METHOD

VALIDATION
ON
CHEMICAL
TESTING
By: M. A. Mercado
30 Sept – 01 Oct. 2019

OBJECTIVES
 To learn the basic concepts and processes on
method validation.
 To understand and guide the chemists/analysts
establish single-laboratory method validation
procedure.

COURSE OUTLINE

I. Introduction to method validation and

method verification
II. Performance characteristics
III. Performing method validation
IV. Fitness for purpose

1
WHY CONDUCT METHOD VALIDATION?
 To ensure that the method is fit for use.
 Provides greater confidence in the laboratory’s
results.
 Provides a solid knowledge and experience of the
practical details of performing the method,
including awareness of any critical steps in the
process.
 Identifies and quantifies sources of potential
errors

METHOD VALIDATION AND

METHOD VERIFICATION

 Validation: verification, where the

specified requirements are adequate for
an intended use. (ISO/IEC 17025:2017)
 Verification: provision of objective
evidence that a given item fulfils specified
requirements. (ISO/IEC 17025:2017)

METHOD VALIDATION AND

METHOD VERIFICATION
Method Validation
Non-standard methods
Laboratory-developed/designed
methods
Standard methods outside the
intended scope
Modifications of standard
methods
Method Verification
To confirm that the laboratory
can properly operate standard
methods before introducing the
tests or calibration
When there is an important
change such as a new but similar
instrument, relocation of
equipment etc.
When quality control indicates
that the performance of an
established method is changing
with time

2
FULL VALIDATION AND
SINGLE-LABORATORY VALIDATION
 Full validation / Collaborative Study:
- an examination of the characteristics of the
method in an interlaboratory study.
 Single-laboratory validation:

- a method performed in a single laboratory by a

single analyst

METHOD VALIDATION PROCESS

 Scope and extent of the validation

 Purpose
 Performance criteria

 Method selection

 Conduct Validation

 Evaluation and Conclusion

METHOD VALIDATION PROCESS

 Scope and extent of the validation

- Balance between costs, risks and technical
possibilities
- Define the necessary possible method
performance parameters
- Define when to conduct revalidation

3
METHOD VALIDATION PROCESS
 Extent of the validation work:
Example for pharmaceutical
Type of analytical application
Performance Quantitative Quantification
characteristics Identification test for Limit test for of main
test impurity impurity component
Selectivity √ √ √ √
Limit of detection √
Limit of quantification √
Working range
including linearity √ √
Trueness (bias) √ √
Precision (repeatability
and intermediate
precision) √ √

METHOD VALIDATION PROCESS

 Purpose of the validation:

- Key activity in chemical analysis and
indispensable for obtaining reliable results.
- The higher the complexity of the method, the
more important and voluminous, as a rule, is
validation.
- Important to demonstrate that methods are
working as expected (validation) and the
obtained results are reliable.

PERFORMANCE CHARACTERISTICS

 Performance characteristics commonly evaluated during

method validation
 Selectivity and specificity
 Limit of detection (LOD) and limit of quantification (LOQ)
 Working range
 Trueness (bias, recovery)
 Precision (repeatability, intermediate precision and
reproducibility)
 Ruggedness (robustness)
 Measurement uncertainty (not a performance
characteristic of a particular measurement procedure but a
property of the results obtained using that measurement
procedure).

4
PERFORMANCE CHARACTERISTICS:
SELECTIVITY AND SPECIFICITY
 Selectivity: the degree to which a method can
quantify the analyte accurately in the presence of
interferences.
 Ability to measure the analyte of interest in
samples to which specific interferences have been
deliberately introduced (those likely to be present
in samples) and likely, on chemical principles, to
respond to the test.

PERFORMANCE CHARACTERISTICS:
SELECTIVITY/SPECIFICITY
 Specificity: the ability of the method to measure
analyte in the presence of components which may
be expected to be present.
 Often used interchangeably with “selectivity”; the
term “selectivity” is often preferred than
“specificity”
 Specificity can be considered as “100%
selectivity”
 Should be conducted during the validation of
identification tests, determination of impurities
and assay.

PERFORMANCE CHARACTERISTICS: LIMIT OF

DETECTION AND LIMIT OF QUANTIFICATION

 Limit of detection (LOD):

The lowest concentration of analyte in the
test sample that can be detected or that can
be reliably distinguished from zero.
 Limit of Quantification (LOQ):

The minimum concentration of analyte in

the test sample that can be quantified with
acceptable precision.

