GOOD LABORATORY PRACTICEIN CHEMISTRY

By Mohamed Salama

CONTENTSCONTENTS

Good Laboratory Practice (GLP)

Difference between Good Laboratory Practice and

ISO/IEC 17025ISO/IEC 17025.

Codex Alimentarius.

G d L b t P tiGood Laboratory Practice

What is GLP?GLP is a quality system concerned with theGLP is a quality system concerned with the

organizational process and the conditions underwhich non-clinical health and environmentalsafety studies are planned, performed,monitored, recorded, archived and reported.

Rationale:Promote the quality and validity of non-clinical

data on which hazard assessments areb dbased.

Processing & non-Regulatedg gvs. Regulated areas

Hi t f GLPHistory of GLP

First evolved in the USA in 1970s by the Food andDrug Administration (FDA) because of concernsDrug Administration (FDA) because of concernsabout the validity of preclinical safety data.

O C fOECD assembled an expert group who formulatedthe first OECD principles of GLP in order toavoid non tariff barriers to trade in chemicals toavoid non-tariff barriers to trade in chemicals, topromote mutual acceptance of non-clinical safetytest data, and to eliminate unnecessary, yduplication of experiments.

Wh t i OECDWhat is OECD

The Organization for Economic Co-operation andDevelopment.

It is an intergovernmental organization. It is an intergovernmental organization.

30 industrialized countries meet to co-ordinateand harmonize policies.

Discuss issues of mutual concern.

Work together to respond to internationalproblems.

30 I d t i li d C t i30 Industrialized Countries

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 i OECDWhat is OECD

OECD guidelines are NOT law.

OECD standards are internationally accepted.

Fi t d l d i 1978 i US FDA GLP First developed in 1978 using US FDA GLP

regulations as a basis.

Revised edition adopted 1977.

W GLPWHY GLP

To promote the development of quality test data.

To provide a managerial tool to ensure a sound

approach to the management including conductapproach to the management, including conduct,

reporting and archiving of laboratory studies.

Wh t GLP i NOTWhat GLP is NOT

GLP has nothing to do with efficacy.

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

If l b l i t b GLP b t d t If a lab claims to be GLP, but dose not run

animal studies, they are misguided.

GLP does not apply to analytical development.

GLP dose not apply to animal field studies.

A li bilit f GLPApplicability of GLP

Non-Clinical safety testing of test items

contained in:

Ph ti l d t Pharmaceutical products.

Pesticide products.p

Veterinary drugs.

Food additives.

Feed additives Feed additives.

C l iConclusion

It is important to clearly differentiate betweenthe formal regulatory use of the term “Goodthe formal regulatory use of the term GoodLaboratory Practice” as opposed to the generalapplication of “Good Practices” in scientificinvestigations.

Since the term “Good Laboratory Practice” is nota trade mark protected term, any laboratorywhich may consider itself to be following goodpractices in its daily work might be tempted topractices in its daily work might be tempted todescribe its adherence to these (possibly evenself-defined) quality standards by this) q y yterminology

GOOD LABORATORY PRACTICE

I t d tiIntroduction

The backbone of GLPs is documentation ofprotocols reports data collection techniques andprotocols, reports, data collection techniques andarchival capabilities.

GLP is needed for: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 tiIntroduction

GLP is not needed for: Basic research Basic research. Studies to develop new analytical methods. Chemical tests used to drive the specifications of Chemical tests used to drive the specifications of

a marketed food product.Good Laboratory Practice (GLP) deals with:y ( )1. Organization.2. Process.3. Conditions under which laboratory studies are

planned, performed, monitored, recorded andreportedreported.

I t d tiIntroduction

To comply with regulations can be quite

expensive (can increase the cost of a laboratory

up to 30%) and sometimes it is just impossible toup 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, training plans should include basic GLP

knowledge for everybody working in a GLP

environment.

M i P i t f GLPMain Points of GLP

Resources: organization, personnel, facilities,equipmentequipment.

Rules: protocols, standard operating procedures,t f th t d di t th i t l i tconcept of the study director as the pivotal point

of study control.

Characterization: test items, test systems.

Documentation: raw data, final report, archives., p ,

Quality assurance: independence from studyconductconduct.

GLP P i i lGLP Principles

A laboratory which intends to conduct studiesthat are GLP compliant will have to be organizedthat are GLP compliant will have to be organizedso that the following conditions apply: A study director (in case of toxicological studies) A study director (in case of toxicological studies).

A quality assurance unit (QAU).

Qualified personnel.

Standard operating procedures (SOPs).p g p ( )

Control and test articles.

E i Equipment.

GLP P i i lGLP Principles

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

A Quality Assurance Unit: A Quality Assurance Unit:audit the laboratory studies and theaccompanying data It may be a separateaccompanying data. It may be a separatedepartment or an individual person, either full orpart time. (any person except the study director).

GLP P i i lGLP Principles

Qualified Personnel:must be qualified through education, trainingmust be qualified through education, trainingand/or experience to follow directions andperform test procedures properly.

Standard Operating Procedures (SOPs):all laboratory activities must be performed in

d i h l i d laccordance with correctly written and properlyfiled, management approved SOPs.These must be readily available to the personnelThese must be readily available to the personnelconcerned. They should cover policies,administration, technical operation, equipment

i d l i l h doperating and analytical methods.

