction of the result can be carried out using the recovery

=> compensation for an estimated systematic effect
Traceability to reference material: trueness
Assessment of trueness using CRM

SAMPLE
RESULT
CRM
(matrix) Compare with
certified value

t- test :
texp < tcrit (P, n-1)
Certified Reference Material (CRM, SRM)

http://www.erm-crm.org/html/homepage.htm
 TRUENESS
HOW TO ESTIMATE TRUENESS (RECOVERY)

(Certified) Reference materials are available…

Sample 1 Value 1
ANALYTICAL PROCESS
Sample 2 Value 2
Sample 3 Value 3
CRM Sample 4 Value 4
Y±y Sample 5 Value 5

n≥6

MEAN VALUE
COMPARISON X±x
STATISTICAL TESTING

(C)RMs at concentration levels close to expected analyte levels should be used

 TRUENESS
HOW TO ESTIMATE TRUENESS (RECOVERY)

(Certified) Reference materials are not available…THE MOST COMMON CASE

A) BLANK SAMPLE IS AVAILABLE

Sample 1 Value 1
ANALYTICAL PROCESS
Sample 2 Value 2

INCUBATION
Sample 3 Value 3
SAMPLE Sample 4 Value 4
Sample 5 Value 5
BLANK
n≥6

Addition of known
amount of an analyte MEAN VALUE
to each replicate X±x
(SPIKE)

Blank samples should be checked, multiple spiking levels should be used

After spike addition sample should be incubated (analyte incorporation into the matrix)
Certified Reference Material (CRM, SRM)

http://www.erm-crm.org/html/homepage.htm
 TRUENESS
HOW TO ESTIMATE TRUENESS (RECOVERY)

(Certified) Reference materials are not available…

A) BLANK SAMPLE IS AVAILABLE

MEAN VALUE
RECOVERY (%) = . 100
ADDED AMOUNT

Recovery values can be both below or above 100%

Recoveries between 80 and 120 are usually acceptable.
 TRUENESS
HOW TO ESTIMATE TRUENESS (RECOVERY)
(Certified) Reference materials are not available…
B) BLANK SAMPLE IS NOT AVAILABLE
Sample 1 Value 1
Sample 2 ANALYTICAL PROCESS Value 2
Sample 3 Value 3
Sample 4 Value 4
SAMPLE Sample 5 Value 5

n≥6 MEAN VALUE

Sample 1 Value 1
Sample 2 Value 2
INCUBATION

Sample 3 Value 3

SAMPLE Sample 4
Sample 5
Value 4
Value 5

n≥6 MEAN VALUE

SPIKE
TRUENESS
Codex Alimentarius: numerical values for the criteria
 PRECISION
PRECISION is related to RANDOM ERRORS
► Component of measurement error that - in replicate
measurements - varies in an unpredictable manner
► Random error = error - systematic error
► Correction of random error can not be done

Some sources of random errors:

► Methods (procedure, calibration,...)
► Laboratory (facility, environment)
► Equipment and materials / reagents / calibrants
► Personnel
► Time
 PRECISION
PRECISION represents random errors of a set of replicate
measurements

PRECISION is calculated as a (relative) standard deviation

of replicate measurements σx

Less precision is reflected by a larger standard deviation

Precision depends critically on the conditions !

REPEATABILITY and REPRODUCIBILITY conditions are
particular sets of extreme conditions.
...nothing to do with true or reference value !
 PRECISION
REPEATABILITY AND REPRODUCIBILITY

Repeatability: a set of conditions that includes

► the same measurement, procedure, operators, same
measuring system, operating conditions and location,
and replicate measurements on the same or similar
objects over a short period of time
Reproducibility: a set of conditions that includes
► different locations, operators, measuring systems, or
even methods on the same or similar objects.
Intermediate precision (intra-laboratory reproducibility):
► the same laboratory, method, procedure but within an
extended period of time - may include new calibrations,
calibrants, operators, measuring systems, etc.
 PRECISION
INCREASING NUMBER OF CONSIDERED RANDOM ERROR SOURCES