5
PERFORMANCE CHARACTERISTICS: LIMIT OF
DETECTION AND LIMIT OF QUANTIFICATION

 Number of determinations/observations:
- The number of replicates (n) should be sufficient
to obtain an adequate estimate of the standard
deviation.
- Typically between 6 and 15 replicates are
considered necessary; 10 replicates are often
recommended in validation procedures/protocols.

PERFORMANCE CHARACTERISTICS:
LIMIT OF DETECTION
 Determination of Limit of Detection (LOD):
1. Analysis of samples with known concentrations
of analyte and by establishing the minimum level
at which the analyte can be reliably detected.
* May be used for non-instrumental methods
but may also be used with instrumental
methods.

PERFORMANCE CHARACTERISTICS:
LIMIT OF DETECTION
 Determination of Limit of Detection (LOD):
2. Based on signal-to-noise ratio
- Performed by comparing measured signals from
samples with known low concentrations of
analyte with those of blank samples and
establishing the minimum concentration at
which the analyte can be reliably detected.
- A signal-to-noise ratio between 3 or 2:1 is
generally considered acceptable for estimating
the detection limit.
*Can only be applied to analytical procedures
which exhibit baseline noise.

6
PERFORMANCE CHARACTERISTICS:
LIMIT OF DETECTION
 Determination of Limit of Detection (LOD):
3. Based on the Standard Deviation of the
Response and the Slope
 Standard Deviation of the Blank

 Calibration Curve

 may be expressed as:

DL = 3.3σ divided by S
where σ = the standard deviation of the response
S = the slope of the calibration curve

PERFORMANCE CHARACTERISTICS:
LIMIT OF DETECTION
 Determination of Limit of detection (LOD):
3a. Standard Deviation of the Blank:
measurement of the magnitude of analytical
background response is performed by
analyzing an appropriate number of blank
samples and calculating the standard
deviation of these responses.

PERFORMANCE CHARACTERISTICS:
LIMIT OF DETECTION
 Determination of Limit of Detection (LOD):
3b. Based on the Calibration Curve:
a specific calibration curve should be studied
using samples containing an analyte in the
range of DL. The residual standard deviation
of a regression line or the standard deviation
of y-intercepts of regression lines may be
used as the standard deviation.

7
PERFORMANCE CHARACTERISTICS:
LIMIT OF DETECTION
Determination of Limit of Detection (LOD):
Example:
Trial No. Blank, mg/L
1 0.05
2 0.05
3 0.1
4 0.05
5 0.1
6 0.05
7 0.05
mean 0.064
Std dev 0.024
(t ) student coefficient
Note: t = at 95%
df= 6 1.943
confidence level
LOD =
mean + t (std dev) 0.112 mg/L

PERFORMANCE CHARACTERISTICS:
LIMIT OF DETECTION
Determination of Limit of Detection (LOD):
Example: Abs Conc, mg/L
0.002 0.01
0.0039 0.02
0.0058 0.03
0.0082 0.04
0.014 0.05

Coefficients Standard Error

Intercept -0.00171 0.001502764
X Variable 1 0.283 0.045310043

LOD = 3 x std dev / slope

= 3 (0.001502764) / 0.283
= 0.018 mg/L

PERFORMANCE CHARACTERISTICS:
LIMIT OF QUANTIFICATION
 Determination of Limit of Quantification (LOQ):
1. Analysis of samples with known concentrations
of analyte and by establishing the minimum level
at which the analyte can be quantified with
acceptable accuracy and precision.
* May be used for non-instrumental methods
but may also be used with instrumental
methods

8
PERFORMANCE CHARACTERISTICS:
LIMIT OF QUANTIFICATION
 Determination of Limit of Quantification (LOQ):
2. Based on signal-to-noise ratio
- Performed by comparing measured signals from
samples with known low concentrations of
analyte with those of blank samples and
establishing the minimum concentration at
which the analyte can be reliably quantified.
- A signal-to-noise ratio between 10:1.

*Can only be applied to analytical procedures

which exhibit baseline noise.

