GOOD LABORATORY PRACTICE

IN CHEMISTRY

By Mohamed Salama
 CONTENTS

 Good Laboratory Practice (GLP)

 Difference between Good Laboratory Practice and

ISO/IEC 17025.
17025

 Codex Alimentarius.
 G dL
Good Laboratory
b t P
Practice
ti

What is GLP?
GLP is a quality system concerned with the
organizational process and the conditions under
which non-clinical health and environmental
safety studies are planned, performed,
monitored, recorded, archived and reported.

Rationale:
Promote the quality and validity of non-clinical
data on which hazard assessments are
b
based.
d
Processingg & non-Regulated
g
vs. Regulated areas
 Hi t
History off GLP

First evolved in the USA in 1970s by the Food and

Drug Administration (FDA) because of concerns
about the validity of preclinical safety data.

OECD
O C assembled an expert group who formulated
f
the first OECD principles of GLP in order to
avoid non-tariff
non tariff barriers to trade in chemicals,
chemicals to
promote mutual acceptance of non-clinical safety
test data,, and to eliminate unnecessary y
duplication of experiments.
 Wh t is
What i OECD

 The Organization for Economic Co-operation and

Development.
 It is an intergovernmental organization.
 30 industrialized countries meet to co-ordinate
and harmonize policies.
 Discuss issues of mutual concern.
 Work together to respond to international
problems.
 30 Industrialized
I d t i li d Countries
C t i

1 Australia 11 Hungary 21 Poland

2 Austria 12 Iceland 22 Portugal
3 Belgium 13 Ireland 23 Slovak Republic
4 Canada 14 Italy 24 Spain
5 Czech Republic 15 Japan 25 Sweden
6 Denmark 16 Korea 26 Switzerland
7 Finland 17 Luxembourg 27 Turkey
8 France 18 Mexico 28 UK
9 Germany 19 Netherlands 29 USA
10 Greece 20 New Zealand 30 Norway
 Wh t is
What i OECD

 OECD guidelines are NOT law.

 OECD standards are internationally accepted.

 Fi t developed
First d l d in
i 1978 using
i US FDA GLP
regulations as a basis.

 Revised edition adopted 1977.

 WHY GLP

 To promote the development of quality test data.

 To provide a managerial tool to ensure a sound

approach to the management,
management including conduct,
conduct
reporting and archiving of laboratory studies.
 Wh t GLP is
What i NOT

 GLP has nothing to do with efficacy.

 If a study does not involve animals, it is not GLP.

 If a lab
l b claims
l i t be
to b GLP,
GLP but
b t dose
d nott run
animal studies, they are misguided.

 GLP does not apply to analytical development.

 GLP dose not apply to animal field studies.

 A li bilit off GLP
Applicability

Non-Clinical safety testing of test items

contained in:

 Ph
Pharmaceutical
ti l products.
d t

 Pesticide products.
p

 Veterinary drugs.

 Food additives.

 Feed additives.
additives
 C
Conclusion
l i

 It is important to clearly differentiate between

the formal regulatory use of the term “Good Good
Laboratory Practice” as opposed to the general
application of “Good Practices” in scientific
investigations.
 Since the term “Good Laboratory Practice” is not
a trade mark protected term, any laboratory
which may consider itself to be following good
practices in its daily work might be tempted to
describe its adherence to these (possibly even
self-defined)) quality
q y standards by
y this
terminology
GOOD LABORATORY PRACTICE
 I t d ti
Introduction

The backbone of GLPs is documentation of

protocols reports,
protocols, reports data collection techniques and
archival capabilities.
GLP is needed for:
 Non clinical safety studies of development of
drugs.
g
 Agricultural pesticide development.
 Development of toxic chemicals.
 Food control (food additives)
 Test of substance with regard to explosive
hazards
 I t d ti
Introduction

GLP is not needed for:

 Basic research.
research
 Studies to develop new analytical methods.
 Chemical tests used to drive the specifications of
a marketed food product.
Good Laboratory
y Practice ((GLP)) deals with:
1. Organization.
2. Process.
3. Conditions under which laboratory studies are
planned, performed, monitored, recorded and
reported.
reported
 I t d ti
Introduction

 To comply with regulations can be quite

expensive (can increase the cost of a laboratory
up to 30%) and sometimes it is just impossible to
comply 100% even when willing, especially when
new regulations are released.

 Hence training plans should include basic GLP

Hence,
knowledge for everybody working in a GLP
environment.
 M i P
Main Points
i t off GLP

 Resources: organization, personnel, facilities,

equipment.
equipment
 Rules: protocols, standard operating procedures,
conceptt off the
th study
t d director
di t as the
th pivotal
i t l point
i t
of study control.
 Characterization: test items, test systems.
 Documentation: raw data,, final report,
p , archives.
 Quality assurance: independence from study
conduct.
conduct
 GLP Principles
P i i l

 A laboratory which intends to conduct studies

that are GLP compliant will have to be organized
so that the following conditions apply:
 A study director (in case of toxicological studies).
studies)

 A quality assurance unit (QAU).

 Qualified personnel.

 Standard operating
p gpprocedures ((SOPs).
)

 Control and test articles.