REPEATABILITY INTRA-LABORATORY REPRODUCIBILITY

REPEATABILITY

SAMPLE: SAME SAMPLE: SAME SAMPLE: SAME

OPERATOR: SAME OPERATOR: DIFFERENT OPERATOR: DIFFERENT
INSTRUMENT: SAME INSTRUMENT: SAME / DIFF. INSTRUMENT: DIFFERENT
TIME PERIOD: SHORT TIME PERIOD: LONG TIME PERIOD: LONG
CALIBRATION: SAME CALIBRATION: DIFFERENT CALIBRATION: DIFFERENT
LAB: SAME LAB: SAME LAB: DIFFERENT

Precision value is related to a certain analyte and concentration level

 PRECISION
VARIABILITY OF RESULTS: 2 DIFFERENT OPERATORS

Standard Integration 1 Integration 2

FLD1 A, Ex=248, Em=374, TT (C:\DATAHP~1\FLD154-4\DATA02\MJKOLONY FLD1 A, Ex=248, Em=374, TT (C:\DATAHP~1\FLD154-4\DATA02\MT021016\048-3301.D) FLD1 A, Ex=248, Em=374, TT (C:\DATAHP~1\FLD154-4\DATA02\MT021016\048-3301.D)
24.530 - 1-MePyr

LU LU

Ar - BaA
23.152 - 1-MePyr
LU

23.152 - 1-MePyr
03

11

88
7.

9.

3.
99

68

10
ea 6
A22.872
60

ea 2
A22.872
:2

:9
Ar 7.7

:2
Ar 5.3
ea
7

4
23.591
25

21
25.004 - B[a]A

200 200

a:

a:
re

re
50

150 150
40

30 100 100

20
50 50

10

0 0

23.75 24 24.25 24.5 24.75 25 25.25 25.5 25.75 m 21.5 22 22.5 23 23.5 24 24.5 m 21.5 22 22.5 23 23.5 24 24.5 m

Difference between results

– 42 % (concentration level ppb)
 PRECISION
VARIABILITY OF RESULTS: 2 DIFFERENT MEASUREMENT SYSTEMS
5.47
100
6.22

6.35
6.07
6.84 Same sample, conditions,
%
7.05
operator,...
7.65
7.54
8.10
9.67

0
5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80

Two different
chromatographs of the
same type

8.00 8.50 10.00 10.50 11.50

9.00 9.50 11.00
 PRECISION
INCREASING NUMBER OF CONSIDERED RANDOM ERROR SOURCES

REPEATABILITY INTRA-LABORATORY REPRODUCIBILITY

REPEATABILITY

► Repeated analyses of a sample containing analyte(s) at:

► level close to expected concentration in analyzed matrix
► level close to regulatory limit
► low level close to limit of quantification of the method
► Appropriate number of repeats: 8 – 15 (at least 5)
► Calculated as standard deviation or relative standard deviation (RSD)
 PRECISION
INCREASING NUMBER OF CONSIDERED RANDOM ERROR SOURCES

REPEATABILITY INTRA-LABORATORY REPRODUCIBILITY

REPEATABILITY

STANDARD DEVIATION MEAN

1 1
(xi – x )2 x= xi
σ = n
n

RELATIVE STANDARD DEVIATION CAN BE EASILY CALCULATED

σ IN MS EXCEL…
RSD (%) = 100
x
 PRECISION
INCREASING NUMBER OF CONSIDERED RANDOM ERROR SOURCES

REPEATABILITY INTRA-LABORATORY REPRODUCIBILITY

REPEATABILITY

RESULT VALUES

σ
RSD (%) = 100
x
 PRECISION
INCREASING NUMBER OF CONSIDERED RANDOM ERROR SOURCES

REPEATABILITY INTRA-LABORATORY REPRODUCIBILITY

REPEATABILITY

► Can be estimated within an inter-laboratory study…

Two components:
σ2r – intra-laboratory variance (typical)
σ2L – between laboratories variance

σ2R = σ2r + σ2L

 PRECISION
INCREASING NUMBER OF CONSIDERED RANDOM ERROR SOURCES

REPEATABILITY INTRA-LABORATORY REPRODUCIBILITY

REPEATABILITY

► Can be estimated within an inter-laboratory study…

…however…
► inter-laboratory study is time-demanding and costly
► it is problematic to find sufficient number of competent
laboratories
► in multi residue analysis it is almost impossible to perform this
kind of study for all analytes / matrices / concentration levels
 PRECISION
REPRODUCIBILITY - HORWITZ