GLP P i i lGLP Principles

Control and Test Articles:must be identified and characterized by Strength,Purity and Stability.Reagents and solutions must be labeled withinformation on origin, identity, concentration,storage conditions and expiration datestorage conditions, and expiration date.

Equipment:instruments must be designed to meet analyticalrequirements and regularly maintained andcalibrated.

GLP B fitGLP Benefits

From the point of view of international trade:

The ultimate goal in fair practice depends on:Reliability of analytical resultsb y y

This in turn, depends on:s , epe s o :

Availability of reliable analytical methods.

Experience of the analyst.

Maintenance of ‘good practice’.

GLP B fitGLP Benefits

Why Reliable Analytical Results?

Reliable analytical results are essential for:y

Protecting the health of consumers.

Facilitating international trade.

DIFFERENCE BETWEEN GOODDIFFERENCE BETWEEN GOODLABORATORY PRACTICE &

ISO/IEC 17025

Difference betweenGLP & ISO/IEC 17025

ISO Members.

OECD Members.

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

Different regulations in different countries.g

Designed for repetitive studies.

Designed for single studies.

Difference betweenGLP & ISO/IEC 17025

Description of quality system in qualitymanual.

Description of quality system in SOPs.

General statements for responsibilities ofpersonnel.

Very specific responsibilities of personnel.

Non specific requirements for storage ofp q grecords and reports.

Specific requirements for storage, retentionp q g ,and archiving.

Difference betweenGLP & ISO/IEC 17025

No study plans required (standard methods

should be used).

Study plan required for each study Study plan required for each study.

Written operating procedures without

specific format.

SOP ith d t il d i t f f t SOPs with detailed requirements for format

and content.

Difference betweenGLP & ISO/IEC 17025

Analysis methods must be verified through

inter-laboratory test (PT).

Validation through inter laboratory test not Validation through inter-laboratory test not

required.

Documented complaints procedures.

I f bl l f l In case of problems only course of law.

Difference betweenGLP & ISO/IEC 17025

Storage of test samples and data until client

accepts results.

Storage of test samples according to local Storage of test samples according to local

regulatory requirements.

GUIDELINES ON GOODGUIDELINES ON GOODLABORATORY PRACTICE IN

R ARESIDUE ANALYSIS

CODEX ALIMENTARIUS

C d Ali t iCodex Alimentarius

Latin for “Food Code” or “Food Book”.

Developed and maintained by the codex

alimentarius commission a body that wasalimentarius commission, a body that was

established in 1963 by the Food and Agriculture

Organization of United Nations (FAO) and the

World Health Organization (WHO)World Health Organization (WHO).

C d Ali t iCodex Alimentarius

Recognized by the world trade organization as an

international reference point for the resolution of

disputes concerning food safety and consumerdisputes concerning food safety and consumer

protection.

Is a collection of standards, codes of practice,

guidelines and other recommendations Some ofguidelines and other recommendations. Some of

these text are very general, and some are very

specific.

C d Ali t iCodex Alimentarius

Some deal with detailed requirements related to

a food or group of foods, others deal with the

operation and management of productionoperation and management of production

processes or the operation of government

regulatory systems for food safety and consumer

protection.p

C d Ali t i C i iCodex Alimentarius Commission

It is intergovernmental standards-setting body

established by FAO and WHO in 1961/63.

11th FAO Conference Resolution no 12/61 (codex 11 FAO Conference Resolution 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 t i C i iCodex Alimentarius Commission

Its Mandate

Dual objective: Protecting the health of consumers.g Facilitating fair practices in food trade. To coordinate all food standards work.

Non-mandatory in nature, codex standards andrelated texts have since 1995 becomeinternational benchmarks for harmonizationunder the SPS and TBT agreements of WTO.

R l f C d St d dRole of Codex Standards

For food safety, codex standards are theinternational benchmarkinternational benchmark.

National regulations consistent with codexstandards meet the requirements of the SPSstandards meet the requirements of the SPSAgreement (i.e. do not have to be justified).

Are not obligatory but are the reference in the Are not obligatory, but are the reference in theevent of a trade dispute.

Where standards are more stringent than codex Where standards are more stringent than codex,there must be a scientific justification (based onassessment of the risk).assess e t o t e s ).

C d Ali t iCodex Alimentarius

Its scientific basis. Liaison & S i

Codex – Risk management. Separation

FAO/WHO Expert Bodies – Risk assessmentp 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 ONGOOD LABORATORY PRACTICE IN

IN RESIDUE ANALYSIS

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

GLP P i i lGLP Principles

A laboratory which intends to conduct studiesthat are GLP compliant will have to be organizedthat are GLP compliant will have to be organizedso that the following conditions apply: A study director (in case of toxicological studies) A study director (in case of toxicological studies).

A quality assurance unit (QAU).

Qualified personnel.

Standard operating procedures (SOPs).p g p ( )

Control and test articles.

E i Equipment.

M i P i i lMain Principles

Good analytical practice may be considered in three

inter related parts:inter related parts:

1. Analyst.

2. Basic resources.

3. Analysis.

C St dCase Study

Why GLP in residue analysis.

Because the analyte concentrations are in the

range µg/kg to mg/kgrange µg/kg to mg/kg.