 E i
Equipment.
 GLP Principles
P i i l

 A Study Director:
responsible for the technical conduct of the study,
as well as for interpretation, analysis,
d
documentation
t ti and d reporting
ti off the
th results.
lt

 A Quality Assurance Unit:

audit the laboratory studies and the
accompanying data.
data It may be a separate
department or an individual person, either full or
part time. (any person except the study director).
 GLP Principles
P i i l

 Qualified Personnel:
must be qualified through education, training
and/or experience to follow directions and
perform test procedures properly.
 Standard Operating Procedures (SOPs):
all laboratory activities must be performed in
accordance
d with
i h correctly
l written
i andd properly
l
filed, management approved SOPs.
These must be readily available to the personnel
concerned. They should cover policies,
administration, technical operation, equipment
operating
i andd analytical
l i l methods.
h d
 GLP Principles
P i i l

 Control and Test Articles:

must be identified and characterized by Strength,
Purity and Stability.
Reagents and solutions must be labeled with
information on origin, identity, concentration,
storage conditions,
conditions and expiration date.
date
 Equipment:
instruments must be designed to meet analytical
requirements and regularly maintained and
calibrated.
 GLP B
Benefits
fit

 From the point of view of international trade:

The ultimate goal in fair practice depends on:

Reliability
b y of analytical
y results

Thiss in turn,, depends

epe s oon::
 Availability of reliable analytical methods.

 Experience of the analyst.

 Maintenance of ‘good practice’.

 GLP B
Benefits
fit

Why Reliable Analytical Results?

Reliable analytical
y results are essential for:

 Protecting the health of consumers.

 Facilitating international trade.

DIFFERENCE BETWEEN GOOD
LABORATORY PRACTICE &
ISO/IEC 17025
 Difference between
GLP & ISO/IEC 17025
 ISO Members.

 OECD Members.

 Th same standard
The t d d for
f all
ll ISO.
ISO

 Different regulations
g in different countries.

 Designed for repetitive studies.

 Designed for single studies.

 Difference between
GLP & ISO/IEC 17025
 Description of quality system in quality
manual.
 Description of quality system in SOPs.
 General statements for responsibilities of
personnel.
 Very specific responsibilities of personnel.
 Non specific
p requirements
q for storage
g of
records and reports.
 Specific
p requirements
q for storage,
g , retention
and archiving.
 Difference between
GLP & ISO/IEC 17025
 No study plans required (standard methods
should be used).

 Study plan required for each study.

study

 Written operating procedures without

specific format.

 SOP with
SOPs ith detailed
d t il d requirements
i t for
f format
f t
and content.
 Difference between
GLP & ISO/IEC 17025
 Analysis methods must be verified through
inter-laboratory test (PT).

 Validation through inter-laboratory

inter laboratory test not
required.

 Documented complaints procedures.

 I case off problems

In bl only
l course off law.
l
 Difference between
GLP & ISO/IEC 17025
 Storage of test samples and data until client
accepts results.

 Storage of test samples according to local

regulatory requirements.
 GUIDELINES ON GOOD
LABORATORY PRACTICE IN
RESIDUE ANALYSIS

CODEX ALIMENTARIUS
 C d Ali
Codex Alimentarius
t i

 Latin for “Food Code” or “Food Book”.

 Developed and maintained by the codex

alimentarius commission,
commission a body that was
established in 1963 by the Food and Agriculture
Organization of United Nations (FAO) and the
World Health Organization (WHO).
(WHO)
 C d Ali
Codex Alimentarius
t i

 Recognized by the world trade organization as an

international reference point for the resolution of
disputes concerning food safety and consumer
protection.

 Is a collection of standards, codes of practice,

guidelines and other recommendations.
recommendations Some of
these text are very general, and some are very
specific.
 C d Ali
Codex Alimentarius
t i

 Some deal with detailed requirements related to

a food or group of foods, others deal with the
operation and management of production
processes or the operation of government
regulatory systems for food safety and consumer
p
protection.
C d Ali
Codex Alimentarius
t i C Commission
i i

 It is intergovernmental standards-setting body

established by FAO and WHO in 1961/63.
 11th FAO Conference Resolution no.
no 12/61 (codex
alimentarius).

 WHA 16.42 Joint AO/WHO programme on food

standards (codex alimentarius).

174 member countries + 1 member organization (EC).

C d Ali
Codex Alimentarius
t i C Commission
i i

 Its Mandate

 Dual objective:
 Protectingg the health of consumers.
 Facilitating fair practices in food trade.
 To coordinate all food standards work.

 Non-mandatory in nature, codex standards and

related texts have since 1995 become
international benchmarks for harmonization
under the SPS and TBT agreements of WTO.
 R l off C
Role Codex
d StStandards
d d

 For food safety, codex standards are the

international benchmark.
benchmark
 National regulations consistent with codex
standards meet the requirements of the SPS
Agreement (i.e. do not have to be justified).
 Are not obligatory,
obligatory but are the reference in the
event of a trade dispute.
 Where standards are more stringent than codex,
codex
there must be a scientific justification (based on
assessment
assess e t oof tthe
e risk).
s ).
 C d Ali
Codex Alimentarius
t i

 Its scientific basis. Liaison &

S
Separation
i
 Codex – Risk management.

 FAO/WHO Expert
p Bodies – Risk assessment
 JECFA – food additives, veterinary drug residues,
contaminants in food.
 JMPR – pesticide residues in food.
 JEMRA – microbiological hazards in food.
 Ad hoc Expert Consultations.
 CASE STUDY

GUIDELINES ON
GOOD LABORATORY PRACTICE IN
IN RESIDUE ANALYSIS

CAC/GL 40-1993, REV.1-2003

 GLP Principles
P i i l

 A laboratory which intends to conduct studies

that are GLP compliant will have to be organized
so that the following conditions apply:
 A study director (in case of toxicological studies).
studies)

 A quality assurance unit (QAU).

 Qualified personnel.

 Standard operating
p gpprocedures ((SOPs).
)

 Control and test articles.

