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.

Good Laboratory P ti G dL b t Practice
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 based. b d

Processing & non-Regulated g g vs. Regulated areas

History of GLP Hi t f
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 f formulated the first OECD principles of GLP in order to avoid non tariff barriers to trade in chemicals to non-tariff chemicals, promote mutual acceptance of non-clinical safety test data, and to eliminate unnecessary , y duplication of experiments.

What i Wh t is 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 I d t i li d Countries Industrialized C t i
1 2 3 4 5 6 7 8 9 Australia Austria Belgium Canada Czech Republic Denmark Finland France Germany 11 Hungary 12 Iceland 13 Ireland 14 Italy 15 Japan 16 Korea 17 Luxembourg 18 Mexico 19 Netherlands 20 New Zealand 21 22 23 24 25 26 27 28 29 30 Poland Portugal Slovak Republic Spain Sweden Switzerland Turkey UK USA Norway

10 Greece

What i Wh t is OECD
  

OECD guidelines are NOT law. OECD standards are internationally accepted. First developed i 1978 using US FDA GLP Fi t d l d in i 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 including conduct management, conduct, reporting and archiving of laboratory studies.

What Wh t GLP is NOT i
  

GLP has nothing to do with efficacy. If a study does not involve animals, it is not GLP. If a l b claims t b GLP b t d lab l i to be GLP, but dose not run t animal studies, they are misguided.

 

GLP does not apply to analytical development. GLP dose not apply to animal field studies.

Applicability f A li bilit of GLP
Non-Clinical safety testing of test items contained in:
    

Pharmaceutical products. Ph ti l d t Pesticide products. p Veterinary drugs. Food additives. Feed additives additives.

Conclusion C 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) ) q quality y standards by y this terminology

GOOD LABORATORY PRACTICE

Introduction I t d ti
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

Introduction I t d ti
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 Practice (GLP) deals with: y ( ) 1. Organization. 2. Process. 3. Conditions under which laboratory studies are planned, performed, monitored, recorded and reported. reported

Introduction I t d ti

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, Hence training plans should include basic GLP knowledge for everybody working in a GLP environment.

Main Points f M i P i t of GLP

Resources: organization, personnel, facilities, equipment. equipment Rules: protocols, standard operating procedures, concept of th study di t as th pivotal point t f the t d director the i t l i t of study control. Characterization: test items, test systems. Documentation: raw data, final report, archives. , p , 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 procedures (SOPs). p gp ( ) Control and test articles. Equipment. E i

GLP Principles P i i l

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

A Quality Assurance Unit: audit the laboratory studies and the accompanying data It may be a separate data. 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 with correctly written and properly d ih l i d l filed, management approved SOPs. These must be readily available to the personnel concerned. They should cover policies, administration, technical operation, equipment operating and analytical methods. i d l i l 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 and expiration date conditions, date.

Equipment: instruments must be designed to meet analytical requirements and regularly maintained and calibrated.

GLP B fit Benefits

From the point of view of international trade: The ultimate goal in fair practice depends on: Reliability of analytical results b 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 fit Benefits

Why Reliable Analytical Results?

Reliable analytical results are essential for: y
 

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. The Th same standard f all ISO t d d for ll ISO. Different regulations in different countries. g 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 requirements for storage of p q g records and reports. Specific requirements for storage, retention p q g , 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.

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

Difference between GLP & ISO/IEC 17025

Analysis methods must be verified through inter-laboratory test (PT).

Validation through inter laboratory test not inter-laboratory required.

 

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

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

Codex Alimentarius C d Ali t i
 

Latin for “Food Code” or “Food Book”. Developed and maintained by the codex

alimentarius commission a body that was commission, established in 1963 by the Food and Agriculture Organization of United Nations (FAO) and the World Health Organization (WHO) (WHO).