Because the analyses can be challenging.

Attention to details is essential

C St dCase Study

These details are summarized in:1 The Analyst:1. The Analyst:The analyst who undergoes residue analysis:

Should have appropriate professional Should have appropriate professionalqualification.

Should be experienced in the correct use ofpapparatus and lab skills.

Should be competent in residue analysis.Sh ld b f ll t i d Should be fully trained.

Should have understanding of the principlesof residue analysisof residue analysis.

C St dCase Study

1. The Analyst:ContinuedContinued…..

Should have understanding of therequirements of Analytical quality assurancerequirements of Analytical quality assurance(AQA) Systems.

Should understand the purpose of each stageShould understand the purpose of each stagein the method and notice and deviation.

Should be trained in the evaluation andinterpretation of data.

C St dCase Study

1. The Analyst:

Continued…..

The staff should spend some of their training The staff should spend some of their trainingperiod in a well established (expert) laboratorywhere

experienced advice and training is available.p g

A record of training and experience must be keptf fffor all laboratory staff.

C St dCase Study

2. Basic Resources:A. The laboratory The laboratory and its facilities must be

designed to allow tasks to well-defined areaswhere maximum safety and minimum chanceof contamination of samples prevailof contamination of samples prevail.

Separate rooms (well ventilated) should bedesignated for sample receipt and storage fordesignated for sample receipt and storage, forsample preparation, for extraction and clean-up and for instrumentation used in thepdeterminative step.

C St dCase Study

A. The laboratory All materials used within the lab should be All materials used within the lab should be

resistant to chemicals. The area used for extraction and clean-up The area used for extraction and clean-up

must meet solvent laboratory specifications. All fume extraction facilities must be of highAll fume extraction facilities must be of high

quality. Sample receipt, storage and preparationp p , g p p

should be handled in areas away from areas ofresidue analysis.

Ensure sample integrity.

C St dCase Study

A. The laboratory Laboratory safety must be considered in terms 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 workingarea.

Small volume of solvents should be held in theworking area and the bulk of the solventsstored separately away from the main workingareaarea.

C St dCase Study

A. The laboratory Minimize the use of highly toxic solvents and Minimize the use of highly toxic solvents and

reagents should whenever possible.

All l h ld b d f l d All waste solvent should be stored safely anddisposed of both safely and in anenvironmentally friendly manner taking intoenvironmentally friendly manner taking intoaccount specific national regulations whereavailable.

All equipment such as lights, and refrigeratorsshould be “spark free” or “explosion proof”should be spark free or explosion proof

C St dCase Study

A. The laboratoryA l f f l h ld b il bl A supply of safety tools should be availablesuch as safety glasses, gloves and otherprotective clothing emergency washingprotective clothing, emergency washingfacilities and a spillage treatment kit.

Appropriate and adequate fire fighting Appropriate and adequate fire fightingequipment must be available.

A great care should be taken in the handling of A great care should be taken in the handling ofstandard reference compounds due to theirtoxic properties.to c p ope t es.

C St dCase Study

B. Equipment and Supplies Adequate supplies of electricity and water. Adequate supplies of reagents, solvents, gas,

glassware, chromatographic materials, etc..,of suitable quality.Ch t hi i t b l Chromatographic equipment, balances,spectrophotometers etc.., must be servicedand calibrated regularlyand calibrated regularly.

Record of all servicing/repairs must bemaintained for every item of equipment.maintained for every item of equipment.

C St dCase Study

B. Equipment and Supplies Regular calibration is essential for equipment

performing measurements. This factori ifi tl t ib t t th t i t fsignificantly contribute to the uncertainty of

measurement. Balances and automated pipettes dispensers Balances and automated pipettes, dispensers

and similar equipment must be calibratedregularly.eg a y.

The operating temperatures of refrigeratorsand freezers should be continually monitoredyor be checked at specified intervals.

C St dCase Study

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

d t bl hi h itand acceptably high purity. Analytical standards should be available for

all a e t co o d the lab o ito i g aall parent compounds, the lab monitoring aswell as those metabolites that are included inMRLs.MRLs.

C St dCase Study

B. Equipment and Supplies

All analytical standards, stock solutions andreagents should be properly labeled.g p p yPreparation date, analyst initials, solvent used,

storage conditions and expiry date.g p y

Compounds influenced be degradativeprocesses (light heat ) must be clearlyprocesses (light, heat,..) must be clearlylabeled and stored under appropriateconditions.

C St dCase Study

B. Equipment and Supplies

Reference standards must be kept underconditions that will minimize the rate ofdegradation.Low temperature.Low temperature.

Exclusion of moisture and light.

Care should be taken that standard solutionsare not concentrated by solvent evaporation.

C St dCase Study

3. Analysis The methods applied for the determination of

pesticide residue should satisfy the followingit icriteria:

Conc. Repeatability Reproducibility Trueness

CV %(3) CV %(4) CV %(3) CV %(4) % Rec (2)CVA%(3) CVL%(4) CVA%(3) CVL%(4) % 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

C St dCase Study

3. Analysis

1) The acceptability of the data produced

depends on the purpose of the analysisdepends on the purpose of the 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 uncertainty.g y

C St dCase Study

3. Analysis

2) These recovery ranges are appropriate for

multi residue methodsmulti-residue methods. For single analyte methods or veterinary drug

residues, more restricted criteria are required.