Reproducibility can be alternatively estimated from an

empirical model developed based on numerous inter-
laboratory studies (more than 150)

Horwitz (Thompson) empirical model of

precision
…the RSD can be expressed as a function of
the concentration …

„William Horwitz“ …
 PRECISION
REPRODUCIBILITY - HORWITZ

Relative standard deviation – variation coefficient:

 lower concentration of analyte → increasing RSD
 nature of analyte, matrix, analytical method etc.:
less important – even can be ignored !

RSD = 2(1- 0.5*logX)

X is an analyte concentration expressed

as a mass ratio
 PRECISION
REPRODUCIBILITY - HORWITZ
50
RELATIVE STANDARD DEVIATION (%)

HORWITZ CURVE
H o r w it z o v a k ř iv k a
40

30
CV (%)

20

10

0
1 10 0 10 0 0 0 1 000 000 10 0 0 0 0 0 0 0

ANALYTE CONCENTRATION (ppb)

ppb
 PRECISION
REPRODUCIBILITY - HORWITZ
Official requirements on precision – trace analysis:

Concentration CVr (%) RSDR (%)

(ppb) Codex Alimentarius Horwitz

<1 35 > 45

1 - 10 30 32 -45

10 - 100 20 22 – 32

> 100 15 < 22

 TRUENESS AND PRECISION = ACCURACY
RELATIONSHIPS BETWEEN TYPE OF ERROR, RELATED
CHARACTERISTICS AND THEIR QUANTITATIVE EXPRESSION
LINEARITY AND CALIBRATION
LINEARITY can be tested by linear regression of the
responses on the concentrations in an appropriate
calibration set

3500000

3000000
2
R = 0,9977
2500000
Peak area

2000000

1500000

1000000

500000

0
0 1000 2000 3000 4000 5000
-1
DON concentration (µg kg )
 LINEARITY, CALIBRATION AND RANGE
GENERAL RECOMENDATIONS FOR LINEAR CALIBRATION

There should be six or more calibration points (standards)

Even spacing over the concentration range of interest
The calibration range should encompass 0–150% or 50–150% of
the concentration likely to be encountered in samples
Calibration standards should be run at least in duplicate in
random order

VALIDATION RANGE is the interval of analyte concentration within

which the method can be regarded as validated
Typically narrower than linear range
In practice, most methods will be validated at only one or two
levels of concentration. The validated range may be taken as a
reasonable extrapolation from these points at concentration scale
 LINEARITY, CALIBRATION AND RANGE

VALIDATED
1·0 CONCENTRATION
LEVEL
VALIDATED
0·8 RANGE

0·6 VALIDATED
Signal

CONCENTRATION
LEVEL
0·4

0·2
Linear range
0
0 0·2 0·4 0·6 0·8 1·0 1·2
Concentration
LIMIT OF DETECTION / QUANTIFICATION
 LIMIT OF DETECTION / QUANTIFICATION
Limit of Detection (LOD): the smallest concentration of analyte
in the test sample which can be reliably distinguished from zero.
►LOD is concentration of analyte which induce signal (S) that
is 3 times higher than the background noise level (N). S/N=3

Limit of Quantification (LOQ): the smallest concentration of

analyte in the test sample which can be reliably quantified.
►LOQ is concentration of analyte which induce signal (S)
that is 10 times higher than the background noise level (N).
S/N=10, LOQ usually corresponds to lowest calibration point.