PERFORMANCE CHARACTERISTICS:
LIMIT OF QUANTIFICATION
 Determination of Limit of Quantification (LOQ):
3. Based on the Standard Deviation of the
Response and the Slope
 Standard Deviation of the Blank

 Calibration Curve
 may be expressed as:

DL = 10σ divided by S
where σ = the standard deviation of the response
S = the slope of the calibration curve

PERFORMANCE CHARACTERISTICS:
LIMIT OF QUANTIFICATION
 Determination of Limit of Quantification (LOQ):
3a. Standard Deviation of the Blank:
measurement of the magnitude of analytical
background response is performed by
analyzing an appropriate number of blank
samples and calculating the standard
deviation of these responses.

9
PERFORMANCE CHARACTERISTICS:
LIMIT OF QUANTIFICATION
 Determination of Limit of Quantification (LOQ):
3b. Based on the Calibration Curve:
a specific calibration curve should be studied
using samples containing an analyte in the
range of QL. The residual standard deviation
of a regression line or the standard deviation
of y-intercepts of regression lines may be
used as the standard deviation.

PERFORMANCE CHARACTERISTICS:
LIMIT OF QUANTIFICATION
Determination of Limit of Quantification (LOQ):
Example:
Trial No. Blank, mg/L
1 0.05
2 0.05
3 0.1
4 0.05
5 0.1
6 0.05
7 0.05
mean 0.064
Std dev 0.024

LOQ = mean + 10 x std dev

= 0.064 + (10 x 0.024)
= 0.308 mg/L

PERFORMANCE CHARACTERISTICS:
LIMIT OF QUANTIFICATION
Determination of Limit of Quantification (LOQ):
Example: Abs Conc, mg/L
0.002 0.01
0.0039 0.02
0.0058 0.03
0.0082 0.04
0.014 0.05

Coefficients Standard Error

Intercept -0.00171 0.001502764
X Variable 1 0.283 0.045310043

LOQ = 10 x std dev / slope

= 10 (0.001502764) / 0.283
= 0.053 mg/L

10
METHOD DETECTION LEVEL
(MDL - BASED ON SMEWW)
 Determine the method detection level (MDL) for
each analyte of interest and method to be used
before data from any samples are reported.
 As a starting point, use an estimate of five times
the estimated detection limit.
 If calculated MDL is not within a factor of l0 of the
value for the known addition, repeat at a more
suitable concentration.
 Conduct MDL determinations at least annually (or
other specified frequency) for each analyte and
method in use at the laboratory.

METHOD DETECTION LEVEL

(MDL - BASED ON SMEWW)
 Perform MDL determinations over a period of at
least 3 days for each part of the procedure.
 Calculate recoveries for MDL samples. Recoveries
should be between 50 and 150% and %RSD values
≤ 20% or repeat the MDL determination.
 Reporting:
- Results below the MDL as ‘‘not detected.’’
- Results between the MDL and MQL with
qualification for quantitation.
- Report results above the MQL with a value and its
associated error.

METHOD DETECTION LEVEL

(MDL - BASED ON SMEWW)
 Perform MDL determination – Example:
Day and Tri al Estimated DL =
No. 0.10 ppm;
spiked bl ank
wi th 0.50ppm
Day 1, trial 1 0.30
Day 1, trial 2 0.35
Day 2, trial 1 0.30
Day 2, trial 2 0.30
Day 3, trial 1 0.35
Day 3, trial 2 0.40
Day 3, trial 3 0.35
mean 0.336
Std dev 0.038
(t ) student
coeffi cient df=
6, 99% conf.
l evel 3.14 MDL = 0.12ppm

11
 PERFORMANCE CHARACTERISTICS:
LINEARITY

 Linearity: the ability of the method, within a

certain range, to provide an instrumental
response or test results proportional to the
quantity of analyte to be determined in the test
sample.
 Should be evaluated across the range of the
analytical procedure.
 A minimum of 5 concentrations is recommended.

PERFORMANCE CHARACTERISTICS:
LINEARITY

 Evaluation of linearity:
1. By visual inspection of a plot of signals as a
function of analyte concentration or content.
2. If there is a linear relationship, test results
should be evaluated by appropriate statistical
methods: example: by calculation of a regression
line by the method of least squares.
Note: In some cases, to obtain linearity between
assays and sample concentrations, the test data
may need to be subjected to a mathematical
transformation prior to the regression analysis.