3) CVA: Coefficient of variation for analysis) A y

excluding sample processing.

C St dCase Study

3. Analysis

4) CVL: Overall coefficient of variation of

laboratory results including up to 10%laboratory results, including up to 10%

variability of residues between analytical

portions (CVSp).

hwhere:

CVL2 = CVSp

2 + CVA2

L Sp A

C St dCase Study

3. AnalysisA. 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 determinationtstage.

• May lead to loss of sensitivity that mayprevent the residue from being detectedprevent the residue from being detected.

C St dCase Study

A. Avoidance of contamination

For these reasons

All l t i l t d• All glassware, reagents, organic solvents and

water should be checked for possible

interfering contaminants before use, by

analysis of a reagent blankanalysis of a reagent blank.

C St dCase Study

A. Avoidance of contamination

Polishes, barrier creams, soaps containing

germicides insect sprays perfumes andgermicides, insect sprays, perfumes and

cosmetics can give rise to interference

problems and are especially significant when

an electron capture detector is being usedan electron capture detector is being used.Their use is banned by the staff while in the

l blaboratory.

C St dCase Study

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.

C St dCase Study

A. Avoidance of contamination Contamination of glassware, syringes and gas

chromatographic columns can arise fromcontact with previous samples or extracts.contact with previous samples or extracts.

Thus… All glassware should be cleaned with detergentAll glassware should be cleaned with detergent

solution, rinsed thoroughly with distilledwater and then rinsed with the solvent to be

dused. Glassware to be used for trace analysis must

be kept separate and must not be used for anybe kept separate and must not be used for anyother purpose.

C St dCase Study

A. Avoidance of contamination Cross-contamination may occur between

reference standard materials and samplet textracts.

Thus…

Pesticide reference standards should always bestored at a suitable temperature in a roomstored at a suitable temperature in a roomseparate from the main residue laboratoryaway from sample storage & extraction areas.away from sample storage & extraction areas.

C St dCase Study

A. Avoidance of contamination The nature and importance of contamination

can vary according to the type ofd t i ti t h i d d th l l fdetermination technique used and the level ofpesticide residue to be determined.

Contamination problems with methods based Contamination problems with methods basedon gas chromatography or high performanceliquid.q .

Chromatography may be less significant if aspectrophotometric determination is used, andp pvice versa.

C St dCase Study

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 Potential hazards associated with its handling.

On receipt, a sample must immediately beassigned a unique identification code whichassigned a unique identification code whichshould accompany it through all stages of theanalysis to the reporting of the results.

C St dCase Study

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 representativeanalytical portions without affecting theanalytical portions without affecting theresidues concentration levels.

Fresh samples should be stored at 1-5oC away Fresh samples should be stored at 1-5 C, awayfrom direct sunlight, while frozen samplesshould be kept frozen and stored at -16oC.p

C St dCase Study

B. Reception & Storage of Samples The effect of storage should be checked by

analyzing fortified samples stored under thediti f i il i dsame conditions for a similar period.

When samples are to be frozen it isrecommended that analytical test portions berecommended that analytical test portions betaken prior to freezing in order to minimizethe possible effect of water separation as icee poss e e ec o wa e sepa a o as cecrystals during storage.

Care must be taken to avoid containers leak.

C St dCase Study

C. Standard Operating Procedures (SOPs)

SOPs Should be for all operations.

SOP h ld t i f ll ki SOPs should contain full working

instructions, expected performance, internal

quality control, safety precautions and

calculation of resultscalculation of results.

Any deviation from SOPs should be recorded.

C St dCase Study

D. Validation of methods

Validation is the process of verifying that amethod is fit for the intended purpose.

The method could be in house, fromliterature, or official method.

Decide the degree of validation required todemonstrate that the method is fit for thei t d dintended purpose.

Produce the necessary validation dataaccordinglyaccordingly.

C St dCase Study

D. Validation of methods

Validation is the process of verifying that amethod is fit for the intended purposemethod is fit for the intended purpose.

Validation will precede practical application ofh h d h l i f i lthe method to the analysis of routine samples.

The method to be validated could be in-houseThe method to be validated could be in house,from literature, or official method, adapted tomatch the capabilities of the lab and thepurpose for which it will be used.

C St dCase Study

D. Validation of methods Decide the degree of validation required to

demonstrate that the method is fit for the intendedpurposepurpose.

Produce the necessary validation data accordingly. Proficiency testing (or other inter-laboratory Proficiency testing (or other inter-laboratory

comparisons), provide an important means forverifying the accuracy of results or between-laboratory variances.

The use of representative analytes or matrices isimportant in validating methodsimportant in validating methods.

C St dCase Study

D. Validation of methodsCommodities are classified according to the CodexCommodities are classified according to the CodexClassification (CA volume2, 2nd ed.).

Commodity Common ti

Commodity l

Representative igroup properties class species

Plant productI High water & Leafy veg. Brassica Spinach, Lettuce

chlorophyll content

Leafy veg.Legume veg.

Broccoli, cabbageGreen beans

II High water & low hl h ll

Pome fruitsS f i

Apple, pearP h hor no chlorophyll

contentStone fruitsBerriesSmall fruitsFruiting veg.