 E i
Equipment.
 M i Principles
Main P i i l

Good analytical practice may be considered in three

inter related parts:

1. Analyst.

2. Basic resources.

3. Analysis.
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Why GLP in residue analysis.

 Because the analyte concentrations are in the

range µg/kg to mg/kg.
mg/kg

 Because the analyses can be challenging.

Attention to details is essential

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These details are summarized in:

1 The Analyst:
1.
The analyst who undergoes residue analysis:
 Should have appropriate professional
qualification.
 Should be experienced
p in the correct use of
apparatus and lab skills.
 Should be competent in residue analysis.
 Should
Sh ld beb fully
f ll trained.
t i d
 Should have understanding of the principles
of residue analysis.
analysis
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1. The Analyst:
Continued…..
Continued
 Should have understanding of the
requirements of Analytical quality assurance
(AQA) Systems.
 Should understand the purpose of each stage
in the method and notice and deviation.
 Should be trained in the evaluation and
interpretation of data.
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1. The Analyst:
Continued…..
 The staff should spend some of their training
period in a well established (expert) laboratory
where
p
experienced advice and training
g is available.
 A record of training and experience must be kept
f all laboratory staff.
for ff
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2. Basic Resources:
A. The laboratory
 The laboratory and its facilities must be
designed to allow tasks to well-defined areas
where maximum safety and minimum chance
of contamination of samples prevail.
prevail
 Separate rooms (well ventilated) should be
designated for sample receipt and storage,
storage for
sample preparation, for extraction and clean-
up
p and for instrumentation used in the
determinative step.
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A. The laboratory
 All materials used within the lab should be
resistant to chemicals.
 The area used for extraction and clean-up
must meet solvent laboratory specifications.
 All fume extraction facilities must be of high
quality.
 Sample
p receipt,
p , storage
g and ppreparation
p
should be handled in areas away from areas of
residue analysis.
 Ensure sample integrity.
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A. The laboratory
 Laboratory safety must be considered in terms
of what is essential and what is preferable
((realistic conditions).
)
 No smoking, eating, drinking or application of
cosmetics should be permitted in the working
area.

 Small volume of solvents should be held in the

working area and the bulk of the solvents
stored separately away from the main working
area.
area
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A. The laboratory
 Minimize the use of highly toxic solvents and
reagents should whenever possible.
 All waste solvent
l should
h ld be
b stored
d safely
f l and
d
disposed of both safely and in an
environmentally friendly manner taking into
account specific national regulations where
available.
 All equipment such as lights, and refrigerators
should be “spark
spark free
free” or “explosion
explosion proof
proof”
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A. The laboratory
 A supply l off safety
f tools
l should
h ld be
b available
il bl
such as safety glasses, gloves and other
protective clothing,
clothing emergency washing
facilities and a spillage treatment kit.
 Appropriate and adequate fire fighting
equipment must be available.
 A great care should be taken in the handling of
standard reference compounds due to their
toxicc p
to properties.
ope t es.
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B. Equipment and Supplies

 Adequate supplies of electricity and water.
 Adequate supplies of reagents, solvents, gas,
glassware, chromatographic materials, etc..,
of suitable quality.
 Chromatographic
Ch t hi equipment,
i t b l
balances,
spectrophotometers etc.., must be serviced
and calibrated regularly.
regularly
 Record of all servicing/repairs must be
maintained for every item of equipment.
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B. Equipment and Supplies

 Regular calibration is essential for equipment
performing measurements. This factor
significantly
i ifi tl contribute
t ib t to
t the
th uncertainty
t i t off
measurement.
 Balances and automated pipettes,
pipettes dispensers
and similar equipment must be calibrated
regularly.
eg a y.
 The operating temperatures of refrigerators
and freezers should be continually y monitored
or be checked at specified intervals.
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B. Equipment and Supplies

 All records should be kept up-to-date and
retained.
 Equipment used must be fit for purpose.
 All reference standards should be of known
andd acceptably
t bl high
hi h purity.
it
 Analytical standards should be available for
all parent
a e t compounds,
co o d the lab monitoring
o ito i g as
a
well as those metabolites that are included in
MRLs.
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B. Equipment and Supplies

 All analytical standards, stock solutions and

reagents
g should be p
properly
p y labeled.
 Preparation date, analyst initials, solvent used,
storage
g conditions and expiry
p y date.

 Compounds influenced be degradative

processes (light,
(light heat,..)
heat ) must be clearly
labeled and stored under appropriate
conditions.
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B. Equipment and Supplies

 Reference standards must be kept under

conditions that will minimize the rate of
degradation.
 Low temperature.

 Exclusion of moisture and light.

 Care should be taken that standard solutions

are not concentrated by solvent evaporation.
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3. Analysis
 The methods applied for the determination of
pesticide residue should satisfy the following
criteria:
it i
Conc. Repeatability Reproducibility Trueness
CVA%(3) CVL%(4) CVA%(3) CVL%(4) % Rec.
Rec (2)
≤ 1 µg/kg 35 36 53 54 50 – 120
> 1 µg/kg ≤ 0.01 mg/kg 30 32 45 46 60 – 120
> 0.01 mg/kg ≤ 0.1 mg/kg 20 22 32 34 70 – 120
> 0.1 mg/kg ≤ 1 mg/kg 15 18 23 25 70 – 110
> 1 mg/kg 10 14 16 19 70 – 110
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3. Analysis

1) The acceptability of the data produced

depends on the purpose of the analysis.
analysis
 When checking for MRL compliance, these
quantitative performance criteria should be fulfilled
as far as possible.