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

Codex Alimentarius C d Ali t i

Some deal with detailed requirements related to a food or group of foods, others deal with the operation processes p protection. and or management the operation of of production government

regulatory systems for food safety and consumer

Codex Alimentarius C C d Ali t i Commission i i

It is intergovernmental standards-setting body established by FAO and WHO in 1961/63.

11th FAO Conference Resolution no 12/61 (codex no. alimentarius).

WHA 16.42 Joint AO/WHO programme on food standards (codex alimentarius).

174 member countries + 1 member organization (EC).

Codex Alimentarius C C d Ali t i Commission i i
 

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 and related texts have since 1995 become international benchmarks for harmonization under the SPS and TBT agreements of WTO.

Role f Codex Standards R l of C d St d d

For food safety, codex international benchmark benchmark.

standards

are

the

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

Codex Alimentarius C d Ali t i
 

Its scientific basis. Codex – Risk management.

Liaison & Separation S i

FAO/WHO Expert Bodies – Risk assessment p
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 procedures (SOPs). p gp ( ) Control and test articles. Equipment. E i

Main P i i l M i Principles

Good analytical practice may be considered in three inter related parts:
1. 2. 3.

Analyst. Basic resources. Analysis.

Case St d C Study
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

Case St d C Study
These details are summarized in: 1. 1 The Analyst: The analyst who undergoes residue analysis:  Should have appropriate professional qualification.  Should be experienced in the correct use of p apparatus 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 principles of residue analysis analysis.

Case St d C Study
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.

Case St d C Study
1. The Analyst: Continued…..
 The staff should spend some of their training

period in a well established (expert) laboratory where p g experienced advice and training is available.
 A record of training and experience must be kept

for f all laboratory staff. ff

Case St d C Study
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 for storage, sample preparation, for extraction and cleanup and for instrumentation used in the p determinative step.

Case St d C Study
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 receipt, storage and preparation p p, g p p should be handled in areas away from areas of residue analysis.  Ensure sample integrity.

Case St d C Study
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

Case St d C Study
A. The laboratory  Minimize the use of highly toxic solvents and reagents should whenever possible.

All waste solvent should b stored safely and l h ld be d f l 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 free” or “explosion proof” spark free explosion proof

Case St d C Study
A. The laboratory

A supply of safety tools should b available l f f l h ld be il bl such as safety glasses, gloves and other protective clothing clothing, emergency washing facilities and a spillage treatment kit. Appropriate and adequate equipment must be available. fire fighting

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

Case St d C 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 Chromatographic t hi equipment, i t balances, b l spectrophotometers etc.., must be serviced and calibrated regularly regularly.  Record of all servicing/repairs must be maintained for every item of equipment.

Case St d C Study
B. Equipment and Supplies

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

Case St d C 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 and acceptably hi h purity. d t bl high it Analytical standards should be available for all parent co o d the lab monitoring a a e t compounds, o ito i g as well as those metabolites that are included in MRLs.

Case St d C Study
B. Equipment and Supplies

All analytical standards, stock solutions and reagents should be properly labeled. g p p y

Preparation date, analyst initials, solvent used, storage conditions and expiry date. g p y

Compounds influenced be degradative processes (light heat ) must be clearly (light, heat,..) labeled and stored under appropriate conditions.

Case St d C Study
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.

Case St d C Study
3. Analysis  The methods applied for the determination of pesticide residue should satisfy the following criteria: it i
Conc. ≤ 1 µg/kg > 1 µg/kg ≤ 0.01 mg/kg > 0.01 mg/kg ≤ 0.1 mg/kg > 0.1 mg/kg ≤ 1 mg/kg > 1 mg/kg Repeatability 35 30 20 15 10 36 32 22 18 14 Reproducibility 53 45 32 23 16 54 46 34 25 19 Trueness % Rec (2) Rec. 50 – 120 60 – 120 70 – 120 70 – 110 70 – 110 CVA%(3) CVL%(4) CVA%(3) CVL%(4)

Case St d C Study
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 uncertainty. g y

Case St d C Study
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.