Peach, cherryStrawberryGrapeTomato, pepper, Fruiting veg. Tomato, pepper, melon

C St dCase Study

D. Validation of methodsCommoditygroup

Common properties

Commodity class

Representative species

Plant productII High water & low or

no chlorophyll content

Root veg. Potato, carrot, parsley

III High acid content Citrus fruits Orange, lemonIV High sugar content Raisin, dates

V High oil or fat Oil seeds Avocado sunflower V High oil or fat Oil seeds

Nuts

Avocado, sunflower seedsPistachios, peanut

C St dCase Study

D. Validation of methodsC dit C C dit R t ti Commoditygroup

Common properties

Commodity class

Representative species

Plant productVI Dry materials Cereals

Cereal products

Wheat, rice or maize grainsWheat flour

C di i G li iCommodities requiring individual tests

Garlic, tea, spices

A i l i i d tAnimal-origin productsMeatsFatsMilk

Cattle meat, chickenFat of meatCow milkMilk

EggsCow milkChicken egg

C St dCase Study

D. Validation of methodsThe selection of representative analytes shouldbe made based on the purpose of analysis takingi t t th f ll iinto account the following:

i. They have a wide range of physico-chemicalproperties (hydrolysis oxidation andproperties (hydrolysis, oxidation andphotolysis characteristics to include those ofrepresented analytes.represented analytes.

ii. Be those which are likely to be detectedregularly, or for which critical decisions willg ybe made based on the results.

C St dCase Study

D. Validation of methods

iii. The concentration of the analytes used tocharacterize a method should be selected tocover the accepted limits of all analytesplanned to be sought in all commodities.

• Parameters to be assessed through the validationprocess should be appropriate both to the methodand to the purpose for which it is applied.

V lid ti f M th dValidation of Methods

These parameters may be summarized as:

Specificity

E t t t hi h th d id fExtent to which a method provides responses fromthe detection system which can be consideredexclusively characteristic of the analyte.exclusively characteristic of the analyte.

The ability of a method to determine accuratelyand specifically the analyte of interest in theand specifically the analyte of interest in thepresence of other components in a sample matrixunder the stated conditions of the test.

S l ti it (S ifi it )Selectivity (Specificity)

The ability of a method to measure only what it is

intend to measure.

The ability to assess unequivocally the analyte inThe ability to assess unequivocally the analyte in

the presence of components which may expected

to be present. Typically these might include

impurities degradants matrix etcimpurities, degradants, matrix, etc…..

S l ti it (S ifi it )Selectivity (Specificity)

Selectivity – discriminates between analyte andother non analyte signals from other compoundsother non analyte signals from other compounds.

Specificity – provide evidence of the identity ofthe analyte.y

Selectivity and Specificity are often usedinterchangeably.g y

Specificity is the ultimate of Selectivity. It is recommended that the term selectivity bey

promoted and that the use of term specificity bediscouraged. (IUPAC recommendation 2001 –IUPAC l N )IUPAC,vol.73, No.8, 1381 – 1386).

M f S l ti itMeasures of Selectivity

Selectivity is essentially a qualitative assessmentbased on the significance or otherwise suitablebased on the significance or otherwise suitabletests for interference.

1. Selectivity Index (ban/bint) (IUPAC requirements)y ( an int) ( C q )ban is the sensitivity of the method (slope of thecalibration curve))bins is the sensitivity of the potentialinterference.It b d t i d i t l b ti fIt can be determined approximately by execution ofprocedure on matrix blank and the same blank spikedwith a potential interfering at one appropriate level(aflatoxin M as interfering for aflatoxin B1).

M f S l ti itMeasures of Selectivity

2. Resolution (Rs) (AOAC requirements)Rs is expressed as a function of both theabsolute separation distance expressed as

t ti ti ( i t ) f th t k tretention times (minutes) of the two peaks, t1and t2, and the baseline widths, W1 and W2, ofthe analyte and nearest peak also expressed inthe analyte and nearest peak, also expressed interms of times, as

Rs = 2(t2-t1)/(W1+W2)Rs 2(t2 t1)/(W1 W2)A resolution of at least 1.5 is usually sought and

1.0 is the minimum usable separation.p

M f S l ti itMeasures of Selectivity

V lid ti f M th dValidation of Methods

These parameters may be summarized as: Analytical Range Analytical RangeRecovery through extraction, clean-up, and

measurement.These tests could be combined with LOD, LOQ

and matrix effect tests.C lib ti R Calibration Range

Could be combined with linearity,reproducibility and signal/noise experimentsreproducibility and signal/noise experiments.

Reporting Limit (LCL)The lowest calibrated level employed during

analysis to detect residues.

Li it f D t tiLimit of Detection

• The lowest content that can be measured with reasonablestatistical certainty.