S/N can be usually calculated in processing software

LIMIT OF DETECTION / QUANTIFICATION

S/N > 6
S/N > 2
signal

noise
 DETECTABILITY – how to lower LOD ?
Increase of S/N ratio in GC/MS-NCI analysis of
nitronaphtalenes (parsley sample) due to increasing
electromultiplyier voltage in detector (1-nitroNAP = 0.1
ppb, 2-nitroNap = 0.08 ppb)

A - EMV = 1300 V B - EMV = 1800 V

84
1-nNap 280
A 1-nNap
82 260 B
2-nNap 240 2-nNap
80
220
200
78
180
76 160
140
74
120

72 100
80
70 60

Time--> 11.80 12.00 12.20 12.40 12.60 Time--> 11.80 12.00 12.20 12.40
Vladimir.Kocourek@vscht.
 LIMIT OF DETECTION / QUANTIFICATION
HOW TO ESTIMATE LOD & LOQ

SPIKING OF BLANK MATRIX WITH DECREASING AMOUNT OF ANALYTES

SAMPLE PREPARATION & MEASUREMENTS
CALCULATION OF S/N VALUES

STANDARD SOLUTION WITH DECREASING AMOUNT OF ANALYTES

MEASUREMENTS
CALCULATION OF S/N VALUES

LOD → LOQ
Signal at LOD vs. background noise

1,64*sblank 1,64*sblank

=5% =5%

s b la n k

y blank YLOD

Response to blank Analyte signal

 Codex Alimentarius: numerical values for the criteria
Applicability:
The method has to be applicable for the specified provision, specified
commodity and the specified level: maximum and/or minimum - ML.
The minimum applicable range of the method depends on the
specified level (ML) to be assessed, and can either be expressed in
terms of the reproducibility standard deviation (sR) or in terms of LOD
and LOQ. CL 2008/7-MAS, March 2008

Minimum applicable range:

ML ≥ 0.1 mg/kg → [ML - 3 sR , ML + 3 sR ]
ML < 0.1 mg/kg → [ML - 2 sR , ML + 2 sR ]

Limit of Detection (LOD):

ML ≥ 0.1 mg/kg → LOD ≤ ML · 1/10
ML < 0.1 mg/kg → LOD ≤ ML · 1/5

Limit of Quantification (LOQ):

ML ≥ 0.1 mg/kg → LOQ ≤ ML · 1/5
ML < 0.1 mg/kg → LOQ ≤ ML · 2/5
 SELECTIVITY AND SPECIFITY

Selectivity: the degree to which a method can quantify the

analyte accurately in the presence of interferents.
► Selective method – the results are influenced by the sample
matrix (interferents, cross-reactivity, matrix effects,…)
► Specific methods – the results are not influenced by the
presence of sample matrix

The presence and influence of any sample matrix

interference on method results should be tested and
described
 SELECTIVITY AND SPECIFITY
MATRIX EFFECTS:
DECREASE (INCERASE) AND
PEAK SHAPE DETERIORATION

High matrix content Low matrix content

 RUGGEDNESS
Ruggedness of an analytical method is the resistance to
change in results when minor deviations are made from the
experimental conditions described in the SOP.
► The aspects of the method that are likely to affect results
should be identified and described in SOP

Examples of factors relevant to ruggedness:

► pH of a solution
► stability of the instrumental system
► extraction time
► concentration of (derivatization) reagents
► temperature/time of (derivatization) reaction
► time allowed for completation of whole analytical process
VALIDATION
Development and
PROCEDURE optimization of the method

Spiked samples

Training
Reference material

Repeated Independent method

measurement

Inter-laboratory study

REPEATABILITY TRUENESS

REPRODUCIBILITY

LOD/LOQ Selectivity
Performance
characteristics
Range Ruggedness

Fit-for-purpose
USEFUL DOCUMENTS
 USEFUL DOCUMENTS
Criteria for validation of methods used in official control of
contaminants and residues in food and feed:
All documents and methods issued to personnel in the laboratory shall be
reviewed and approved for use by authorized personnel prior to issue.

Documents are periodically reviewed and, where necessary, revised to

ensure continuing suitability and compliance with applicable
requirements;
Invalid or obsolete documents are promptly removed from all points of
issue or use, or otherwise assured against unintended use;
Procedures shall be established to describe how changes in documents
maintained in computerized systems are made and controlled.
All records and data shall be stored and retained in such a way that they
are readily retrievable in facilities that provide a suitable environment to
prevent damage or deterioration and to prevent loss.

The laboratory shall have procedures to protect and back-up records

stored electronically and to prevent unauthorized access to or
amendment of these records.
The laboratory shall retain records of primary data, calculations, calibration
records, staff records and a copy of each test report issued.
The records for each test shall contain information to facilitate identification
of factors affecting the uncertainty and to enable the test to be repeated
under conditions as close as possible to the original.
The records shall include the identity of personnel responsible for the
performance of each test and checking of results.