 When data is below the MRL, it could be accepted

with the higher
g uncertainty.
y
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3. Analysis

2) These recovery ranges are appropriate for

multi residue methods.
multi-residue methods
 For single analyte methods or veterinary drug
residues, more restricted criteria are required.

3)) CVA: Coefficient of variation for analysis

y
excluding sample processing.
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3. Analysis

4) CVL: Overall coefficient of variation of

laboratory results,
results including up to 10%
variability of residues between analytical
portions (CVSp).

h :
where

CVL2 = CVSp2 + CVA2

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3. Analysis
A. Avoidance of contamination
 Contamination
• Very specific to residue analysis due to its
effect on interference.
• May lead to false positive or false negative
results if it occurs in the determination
stage.
t
• May lead to loss of sensitivity that may
prevent the residue from being detected.
detected
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A. Avoidance of contamination

 For these reasons

• All glassware,
l reagents,
t organic
i solvents
l t and
d
water should be checked for possible
interfering contaminants before use, by
analysis of a reagent blank.
blank
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A. Avoidance of contamination

 Polishes, barrier creams, soaps containing

germicides
germicides, insect sprays
sprays, perfumes and
cosmetics can give rise to interference
problems and are especially significant when
an electron capture detector is being used.
used
 Their use is banned by the staff while in the
l b
laboratory.
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A. Avoidance of contamination
 Other sources which may give rise to
contamination and interferences:
 Lubricants.
 Natural & synthetic rubbers.
 Oil from compressed air lines.
 Manufacturing impurities in filter paper,
cotton wool,…..
 Chemical reagents & general lab solvents.
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A. Avoidance of contamination
 Contamination of glassware, syringes and gas
chromatographic columns can arise from
contact with previous samples or extracts.
Thus…
 All glassware should be cleaned with detergent
solution, rinsed thoroughly with distilled
water and then rinsed with the solvent to be
used.
d
 Glassware to be used for trace analysis must
be kept separate and must not be used for any
other purpose.
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A. Avoidance of contamination
 Cross-contamination may occur between
reference standard materials and sample
extracts.
t t

Thus…
 Pesticide reference standards should always be
stored at a suitable temperature in a room
separate from the main residue laboratory
away from sample storage & extraction areas.
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A. Avoidance of contamination
 The nature and importance of contamination
can vary according to the type of
d t
determination
i ti technique
t h i used
d and
d the
th level
l l off
pesticide residue to be determined.
 Contamination problems with methods based
on gas chromatography or high performance
liquid.
q .
 Chromatography may be less significant if a
spectrophotometric
p p determination is used, and
vice versa.
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B. Reception & Storage of Samples

 Every received sample should be
accompanied by complete information about:
 Source of the sample.
 Required analysis.
 Potential hazards associated with its handling.
handling
 On receipt, a sample must immediately be
assigned a unique identification code which
should accompany it through all stages of the
analysis to the reporting of the results.
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B. Reception & Storage of Samples

 Samples should have disposal review system
and records should be kept.
 Carry out sample processing and sub-sampling
using procedures which provide representative
analytical portions without affecting the
residues concentration levels.
 Fresh samples should be stored at 1-5oC,
C away
from direct sunlight, while frozen samples
should be kept
p frozen and stored at -16oC.
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B. Reception & Storage of Samples

 The effect of storage should be checked by
analyzing fortified samples stored under the
same conditions
diti f a similar
for i il period.
i d
 When samples are to be frozen it is
recommended that analytical test portions be
taken prior to freezing in order to minimize
thee poss
possible
e eeffect
ec oof wa
water
e sepa
separation
a o as ice
ce
crystals during storage.
 Care must be taken to avoid containers leak.
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C. Standard Operating Procedures (SOPs)

 SOPs Should be for all operations.

 SOPs
SOP should
h ld contain
t i f ll
full working
ki
instructions, expected performance, internal
quality control, safety precautions and
calculation of results.
results

 Any deviation from SOPs should be recorded.

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D. Validation of methods
 Validation is the process of verifying that a
method is fit for the intended purpose.
 The method could be in house, from
literature, or official method.
 Decide the degree of validation required to
demonstrate that the method is fit for the
i t d d purpose.
intended
 Produce the necessary validation data
accordingly.
accordingly
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D. Validation of methods