• The lowest analyte content, if actually present, that will bedetected and can be identified. (AOAC)( )

• The lowest conc. of analyte in a sample that can bedetected but not necessarily quantitated under the stateddetected, but not necessarily quantitated under the statedconditions of the test. (NATA)

The true net conc or amount of the analyte in the material• The true net conc. or amount of the analyte in the materialto be analyzed which will lead with probability (1-b), to theconclusion that the conc. of the analyte in the analyzed

t i l i l th th t f th bl k t i (ISO/DIS)material is larger than that of the blank matrix. (ISO/DIS)

Li it f D t tiLimit of Detection

Li it f D t tiLimit of Detection

Li it f Q tifi tiLimit of Quantification

The content equal to or greater than the lowest

conc. point on the calibration curve. (AOAC)

The lowest conc Of an analyte that can be The lowest conc. Of an analyte that can be

determined with acceptable precision

(repeatability) and accuracy under the stated

conditions of the test (NATA)conditions of the test. (NATA)

it is also known as Limit of Reporting

Li it f Q tifi tiLimit of Quantification

Li it f Q tifi tiLimit of Quantification

LOD & LOQLOD & LOQ

L t C lib ti L lLowest Calibration Level

R ti Li itReporting Limit

V lid ti f M th dValidation of Methods

These parameters may be summarized as: Analyte Stability Analyte StabilityIn sample processing and under storage

conditionsconditions. Homogeneity of analytical samplesUniformity of dispersion of the analyte inUniformity of dispersion of the analyte in

matrix. Repeatability & ReproducibilityRepeatability & ReproducibilityIt could be considered as measure of the previous

two parameters.p

P f V ifi tiPerformance Verification

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 analystperformanceperformance.

Demonstrating that the method is under‘statistical control’statistical control .

i.e. Accuracy & uncertainty of the method aresimilar to those during method validation.similar to those during method validation.

P f V ifi tiPerformance Verification

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 analyzedfor the same analytesfor the same analytes.

Control chart is constructed with the averagerecovery of representative analytes inrecovery of representative analytes inrepresentative matrices, used when a smallno. of different types of samples are analyzedyp p yfor a great no. of analytes.

C t l Ch tControl Chart

A statistical tool to determine if a process is incontrolcontrol.

In 1931, Dr. Walter Shewhart, a scientist at the In 1931, Dr. Walter Shewhart, a scientist at theBell Telephone laboratories, proposed applyingstatistical based control chart to interpretindustrial manufacturing processes.

I S L d E R J i d In 1950, S. Levey and E.R. Jennings suggestedthe use of Dr.Shewhart’s control chart in theclinical laboratoryclinical laboratory.

C t l Ch tControl Chart

What is control charting or statistical processcontrolcontrol.

A means of estimating variation in an analysisprocess due to: Random or common variation. Unusual or special causes.

Control charts or SPC tell us: Control charts or SPC tell us: When to adjust a process. When to leave it alone When to leave it alone.

C t l Ch tControl Chart

A graphical plot of test results overtime.limits drawn are based in the statistical analysis(sigma or standard deviation, ….) of the plottedd tdata.

C l li i ● Mean

+2s

+3s

trol

Val

ue

Control Limit

●●

Control limits: Upper and lower control

● Mean

-2s

-3s

Time

Con

t

Warning Limit

●●●

= ± 3 s.d.Warning limits: Upper and lower warning limits = ± 2 s.d.

C t l Ch tControl Chart

Why use control Charts.

Monitor process variation over time.

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

cause variation.

Assess effectiveness of changes.

Communicate process performance.

C t l Ch tControl Chart

The Idea of QC Chart:

C t l Ch tControl Chart

Different chart are used depending on the nature ofthe charted data. Commonly used charts are:y

For continuous (variables) data: Shewhart sample mean (X bar – chart).S ew a t sa p e ea ( ba c a t). Shewhart sample range (R – chart). Shewhart sample (X – chart).Shewhart sample (X chart). Cumulative sum (CUSUM). Exponentially Weighted Moving Average Exponentially Weighted Moving Average

(EWMA) chart. Moving – average and range charts.Moving average and range charts.

C t l Ch tControl Chart

For Discrete (attributes and countable) data.

Sample proportion defective (p-Chart).

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

Sample number of defects (c-chart).( )

Sample number of defects per unit (u-chart or

c bar-chart).

X h tX-charts

One of the oldest and simplest types of controlchartchart.

It is based on the distribution of the controlvalues around a true or expected value.

It can be used to monitor the combination of It can be used to monitor the combination ofsystematic and random effects for control values,based on single results or on a mean of multipleanalyses.

X h tX-charts

Using reference material similar to a routinesamples as control sample the bias may besamples as control sample, the bias may bemonitored by comparing the mean control valueover time with reference value.

Special applications of the X chart. The blank value chart. Recovery chart

Calibration parameters such as slope andp pintercept, in so far they are determined daily, canalso be tested by means of the X chart.

R Ch tR - Chart

R chart serves repeatability control. The range is defined as the difference between thee a ge s de ed as t e d e e ce betwee t e

largest and smallest single result for two or moreseparate samples.

R chart applications in analytical laboratories R chart applications in analytical laboratoriesappears in duplicate determination (of samples to beanalyzed) in each analysis series.

Test samples selected among the samples to beanalyzed in each analytical run.

Since the range is normally proportional to sample Since the range is normally proportional to sampleconc. (at levels well above the detection limit), it willbe more appropriate to use a control chart where thecontrol value is the relative range r%control value is the relative range r%.