Case St d C Study
3. Analysis 4) CVL: Overall coefficient of variation of

laboratory results including up to 10% results, variability of residues between analytical portions (CVSp). where: h CVL2 = CVSp2 + CVA2

Case St d C Study
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.

Case St d C Study
A. Avoidance of contamination
 •

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

Case St d C Study
A. Avoidance of contamination

Polishes, barrier creams, soaps containing germicides, germicides cosmetics insect can give sprays, sprays rise perfumes to and 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 laboratory. l b

Case St d C 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.

Case St d C Study
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.

Case St d C Study
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.

Case St d C Study
A. Avoidance of contamination

The nature and importance of contamination can vary according to the type of determination t h i d t i ti technique used and th l d d the level of l f 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 determination is used, and p p vice versa.

Case St d C 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 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.

Case St d C 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 representative analytical portions without affecting the residues concentration levels. Fresh samples should be stored at 1-5oC away C, from direct sunlight, while frozen samples should be kept frozen and stored at -16oC. p

Case St d C Study
B. Reception & Storage of Samples

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

Case St d C Study
C. Standard Operating Procedures (SOPs)  SOPs Should be for all operations.  SOP SOPs quality should h ld control, contain t i safety full f ll working ki and

instructions, expected performance, internal precautions calculation of results results.  Any deviation from SOPs should be recorded.

Case St d C Study
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 intended i t d d purpose.  Produce the necessary validation data accordingly. accordingly

Case St d C Study
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 method to the analysis of routine samples. h h d h l i f i l The method to be validated could be in-house in house, from literature, or official method, adapted to match the capabilities of the lab and the purpose for which it will be used.

Case St d C Study
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 betweenlaboratory variances. The use of representative analytes or matrices is important in validating methods methods.

Case St d C Study
D. Validation of methods
Commodities are classified according Classification (CA volume2, 2nd ed.).
Commodity group Plant product I High water & chlorophyll content Leafy veg. Brassica Leafy veg. Legume veg. Spinach, Lettuce Broccoli, cabbage Green beans Apple, pear Peach, cherry P h h Strawberry Grape Tomato, pepper, melon Common properties ti Commodity class l

to

the

Codex

Representative species i

II

High water & low Pome fruits or no chlorophyll S hl h ll Stone fruits f i Berries content Small fruits Fruiting veg.

Case St d C Study
D. Validation of methods
Commodity group Plant product II High water & low or Root veg. no chlorophyll content High acid content High sugar content High oil or fat Oil seeds Nuts Citrus fruits Potato, carrot, parsley Orange, lemon Raisin, dates Avocado, Avocado sunflower seeds Pistachios, peanut Common properties Commodity class Representative species

III IV V

Case St d C Study
D. Validation of methods
Commodity C dit group Plant product VI Dry materials Wheat, rice or maize grains Cereal products Wheat flour Garlic, G li tea, spices i Cereals Common C properties Commodity C dit class Representative R t ti species

Commodities C di i requiring individual tests Animal-origin A i l i i products d t Meats Fats Milk Eggs

Cattle meat, chicken Fat of meat Cow milk Chicken egg

Case St d C Study
D. Validation of methods The selection of representative analytes should be made based on the purpose of analysis taking into i t account th f ll i t the following: 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, or for which critical decisions will g y be made based on the results.

Case St d C Study
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.

Validation f M th d V lid ti of Methods

These parameters may be summarized as:

Specificity

Extent to hi h E t t t which a method provides responses f th d id from 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.

Selectivity (S ifi it ) S l ti it (Specificity)
 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, matrix, etc….. impurities degradants matrix etc

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

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

Measures of Selectivity M f S l ti it
2.