 Validation is the process of verifying that a

method is fit for the intended purpose.
purpose

 Validation will precede practical application of

the
h method
h d to the
h analysis
l i off routine
i samples.l

 The method to be validated could be inin-house

house,
from literature, or official method, adapted to
match the capabilities of the lab and the
purpose for which it will be used.
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D. Validation of methods
 Decide the degree of validation required to
demonstrate that the method is fit for the intended
purpose.
purpose
 Produce the necessary validation data accordingly.
 Proficiency testing (or other inter-laboratory
comparisons), provide an important means for
verifying the accuracy of results or between-
laboratory variances.
 The use of representative analytes or matrices is
important in validating methods.
methods
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D. Validation of methods
Commodities are classified according to the Codex
Classification (CA volume2, 2nd ed.).
Commodity Common Commodity Representative
group properties
ti class
l species
i
Plant product
I High water & Leafy veg. Brassica Spinach, Lettuce
chlorophyll Leafy veg. Broccoli, cabbage
content Legume veg. Green beans
II High water & low Pome fruits Apple, pear
or no chlorophyll
hl h ll Stone
S fruits
f i P
Peach,
h cherry
h
content Berries Strawberry
Small fruits Grape
Fruiting veg. Tomato, pepper,
melon
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D. Validation of methods
Commodity Common Commodity Representative
group properties class species
Plant product
II High water & low or Root veg. Potato, carrot,
no chlorophyll parsley
content
III High acid content Citrus fruits Orange, lemon
IV High sugar content Raisin, dates
V High oil or fat Oil seeds Avocado, sunflower
Avocado
seeds
Nuts Pistachios, peanut
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D. Validation of methods
Commodity
C dit Common
C Commodity
C dit Representative
R t ti
group properties class species
Plant product
VI Dry materials Cereals Wheat, rice or maize
grains
Cereal products Wheat flour
Commodities
C di i G li tea, spices
Garlic, i
requiring
individual tests
A i l i i products
Animal-origin d t
Meats Cattle meat, chicken
Fats Fat of meat
Milk Cow milk
Eggs Chicken egg
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D. Validation of methods
The selection of representative analytes should
be made based on the purpose of analysis taking
i t accountt the
into th following:
f ll i
i. They have a wide range of physico-chemical
properties (hydrolysis
(hydrolysis, oxidation and
photolysis characteristics to include those of
represented analytes.
ii. Be those which are likely to be detected
regularly,
g y or for which critical decisions will
be made based on the results.
 C
Case St
Study
d

D. Validation of methods
iii. The concentration of the analytes used to
characterize a method should be selected to
cover the accepted limits of all analytes
planned to be sought in all commodities.

• Parameters to be assessed through the validation

process should be appropriate both to the method
and to the purpose for which it is applied.
 V lid ti off Methods
Validation M th d

 These parameters may be summarized as:

 Specificity
Extent
E t t to
t which
hi h a method
th d provides
id responses from
f
the detection system which can be considered
exclusively characteristic of the analyte.
 The ability of a method to determine accurately
and specifically the analyte of interest in the
presence of other components in a sample matrix
under the stated conditions of the test.
 S l ti it (Specificity)
Selectivity (S ifi it )

 The ability of a method to measure only what it is

intend to measure.

 The ability to assess unequivocally the analyte in

the presence of components which may expected
to be present. Typically these might include
impurities degradants,
impurities, degradants matrix,
matrix etc…..
etc
 S l ti it (Specificity)
Selectivity (S ifi it )

 Selectivity – discriminates between analyte and

other non analyte signals from other compounds.
compounds
 Specificity – provide evidence of the identity of
the analyte.
y
 Selectivity and Specificity are often used
interchangeably.
g y
 Specificity is the ultimate of Selectivity.

 It is recommended that the term selectivity y be

promoted and that the use of term specificity be
discouraged. (IUPAC recommendation 2001 –
IUPAC l
IUPAC,vol.73, N
No.8, 1381 – 1386).
)
 M
Measures off Selectivity
S l ti it

Selectivity is essentially a qualitative assessment

based on the significance or otherwise suitable
tests for interference.
1. Selectivityy Index ((ban/bint) ((IUPAC
C requirements)
q )
ban is the sensitivity of the method (slope of the
calibration curve))
bins is the sensitivity of the potential
interference.
It can beb determined
d t i d approximately
i t l by b execution
ti off
procedure on matrix blank and the same blank spiked
with a potential interfering at one appropriate level
(aflatoxin M as interfering for aflatoxin B1).
 M
Measures off Selectivity
S l ti it

2. Resolution (Rs) (AOAC requirements)

Rs is expressed as a function of both the
absolute separation distance expressed as
retention
t ti ti
times ( i t ) off the
(minutes) th two
t peaks,
k t1
and t2, and the baseline widths, W1 and W2, of
the analyte and nearest peak, peak also expressed in
terms of times, as
Rs = 2(t2-tt1)/(W1+WW2)
A resolution of at least 1.5 is usually sought and
p
1.0 is the minimum usable separation.
M
Measures off Selectivity
S l ti it
 V lid ti off Methods
Validation M th d

 These parameters may be summarized as:

 Analytical Range
Recovery through extraction, clean-up, and
measurement.
These tests could be combined with LOD, LOQ
and matrix effect tests.
 Calibration
C lib ti Range
R
Could be combined with linearity,
reproducibility and signal/noise experiments.
experiments
 Reporting Limit (LCL)
The lowest calibrated level employed during
analysis to detect residues.
 Li it off Detection
Limit D t ti

• The lowest content that can be measured with reasonable

statistical certainty.

• The lowest analyte content, if actually present, that will be

detected and can be identified. (AOAC)
( )

• The lowest conc. of analyte in a sample that can be

detected but not necessarily quantitated under the stated
detected,
conditions of the test. (NATA)

• The true net conc.

conc or amount of the analyte in the material
to be analyzed which will lead with probability (1-b), to the
conclusion that the conc. of the analyte in the analyzed
material
t i l is
i larger
l th that
than th t off the
th blank
bl k matrix.
t i (ISO/DIS)
Li it off Detection
Limit D t ti
Li it off Detection
Limit D t ti
 Li it off Quantification
Limit Q tifi ti

 The content equal to or greater than the lowest

conc. point on the calibration curve. (AOAC)

 The lowest conc.

conc Of an analyte that can be
determined with acceptable precision
(repeatability) and accuracy under the stated
conditions of the test.
test (NATA)

it is also known as Limit of Reporting

Li it off Quantification
Limit Q tifi ti
Li it off Quantification
Limit Q tifi ti
LOD & LOQ
L
Lowest
tCCalibration
lib ti Level
L l
R
Reporting
ti LiLimit
it
 V lid ti off Methods
Validation M th d

 These parameters may be summarized as:

 Analyte Stability
In sample processing and under storage
conditions.
conditions
 Homogeneity of analytical samples
Uniformity of dispersion of the analyte in
matrix.
 Repeatability & Reproducibility
It could be considered as measure of the previous
two parameters.
p
 P f
Performance Verification
V ifi ti

1. The main purposes

 Monitor the performance of the method under
the actual conditions during its use.
 Studying the effect caused by matrix,
instruments, quality of chemicals and analyst
performance.
performance
 Demonstrating that the method is under
‘statistical
statistical control
control’.
i.e. Accuracy & uncertainty of the method are
similar to those during method validation.
 P f
Performance Verification
V ifi ti

2. Construction & use of control charts

 To demonstrate the performance of a method
and its reproducibility.
 Control chart of recoveries used when a large
no. of the same type of sample are analyzed
for the same analytes.
analytes
 Control chart is constructed with the average
recovery of representative analytes in
representative matrices, used when a small
no. of different types
yp of samples
p are analyzed
y
for a great no. of analytes.
 C t l Ch
Control Chartt

 A statistical tool to determine if a process is in

control.
control

 In 1931, Dr. Walter Shewhart, a scientist at the

Bell Telephone laboratories, proposed applying
statistical based control chart to interpret
industrial manufacturing processes.

 IIn 1950, S.S Levey

L and
d E.R.
E R Jennings
J i suggested
d
the use of Dr.Shewhart’s control chart in the
clinical laboratory.
laboratory
 C t l Ch
Control Chartt

What is control charting or statistical process

control.
control

 A means of estimating variation in an analysis

process due to:
 Random or common variation.
 Unusual or special causes.

 Control charts or SPC tell us:

 When to adjust a process.
 When to leave it alone.
alone
 C t l Ch
Control Chartt

A graphical plot of test results overtime.

limits drawn are based in the statistical analysis
(sigma or standard deviation, ….) of the plotted
d t
data. Control
Limit

Conttrol Value
+3s
+2s
● ●

Controll limits:
C li i
● Mean
● ● -2s
●
-3s

 Upper and lower control Warning

Time Limit

= ± 3 s.d.
Warning limits:
 Upper and lower warning limits = ± 2 s.d.
 C t l Ch
Control Chartt

Why use control Charts.

 Monitor process variation over time.

 Diff
Differentiate
ti t between
b t special
i l cause and
d common
cause variation.

 Assess effectiveness of changes.

 Communicate process performance.

 C t l Ch
Control Chartt

The Idea of QC Chart:

 C t l Ch
Control Chartt

Different chart are used depending on the nature of

the charted data. Commonly y used charts are:
 For continuous (variables) data:

 S
Shewhart
ew a t sa
sample
p e mean
ea ((X ba
bar – cchart).
a t).
 Shewhart sample range (R – chart).
 Shewhart sample (X – chart).
 Cumulative sum (CUSUM).
 Exponentially Weighted Moving Average
(EWMA) chart.
 Moving – average and range charts.
 C t l Ch
Control Chartt

 For Discrete (attributes and countable) data.

 Sample proportion defective (p-Chart).

 S
Sample
l number
b off defectives
d f ti (
(np-chart).
h t)

 Sample number of defects (c-chart).

( )

 Sample number of defects per unit (u-chart or

c bar-chart).
 X h t
X-charts

 One of the oldest and simplest types of control

chart.
chart

 It is based on the distribution of the control

values around a true or expected value.

 It can be used to monitor the combination of

systematic and random effects for control values,
based on single results or on a mean of multiple
analyses.
 X h t
X-charts

 Using reference material similar to a routine

samples as control sample,
sample the bias may be
monitored by comparing the mean control value
over time with reference value.
 Special applications of the X chart.

 The blank value chart.

 Recovery chart
 Calibration p parameters such as slope p and
intercept, in so far they are determined daily, can
also be tested by means of the X chart.
 R - Chart
Ch t

 R chart serves repeatability control.

 Thee range
a ge iss de
defined
ed as tthee d
difference
e e ce betwee
between tthe
e
largest and smallest single result for two or more
separate samples.
 R chart applications in analytical laboratories
appears in duplicate determination (of samples to be
analyzed) in each analysis series.
 Test samples selected among the samples to be
analyzed in each analytical run.
 Since the range is normally proportional to sample
conc. (at levels well above the detection limit), it will
be more appropriate to use a control chart where the
control value is the relative range r%.
r%
 C t tC
Construct Control
t l Ch
Charts
t

 Select the quality characteristic.

 Develop a quality plan.
plan
 Select the type of control chart.
 Choose the proper sub
sub-group
group size.
 Collect the data.
 Determine the trial control limits and chart mid-
point.
 Determine the revised control limits and chart
mid-point.
id i t
 Construct the revised control chart.
 Continue to use the chart.
chart
 Dailyy Interpretation
p of
Control Chart
There are three possible cases:

1. The method is in control.

2. The method is in control but the long-term

evaluation shows that the method is out of
statistical control.

3. The method is out of control.

T i l Out
Typical O t – off – Control
C t lR Rules
l
T i l Out
Typical O t – off – Control
C t lR Rules
l
T i l Out
Typical O t – off – Control
C t lR Rules
l
T i l Out
Typical O t – off – Control
C t lR Rules
l
T i l Out
Typical O t – off – Control
C t lR Rules
l
T i l Out
Typical O t – off – Control
C t lR Rules
l
T i l Out
Typical O t – off – Control
C t lR Rules
l
T i l Out
Typical O t – off – Control
C t lR Rules
l
 Long-term
g evaluation of
Quality Control data
Consider the following questions:
 What is the quality (random and systematic
effects) currently in the laboratory? Has the
quality significantly changed?