C t t C t l Ch tConstruct Control Charts

Select the quality characteristic. Develop a quality plan Develop a quality plan. Select the type of control chart. Choose the proper sub-group size. Choose the proper sub group size. Collect the data. Determine the trial control limits and chart mid-

point. Determine the revised control limits and chart

id i tmid-point. Construct the revised control chart. Continue to use the chart Continue to use the chart.

Daily Interpretation ofy pControl Chart

There are three possible cases:

1. The method is in control.

2. The method is in control but the long-termevaluation shows that the method is out ofevaluation shows that the method is out ofstatistical control.

3. The method is out of control.

T i l O t f C t l R lTypical Out – of – Control Rules

T i l O t f C t l R lTypical Out – of – Control Rules

T i l O t f C t l R lTypical Out – of – Control Rules

T i l O t f C t l R lTypical Out – of – Control Rules

T i l O t f C t l R lTypical Out – of – Control Rules

T i l O t f C t l R lTypical Out – of – Control Rules

T i l O t f C t l R lTypical Out – of – Control Rules

T i l O t f C t l R lTypical Out – of – Control Rules

Long-term evaluation ofgQuality Control data

Consider the following questions: What is the quality (random and systematic

effects) currently in the laboratory? Has thequality significantly changed?quality significantly changed?

Are control limits and central line in the controlchart still optimal fordetection situationsout of control?

R t O t f C t lResponses to Out-of-Control

Invoke the corrective action procedure.

Determine root cause.

Implement corrective action.

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

Repeat sample analyses.

How often should control limitsBe 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.line remain stable over a long period of time.

The central line and control limits should not be The central line and control limits should not be

changed frequently since this will make it

difficult to detect gradual changes in analytical

quality.q y

How often should control limitsBe evaluated

The laboratory should have a policy for how often

control limit are evaluated and how it is decided

if a change is needed.if a change is needed.

Control limits should not be changed based on Control limits should not be changed based on

less than 20 sets of new data since last

evaluation.

S QC SETTING UP A QC PROGRAM

P ti l i tPractical points

Method Validation – use information gainedfrom method validation as basis for routinefrom method validation as basis for routinequality control.

C f f Concentration range – if conc. of an analytevary considerably, use separate X-charts and R-charts for different conc levelscharts for different conc. levels.

R-chart with test samples – to monitorrepeatability using range charts (R-chart or r%-chart), analyzing a test sample in duplicate in

h l ti l i d deach analytical run is recommended.

P ti l i tPractical points

Frequency of control analyses – stability ofthe measurement system can have an influencethe measurement system can have an influenceon the frequency of control analyses.

O One control sample in each analytical run(general rule).

If there are errors caused by calibration drift,the number of control samples to be analyzedin each analytical run may need to be higherthan under very stable measurement

diticonditions.

P ti l i tPractical points

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

all measurements performed after the last

approved sample in the quality control may toapproved 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 analysesthe cost of repeating analyses.

P ti l i tPractical points

Position of control samples in an analytical

run – it is recommended that control samples or

checks are analyzed at least the beginning ofchecks 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 A good balance between QC and test

samples – QC fit for purpose.

Q lit C t lQuality Control

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

Quality control is NOT…. Equipment calibration. Equipment standardization (GC calibration). Equipment monitoring.q p g

Bli d S lBlind Samples

Single Blind Samples Proficiency Testing (PT) samples Proficiency Testing (PT) samples. Real – life samples (retested or purchased). Spiked samples prepared by quality department.p p p p y q y p

Double blind samples Submitted as “customer” samples by a “customer”.p ySamples 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.

PROFICIENCYPROFICIENCY

TTESTS

(PT)

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

Determination of laboratory testing performance bymeans of inter laboratory comparisons.y p

Note – for the purpose of this guide, the termlaboratory proficiency testing is taken in itswidest sense and includes, for example:

1. Qualitative Schemes – for example wherel b i i d id iflaboratories are required to identify acomponent of a test item.D t t f ti i f l2. Data transformation exercises – for examplewhere laboratories are furnished with sets ofdata and are required to manipulate the data toq pprovide further information.

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

3. Single item testing – where one item is sentto a number of laboratories sequentially andto a number of laboratories sequentially andreturned to the organizer at intervals.

4. One off exercises – where laboratories areprovided with a test item on a single occasion.

5. Continuous schemes – where laboratories areprovided with test items at regular intervals ona continuing basis.

6. Sampling – for example where individuals ororganizations are required to take samples forsubsequent analysissubsequent analysis.

P fi i T tiProficiency Testing

PT organizers distribute portions of a homogeneousPT organizers distribute portions of a homogeneous material to each of the participants

Participants analyze the material under t i l diti d t t th itypical conditions and report to the organizer

Organizer reports the results usually in the form of a score relating to the accuracy of the resulta score relating to the accuracy of the result

P fi i T tiProficiency Testing

A score of zero – implies a perfect result. This willhappen quite rarely even in perfectly competentpp q y p y plaboratory.

Laboratory complying with the PT scheme’s fitnessLaboratory complying with the PT scheme s fitnessfor purpose criterion will commonly producescores falling between -2 and +2. they mightexpect to produce a value somewhat outside thisexpect to produce a value somewhat outside thisrange occasionally, roughly about 1 time in 20. soan isolated event of this kind is not of greatmoment. The sign (i.e. + or -) of the scoreindicates a negative or positive error,respectivelyrespectively.