When mistakes occur in records, each mistake shall be crossed out, not
erased, made illegible or deleted, and the correct value entered alongside.
All such alterations to records shall be signed by the person making the
correction.
In the case of records stored electronically, equivalent measures shall be taken to
avoid loss or change of original data.
The laboratory shall use test methods, including methods for sampling,
which meet the needs of the customer and which are appropriate for the
tests it undertakes.
Methods published in international, regional or national standards shall
preferably be used.

Appropriate methods have been also published by reputable technical

organizations, or in relevant scientific texts or journals, or specified by the
manufacturer of the equipment.
Laboratory-developed methods or methods adopted by the laboratory may
also be used if they are appropriate for the intended use and if they are
validated.
The laboratory shall validate nonstandard methods, laboratory developed
methods, standard methods used outside their intended scope, and
modifications of standard methods to confirm that the methods are fit for the
intended use.
Testing laboratories shall apply procedures for estimating uncertainty of
measurement based on the method validation data.
USEFUL DOCUMENTS
 Pesticides standards
“Pure” standards should be of known purity and each must be uniquely identified and
the date of receipt recorded. They should be stored at low temperature, preferably in a
freezer, with light and moisture excluded, i.e. under conditions that minimise
degradation. The identity of freshly acquired “pure” standards should be checked if
the analytes are new to the laboratory.
When preparing stock standards of “pure” standards of analytes and internal
standards, the identity and mass of the “pure” standard and the identity and amount of
the solvent must be recorded. The
solvent(s) must be appropriate to the analyte (solubility, no reaction) and method of
analysis. Moisture must be excluded during equilibration of the “pure” standard to
room temperature before use and concentrations must be corrected for the purity of the
“pure” standard.
Not less than 10 mg of the “pure” standard should be weighed using a 5 decimal place
balance. The ambient temperature should be that at which the glassware is calibrated,
otherwise preparation of the standard should be based on solvent-mass measurement.

Existing stock and working solutions may be tested against newly prepared solutions
by comparing the detector responses obtained from appropriate dilutions of individual
standards or mixtures of standards.
The means from at least three replicate measurements for each of two solutions (old
and new) should not normally differ by more than ±10 %. The mean from the new
solution is taken to be 100%. Differences in apparent concentration between old and
new standards must be investigated.
 Calibration
Responses used to quantify residues must be within the dynamic range of the detector.
Extracts containing high-level analytes may be diluted to bring them within the calibrated
range.
Validation of analytical methods should include determination of recovery at the
proposed reporting limit.
Calibration by interpolation between two levels is acceptable providing the difference
between the 2 levels is not greater than a factor of 4, and where the mean response
factors, derived from replicate determinations at each level, indicate acceptable
linearity of response with the higher being not more than 110 % of the lower response
factor.
Single-level calibration may provide more accurate results than multi-level calibration
if the detector response is variable with time. When single-level calibration is
employed, the sample response should be within ± 20 % of the calibration standard
response if the MRL is exceeded.
The potential for matrix effects to occur should be assessed at method validation.
They are variable in occurrence and intensity but some techniques are particularly
prone to them. If the techniques used are not inherently free from such effects,
calibration should be matrix-matched routinely.
Where a calibration standard is a mixture of isomers, etc., of the analyte, detector
response generally may be assumed to be similar, on a molar basis, for each component.
 Contamination & interference
Samples must be separated from each other, and from other sources of
potential contamination, during transit to, and storage at, the laboratory.
Pest control in, or near, the laboratory must be restricted to pesticides that
will not be sought as residues.

Volumetric equipment, such as flasks, pipettes and syringes must be

cleaned scrupulously, especially for re-use. As far as practicable, separate
glassware, etc., should be allocated to standards and sample extracts, in
order to avoid cross-contamination. Avoid using excessively scratched or
etched glassware.