 Are control limits and central line in the control

chart still optimal for
detection situations
out of control?
 R
Responses tto O
Out-of-Control
t fC t l

 Invoke the corrective action procedure.

 Determine root cause.

 Implement corrective action.

 Repeat QC sample
R l twice
i to demonstrate
d
“in-control”.

 Repeat sample analyses.

 How often should control limits
Be evaluated
 For successful use of control charts it is
important that the control limits and the central
line remain stable over a long period of time.

 The central line and control limits should not be

changed frequently since this will make it
difficult to detect gradual changes in analytical
q
quality.
y
 How often should control limits
Be evaluated
 The laboratory should have a policy for how often
control limit are evaluated and how it is decided
if a change is needed.

 Control limits should not be changed based on

less than 20 sets of new data since last
evaluation.
SETTING UP A QC
PROGRAM
 P ti l points
Practical i t

 Method Validation – use information gained

from method validation as basis for routine
quality control.

 Concentration range – iff conc. off an analyte

C
vary considerably, use separate X-charts and R-
charts for different conc.
conc levels.
levels

 R-chart with test samples – to monitor

repeatability using range charts (R-chart or r%-
chart), analyzing a test sample in duplicate in
each
h analytical
l ti l run is
i recommended.
d d
 P ti l points
Practical i t

 Frequency of control analyses – stability of

the measurement system can have an influence
on the frequency of control analyses.

 One control sample in each analytical run

O
(general rule).

 If there are errors caused by calibration drift,

the number of control samples to be analyzed
in each analytical run may need to be higher
than under very stable measurement
conditions.
diti
 P ti l points
Practical i t

 If the result of the QC sample is out of control,

all measurements performed after the last
approved sample in the quality control may to
be reanalyzed.

 Therefore, the frequency of control is therefore

a balance between the cost of the control and
the cost of repeating analyses.
analyses
 P ti l points
Practical i t

 Position of control samples in an analytical

run – it is recommended that control samples or
checks are analyzed at least the beginning of
each run and before finishing the analytical run,
in cause errors.

 A good balance between QC and test

samples – QC fit for purpose.
 Q lit Control
Quality C t l

 Principles:
 Full . . . Complete analysis.
 Through all steps of the method.

 Quality control is NOT….

 Equipment calibration.
 Equipment standardization (GC calibration).
 Equipment
q p monitoring.
g
 Bli d Samples
Blind S l

 Single Blind Samples

 Proficiency Testing (PT) samples.
samples
 Real – life samples (retested or purchased).
 Spiked
p samples
p prepared
p p by
yqquality
y department.
p
 Double blind samples
 Submitted as “customer” samples
p by
y a “customer”.
Samples appear to be real samples but are usually
prepared by a PT or reference material provider.
 Focus is on accuracy without any analyst bias.
 Acceptance criteria are based on PT data.
PROFICIENCY

TESTS

(PT)
 PT – ISO/IEC G
Guide
id 43:1997
43 1997

Determination of laboratory testing performance by

means of inter laboratory
y comparisons.
p
Note – for the purpose of this guide, the term
laboratory proficiency testing is taken in its
widest sense and includes, for example:
1. Qualitative Schemes – for example where
l b
laboratories
i are requiredi d to identify
id if a
component of a test item.
2. Data
D t transformation
t f ti exercises
i – for
f example l
where laboratories are furnished with sets of
q
data and are required to manipulate
p the data to
provide further information.
 PT – ISO/IEC G
Guide
id 43:1997
43 1997

3. Single item testing – where one item is sent

to a number of laboratories sequentially and
returned to the organizer at intervals.
4. One off exercises – where laboratories are
provided with a test item on a single occasion.
5. Continuous schemes – where laboratories are
provided with test items at regular intervals on
a continuing basis.
6. Sampling – for example where individuals or
organizations are required to take samples for
subsequent analysis.
analysis
 P fi i
Proficiency Testing
T ti

PT organizers distribute portions of a homogeneous

material to each of the participants

Participants analyze the material under

t i l conditions
typical diti and
d reportt to
t the
th organizer
i

Organizer reports the results usually in the form of

a score relating to the accuracy of the result
 P fi i
Proficiency Testing
T ti

A score of zero – implies a perfect result. This will

happen
pp quite rarely
q y even in pperfectly
y competent
p
laboratory.
Laboratory complying with the PT scheme
scheme’ss fitness
for purpose criterion will commonly produce
scores falling between -2 and +2. they might
expect to produce a value somewhat outside this
range occasionally, roughly about 1 time in 20. so
an isolated event of this kind is not of great
moment. The sign (i.e. + or -) of the score
indicates a negative or positive error,
respectively.
respectively
 P fi i
Proficiency Testing
T ti

A score outside the range from -3 to +3 would be

very unusual for a laboratory operating under
the given fitness for purpose criterion, and is
taken to indicate that the accuracy requirement
has not been met (at least on that occasion). The
cause of the event should be investigated
g and
remedied.
MEASUREMENT
UNCERTAINTY
 d fi iti
definition

“a parameter associated with the result of a

measurement that characterizes the dispersion
measurement,
of values that could reasonably be attributed to
the measure”. (ISO-VIM:1993)
The parameter may be σ or the width of confidence
interval.
The number after ±.
MU dose not imply doubt about the validity of a
measurement, on the contrary, knowledge of
uncertainty implies increased confidence in the
validity of a measurement result.
result
 Wh needs
Who d MU

1. The customer needs it together with the result

to make a correct decision. The uncertainty of
the result is important, e.g. when looking to
allowable (legal) concentration limits.