Resolution (Rs) (AOAC requirements)

Rs is expressed as a function of both the absolute separation distance expressed as retention ti t ti times ( i t ) of th t (minutes) f the two peaks, t1 k and t2, and the baseline widths, W1 and W2, of the analyte and nearest peak also expressed in peak, terms of times, as Rs = 2(t2-t1)/(W1+W2) t W A resolution of at least 1.5 is usually sought and p 1.0 is the minimum usable separation.

Measures of Selectivity M f S l ti it

Validation f M th d V lid ti of Methods

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.  C lib ti Calibration R Range Could be combined with linearity, reproducibility and signal/noise experiments experiments.  Reporting Limit (LCL) The lowest calibrated level employed during analysis to detect residues.

Limit f D t ti Li it of Detection

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, detected but not necessarily quantitated under the stated conditions of the test. (NATA) The true net conc or amount of the analyte in the material conc. to be analyzed which will lead with probability (1-b), to the conclusion that the conc. of the analyte in the analyzed material i l t i l is larger th th t of th bl k matrix. (ISO/DIS) than that f the blank t i

Limit f D t ti Li it of Detection

Limit f D t ti Li it of Detection

Limit f Q Li it of Quantification tifi ti

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 conc. determined with acceptable precision (repeatability) and accuracy under the stated conditions of the test (NATA) test. it is also known as Limit of Reporting

Limit f Q Li it of Quantification tifi ti

Limit f Q Li it of Quantification tifi ti

LOD & LOQ

Lowest C lib ti Level L t Calibration L l

Reporting Li it R ti Limit

Validation f M th d V lid ti of Methods

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 p two parameters.

Performance Verification P f 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 control’. statistical control i.e. Accuracy & uncertainty of the method are similar to those during method validation.

Performance Verification P f 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 of samples are analyzed yp p y for a great no. of analytes.

Control Chart C t l Ch t

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

Control Chart C t l Ch t
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.

Control Chart C t l Ch t
A graphical plot of test results overtime. limits drawn are based in the statistical analysis (sigma or standard deviation, ….) of the plotted data. d t
Cont trol Value +3s ● ● +2s Mean -2s -3s Warning Limit

Control Limit

Control li i C l limits:  Upper and lower control = ± 3 s.d. Warning limits:  Upper and lower warning limits = ± 2 s.d.
● ● ● ● Time

Control Chart C t l Ch t
Why use control Charts.
 

Monitor process variation over time. Differentiate b t Diff ti t between special cause and common i l d cause variation.

 

Assess effectiveness of changes. Communicate process performance.

Control Chart C t l Ch t
The Idea of QC Chart:

Control Chart C t l Ch t
Different chart are used depending on the nature of the 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).  Cumulative sum (CUSUM).  Exponentially Weighted Moving Average (EWMA) chart.  Moving – average and range charts.

Control Chart C t l Ch t

For Discrete (attributes and countable) data.
   

Sample proportion defective (p-Chart). Sample number of d f ti S l b f defectives ( (np-chart). h t) Sample number of defects (c-chart). ( ) Sample number of defects per unit (u-chart or c bar-chart).

X-charts X h t

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-charts X h t
Using reference material similar to a routine samples as control sample the bias may be sample, 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 parameters such as slope and p p intercept, in so far they are determined daily, can also be tested by means of the X chart.

R - Ch t Chart
 

 

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

Construct Control Charts C t t C t l Ch t
Select the quality characteristic.  Develop a quality plan plan.  Select the type of control chart.  Choose the proper sub-group size. sub group  Collect the data.  Determine the trial control limits and chart midpoint.  Determine the revised control limits and chart mid-point. id i t  Construct the revised control chart.  Continue to use the chart chart.

Daily Interpretation of y p 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.

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

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

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

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

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

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

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

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

Long-term evaluation of g 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?