Equipment, containers, solvents (including water), reagents, etc., should

be checked as sources of possible interference. Rubber and plastic items
(e.g. seals, protective gloves, wash bottles) and lubricants are frequent
sources.
Vial seals should be PTFE-lined. Extracts should be kept out of contact
with seals by keeping vials upright. Vial seals may have to be replaced
quickly after piercing, if re-analysis of the extracts is necessary. Analysis
of reagent blanks should identify sources of interference in the equipment
or materials used.
 Validation parameters and criteria
Parameter What/How Criterion

Linearity Through calibration curve Residuals < ±20 %

Matrix effect Comparison of response from solvent
standards and matrix-matched standards
LOQ Definition: lowest level for which it has been ≤ MRL
demonstrated that criteria
for accuracy and precision have been met
Specificity Response in reagent blank and control < 30 % of LOQ
samples
Accuracy Determine average recovery for both spike 70-120 %
levels
Precision (RSDr) Determine repeatability RSDr, determine for ≤ 20 %
both spike levels
 Method validation
Within-laboratory method validation should be performed to provide
evidence that a method is fit for the purpose for which it is to be used.
Method validation is a requirement of accreditation bodies, and must be
supported and extended by method performance verification during
routine analysis (analytical quality control).

The method must be tested for sensitivity, mean recovery (as a measure of
trueness or bias), precision, and limit of quantification (LOQ).
This means that spiked recovery experiments to check the accuracy of the
method should be undertaken.
A minimum of 5 replicates is required at both the reporting limit, and at
least another higher level, perhaps an action level, for example the MRL.
The (method) LOQ is defined as the lowest validated spike level meeting
the method performance acceptability criteria:
mean recovery for each pesticide in the range 70-120 %, with a RSDr ≤ 20%.

Exceptionally, where recovery is low but consistent (demonstrating good

precision) and the basis for this is well established (e.g. due to pesticide
distribution in partition), a mean recovery below 70% may be acceptable.
 Acceptability of analytical performance
Acceptable limits for a single recovery result should normally be within the range of
the mean recovery +/- 2x RSD).
The laboratory must participate regularly in relevant proficiency tests.
When a low number of compounds are analysed with respect to the pesticides
present in the test sample, false positive(s) or negative(s) are reported or the
accuracy achieved in any of the tests is questionable or unacceptable, the
problem(s) should be investigated. Unacceptable performance, has to be rectified
before proceeding with further determinations of the analyte involved.

Negative results for represented analytes are supported only indirectly by the
recovery and LCL data for representative analytes and must be interpreted with
caution.

Positive results (residues at or above the RL) usually require additional

confirmation. Suspected MRL exceedances or unusual residues must be identified.
The use of a highly specific detection system, such as mass spectrometry, is
recommended.
Selective detectors employed with GC or LC such as ECD, FPD, NPD, DAD and
fluorescence, offer only limited specificity. Their use, even in combination with
different polarity columns, does not provide unambiguous identification.
 Identification - confirmation
Mass spectrometry in conjunction with chromatographic separation is a
very powerful combination for identification of an analyte. It
simultaneously provides:
i. retention time
ii. ion mass/charge ratio; and
iii. abundance data.
The retention time (or relative retention time) of the analyte in the sample
extract must match that of the calibration standard within a specified window
after taking into consideration the resolving power of the chromatographic
system.
The relative retention time of the analyte, should correspond to that of the
calibration solution with a tolerance of ± 0.5 % for GC and ± 2.5 % for LC.
Reference spectra for the analyte should be generated using the
instruments and techniques employed for analysis of the samples. If major
differences are evident between a published spectrum and that generated
within the laboratory, the latter must be shown to be valid. To avoid
distortion of ion ratios, the response of the analyte ions must not overload
the detector.
 Identification - confirmation
Different types and modes of mass spectrometric detectors provide different
degrees of selectivity, which relates to the confidence in identification:
 Identification - confirmation
Different types and modes of mass spectrometric detectors provide
different degrees of selectivity, which relates to the confidence in
identification:

For a higher degree of confidence in identification, further evidence may be

required. This can be achieved through additional mass spectrometric
information, for example evaluation of full scan spectra, additional accurate
mass (fragment) ions, additional product ions (in MS/MS), or accurate mass
product ions.
 Identification points
Commission decision 2002/657/EC:
Method validation – pesticides: conditions
Thank you for your
attention…

▐ [email protected] ▌