2. The laboratory to know its own quality of

measurement and to improve to the required
quality.
 Wh should
Why h ld th
the llab
b give
i MU

1. The customer needs it to make correct decisions.

2
2. An estimation of the measurement uncertainty
is required in ISO 17025 (5.4.6)
 U
Uncertainty
t i t Sources
S

The uncertainty of the result may arise from many

possible sources, some examples are:

 Sampling
Sampling.

 Storage conditions.

 Preparation of analytical portions.

 Incomplete extraction and clean-up.

 Matrix effects (sample composition)

 U
Uncertainty
t i t Sources
S

 Contamination during sampling or sample

preparation.
preparation
 Effect of environmental condition, measurement
conditions.
conditions
 Computational effects (software, calibration
models…))
 Uncertainty of weighs and volumetric equipment.
 Instrument effects (stability, linearity…).
 Approximations and assumptions incorporated in the
test method. Uncertainties in analytical
y process
p
 Random effects. QUAM :2000.1 (Appendix C)
 E
Errors & Uncertainty
U t i t

Error – “the result of measurement minus a true

value of measurand”. (ISO-VIM:1993).

1. Random errors.

2. Systematic errors.

3. Gross (spurious or blunder) errors.

 E
Errors & Uncertainty
U t i t

Random error (Type A):

“result of measurement minus the mean that would
result from an infinite number of measurements
off the
th same measurand d carried
i d outt under
d
repeatability conditions”. (ISO-VIM:1993)
Note1: random error is equal to error minus
systematic error.
Note2: because only a finite number of
measurements can be made, it is possible to
determine onlyy an estimate of random error.
 E
Errors & Uncertainty
U t i t

Systematic error (type B):

“mean that would result from an infinite number of
measurements of the same measurand carried
outt under
d repeatability
t bilit conditions
diti minus
i a true
t
value of the measurand”. (ISO-VIM:1993)
Note1: systematic error is equal to error minus random error.
error
Note2: the systematic error is independent of the number of
measurements made and can not therefore be reduced by
increasing the number of analyses under constant
measurement conditions.
Note3: the result of a measurement should be corrected for all
recognized significant systematic effects.
E
Errors & Uncertainty
U t i t
 E
Errors & Uncertainty
U t i t

Not synonyms, but different concepts

 Error is a single value, if known it can be applied as a
correction to the measured value

 Uncertainty takes the form of a range, in which the true

value has a known probability of being found.found (it
cannot be applied as a correction)
The result of an analysis may be by chance close to the true value of
the measurand, and hence have a negligible error; however the
uncertainty may still be very large simply because the analyst doesn’t
k
know how
h closel that
h resultl is
i to the
h true value
l
TOP – DOWN APPROACH
 T D
Top-Down A
Approach
h

Theoretical ‘Bottom Up’ approach recommended by the ISO

GUM ‘bible’ on uncertainty:
‘Guide to Expression of Uncertainty of Measurement’, ISO
(1993)

You had a look at how this approach may be used so that you
may decide for yourself. (It
(It’ss a good idea to have some
knowledge of estimating MU from first principles)

This may not be the best way to go about estimating the MU

associated with results generated by a complex chemical test
method.
method
 T D
Top-Down A
Approach
h

The Bottom – Up Approach followed in estimation of

measurement uncertaintyy in chemical analysis
y have drawn
fierce criticism from chemists.

Horwitz ‘The
W Horwitz,
W. The Certainty of Uncertainty’
Uncertainty ,
J. AOAC International, 86,109 (2003)
This absurd and budget
‘This budget-busting
busting approach (for
analytical chemistry) arose from metrological chemists
taking over in entirety the concepts developed by
metrologists
t l i t forf physical
h i l processes measured d with
ith 5-7
57
significant figures ….and applying them to analytical
chemical measurements with 2-3 significant
g figures’
g
 T D
Top-Down A
Approach
h

W. Horwitz, ‘The Certainty of Uncertainty’,

J. AOAC International, 86,109 (2003)

‘This approach also ignores the fact that some

chemical methods are influenced by numerous
factors, some positive and some negative, that
tend to cancel out….and
out and that often other chemical
methods are influenced by factors that overwhelm
the weight and volume uncertainties presented in
published examples’
 T D
Top-Down A
Approach
h

An Alternative Approach...
‘Top-down’ calculation/estimate using available
validation and Q
QC data.

ACCURACY: Trueness (bias) and Precision

a ‘reasonable’ estimate of MU may be obtained by

considering
id i th
the uncertainties
t i ti associated
i t d with
ith
imprecision and bias
 T D
Top-Down A
Approach
h

1. Accuracy : Trueness (bias) and Precision

1 1 Analysis
1.1 A l i off spiked
ik d samples
l (Validation
(V lid ti Data)
D t )
1.2 IQC (Reproducibility within-lab – u(Rw)
1 2 IQC + CRM (Lab
1.2 (L b Bias)
Bi )
1.3 IQC +Inter-lab Comparisons or PT (Lab
Bias)
2. Twice Reproducibility from inter-lab studies

3. Horwitz formula (Anticipated expanded MU = 2 X

PRSDR )
 T D
Top-Down A
Approach
h

 When a stable control sample is covering the whole

analytical process and has a matrix similar to the
samples, the within-laboratory reproducibility at that
concentration level can simply be estimated from the
analyses of the control samples.

 If the analyses performed cover a wide range of

concentration levels, also control samples of other
concentration
t ti levels
l l should
h ld be
b used.
d