Responses t O t f C t l R to Out-of-Control
   

Invoke the corrective action procedure. Determine root cause. Implement corrective action. Repeat QC sample twice to d R l i demonstrate “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

Practical i t P ti l points

Method Validation – use information gained from method validation as basis for routine quality control. Concentration range – if conc. of an analyte C f f vary considerably, use separate X-charts and Rcharts for different conc levels conc. 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 analytical run i recommended. h l ti l is d d

Practical i t P ti l points

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

Practical i t P ti l 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 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.

Practical i t P ti l points

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.

Quality C t l Q lit 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

Blind S Bli d Samples l

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

Double blind samples
Submitted as “customer” samples by a “customer”. p y 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 id 43 1997 Guide 43:1997
Determination of laboratory testing performance by means of inter laboratory comparisons. y 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 laboratories are required to id if l b i i d identify a component of a test item. Data transformation exercises – f example D t t f ti i for l where laboratories are furnished with sets of q p data and are required to manipulate the data to provide further information.

2.

PT – ISO/IEC G id 43 1997 Guide 43:1997
3.

4.

5.

6.

Single item testing – where one item is sent to a number of laboratories sequentially and returned to the organizer at intervals. One off exercises – where laboratories are provided with a test item on a single occasion. Continuous schemes – where laboratories are provided with test items at regular intervals on a continuing basis. Sampling – for example where individuals or organizations are required to take samples for subsequent analysis analysis.

Proficiency Testing P fi i T ti
PT organizers distribute portions of a homogeneous material to each of the participants

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

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

Proficiency Testing P fi i T ti
A score of zero – implies a perfect result. This will happen quite rarely even in perfectly competent pp q y p y p laboratory. Laboratory complying with the PT scheme’s fitness scheme s 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

Proficiency Testing P fi i 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 and g remedied.

MEASUREMENT UNCERTAINTY

definition d fi iti
“a parameter associated with the result of a measurement, measurement that characterizes the dispersion 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.

Who Wh needs MU d
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.

Why h ld the lab i Wh should th l b give MU
1. 2. 2

The customer needs it to make correct decisions. An estimation of the measurement uncertainty is required in ISO 17025 (5.4.6)

Uncertainty Sources U t i t 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)

Uncertainty Sources U t i t 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 process y p Random effects. QUAM :2000.1 (Appendix C)

Errors & Uncertainty E U t i t
Error – “the result of measurement minus a true value of measurand”. (ISO-VIM:1993).

1. 2. 3.

Random errors. Systematic errors. Gross (spurious or blunder) errors.

Errors & Uncertainty E U t i t
Random error (Type A): “result of measurement minus the mean that would result from an infinite number of measurements of th same measurand carried out under f the d i d t 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 y determine only an estimate of random error.

Errors & Uncertainty E U t i t
Systematic error (type B): “mean that would result from an infinite number of measurements of the same measurand carried out under repeatability conditions minus a t t d t bilit diti i true 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.

Errors & Uncertainty E U t i t

Errors & Uncertainty E 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 (it found. 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 know how close that result is to the true value k h l h l i h l

TOP – DOWN APPROACH

Top-Down A T D 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’s a good idea to have some (It s 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

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

Top-Down A T D 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 that often other chemical out….and methods are influenced by factors that overwhelm the weight and volume uncertainties presented in published examples’

Top-Down A T D Approach h
An Alternative Approach...
‘Top-down’ calculation/estimate Q validation and QC data. using available

ACCURACY: Trueness (bias) and Precision
a ‘reasonable’ estimate of MU may be obtained by considering th id i the uncertainties associated with t i ti i t d ith imprecision and bias

Top-Down A T D Approach h
1. Accuracy : Trueness (bias) and Precision

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

Top-Down A T D Approach h

When a stable control sample analytical process and has a samples, the within-laboratory concentration level can simply analyses of the control samples.

is covering the whole matrix similar to the reproducibility at that be estimated from the

If the analyses performed cover a wide range of concentration levels, also control samples of other concentration l l should b used. t ti levels h ld be d

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