P fi i T tiProficiency Testing

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 isthe 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 andg

remedied.

MEASUREMENTUUNCERTAINTY

d fi itidefinition

“a parameter associated with the result of ameasurement that characterizes the dispersionmeasurement, that characterizes the dispersionof values that could reasonably be attributed tothe measure”. (ISO-VIM:1993)

The parameter may be σ or the width of confidenceinterval.

The number after ±.MU dose not imply doubt about the validity of a

measurement, on the contrary, knowledge ofuncertainty implies increased confidence in thevalidity of a measurement resultvalidity of a measurement result.

Wh d MUWho needs 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 tothe 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 requiredmeasurement and to improve to the required

quality.

Wh h ld th l b i MUWhy should the lab give MU

1. The customer needs it to make correct decisions.2 An estimation of the measurement uncertainty2. An estimation of the measurement uncertainty

is required in ISO 17025 (5.4.6)

U t i t SUncertainty Sources

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) Matrix effects (sample composition)

U t i t SUncertainty Sources

Contamination during sampling or samplepreparationpreparation.

Effect of environmental condition, measurementconditionsconditions.

Computational effects (software, calibrationmodels…))

Uncertainty of weighs and volumetric equipment. Instrument effects (stability, linearity…). Approximations and assumptions incorporated in the

test method. Uncertainties in analytical process Random effects.

y pQUAM :2000.1 (Appendix C)

E & U t i tErrors & Uncertainty

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 & U t i tErrors & Uncertainty

Random error (Type A):“result of measurement minus the mean that would

result from an infinite number of measurementsf th d i d t dof the same measurand carried out under

repeatability conditions”. (ISO-VIM:1993)Note1: random error is equal to error minusNote1: random error is equal to error minus

systematic error.Note2: because only a finite number ofNote2: because only a finite number of

measurements can be made, it is possible todetermine only an estimate of random error.y

E & U t i tErrors & Uncertainty

Systematic error (type B):“mean that would result from an infinite number of

measurements of the same measurand carriedt d t bilit diti i tout under repeatability conditions minus a true

value of the measurand”. (ISO-VIM:1993)Note1: systematic error is equal to error minus random errorNote1: systematic error is equal to error minus random error.Note2: the systematic error is independent of the number of

measurements made and can not therefore be reduced byincreasing the number of analyses under constantmeasurement conditions.

Note3: the result of a measurement should be corrected for allNote3: the result of a measurement should be corrected for allrecognized significant systematic effects.

E & U t i tErrors & Uncertainty

E & U t i tErrors & Uncertainty

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 truevalue has a known probability of being found (itvalue has a known probability of being found. (itcannot be applied as a correction)

The result of an analysis may be by chance close to the true value ofThe 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 h l h l i h lknow how close that result is to the true value

TOP – DOWN APPROACH

T D A hTop-Down Approach

Theoretical ‘Bottom Up’ approach recommended by the ISOGUM ‘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 youmay decide for yourself. (It’s a good idea to have somemay decide for yourself. (It s a good idea to have someknowledge of estimating MU from first principles)

This may not be the best way to go about estimating the MUassociated with results generated by a complex chemical testmethodmethod.

T D A hTop-Down Approach

The Bottom – Up Approach followed in estimation ofmeasurement uncertainty in chemical analysis have drawny yfierce criticism from chemists.

W Horwitz ‘The Certainty of Uncertainty’W. Horwitz, The Certainty of Uncertainty ,J. AOAC International, 86,109 (2003)

‘This absurd and budget-busting approach (forThis absurd and budget busting approach (foranalytical chemistry) arose from metrological chemiststaking over in entirety the concepts developed by

t l i t f h i l d ith 5 7metrologists for physical processes measured with 5-7significant figures ….and applying them to analyticalchemical measurements with 2-3 significant figures’g g

T D A hTop-Down Approach

W. Horwitz, ‘The Certainty of Uncertainty’,J. AOAC International, 86,109 (2003)J. AOAC International, 86,109 (2003)

‘This approach also ignores the fact that somechemical methods are influenced by numerousfactors, some positive and some negative, thattend to cancel out and that often other chemicaltend to cancel out….and that often other chemicalmethods are influenced by factors that overwhelmthe weight and volume uncertainties presented inthe weight and volume uncertainties presented inpublished examples’

T D A hTop-Down Approach

An Alternative Approach...

‘Top-down’ calculation/estimate using availablevalidation and QC data.Q

ACCURACY: Trueness (bias) and Precision

a ‘reasonable’ estimate of MU may be obtained byid i th t i ti i t d ithconsidering the uncertainties associated with

imprecision and bias

T D A hTop-Down Approach

1. Accuracy : Trueness (bias) and Precision

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

Bias)Bias)

2. Twice Reproducibility from inter-lab studies

3. Horwitz formula (Anticipated expanded MU = 2 XPRSD )PRSDR )

T D A hTop-Down Approach

When a stable control sample is covering the wholeanalytical process and has a matrix similar to theanalytical process and has a matrix similar to thesamples, the within-laboratory reproducibility at thatconcentration level can simply be estimated from theanalyses of the control samples.

If the analyses performed cover a wide range ofconcentration levels, also control samples of other

t ti l l h ld b dconcentration levels should be used.