PERFORMANCE CHARACTERISTICS:
LINEARITY
 Evaluation of linearity: by visual inspection of a
plot of signals
0.35

0.3

0.35 0.25

0.3 0.2
Abs
0.25 0.15

0.2 0.1
Abs
0.15 0.05
0.1 0
0.05 0.00 0.20 0.40 0.60 0.80 1.00 1.20
Conc, mg/L
0
0.00 0.20 0.40 0.60 0.80 1.00 1.20
Conc, mg/L

12
 PERFORMANCE CHARACTERISTICS:
LINEARITY

 Evaluation of linearity: by regression analysis

0.35
0.3 y = 0.3073x + 0.0097
R² = 0.9696
0.25
0.2
Abs
0.15
0.1
Linearity 0.05
0.35 0
0.3 y = 0.3067x + 7E-05 0.00 0.20 0.40 0.60 0.80 1.00 1.20
R² = 0.9993 Conc, mg/L
0.25

0.2
Abs
0.15

0.1

0.05
0
0.00 0.20 0.40 0.60 0.80 1.00 1.20
Conc, mg/L

PERFORMANCE CHARACTERISTICS:
WORKING RANGE

 Working range: should be stated in the scope of the

validation study.
- Defined as the interval over which the method
provides results with an acceptable uncertainty.
- The lower end of the working range is bounded by the
limit of quantification (LOQ).
- The upper end of the working range is defined by
concentrations at which significant anomalies in the
analytical sensitivity are observed
Calibration range: the interval between the upper and
lower concentration of analyte which can be determined
with demonstrated precision, accuracy and response
function.

PERFORMANCE CHARACTERISTICS:
WORKING RANGE

 Assessment of working range:

- Consider both the method linearity and the
proposed calibration procedure given in the
method.
- The initial assessment of the working range is by
a visual inspection of the response curve.
- The next step is to confirm the relationship
between concentration and instrument response
by examining the regression statistics and
residual plot for the chosen model (e.g. linear,
quadratic).

13
PERFORMANCE CHARACTERISTICS:
TRUENESS
 Trueness:
- Degree of agreement of the mean value from a
series of measurements with the reference value
or true value.
- Normally expressed quantitatively as bias.

- Three (3) general approaches:

1. analysis of reference materials

2. recovery experiments using spiked samples, and

3. comparison with results obtained with another

method.

PERFORMANCE CHARACTERISTICS:
TRUENESS
 Bias: can be expressed simply as:

bias = mean – reference value

 % Recovery:

mean - ref. value

% bias = ----------------------------- x 100
ref value

PERFORMANCE CHARACTERISTICS:
TRUENESS

 % Spike Recovery:

Mean Spiked result – No spiked result

% Spike recovery = ---------------------------------- x 100

added concentration

14
PERFORMANCE CHARACTERISTICS:
TRUENESS
 Example: Expected recovery as a function of analyte
concentration (from AOAC)

PERFORMANCE CHARACTERISTICS:
PRECISION
 Precision:
- Measure of how close results are to one another.
- Usually expressed as standard deviation.
- Three (3) quantitative determination:

1. repeatability

2. intermediate precision, and

3. reproducibility

PERFORMANCE CHARACTERISTICS:
PRECISION
 Repeatability: measurements performed by a
single analyst using the same equipment over a
short timescale.
 Intermediate precision: measurements are made
in a single laboratory but under conditions that
are more variable than repeatability conditions,
(e.g. different analysts, extended timescale,
different set of equipment).
 Reproducibility: measurements are made with
the same test methods on identical test items in
different test facilities with different analysts
and different equipment.

15
PERFORMANCE CHARACTERISTICS:
PRECISION
 Evaluating precision results:
- Requires sufficient replicate measurements to be
made on suitable materials.
- The materials should be representative of test
samples in terms of matrix and analyte
concentration, homogeneity and stability.
- The replicates should also be independent, i.e.
the entire measurement process, including any
sample preparation steps, should be repeated.

PERFORMANCE CHARACTERISTICS:
PRECISION
 Evaluating precision results:
- The minimum number of replicates specified
varies with different protocols, but is typically
between 6 and 15 for each material used in the
study (Eurachem)
- a minimum of 9 determinations covering the
specified range for the procedure; e.g., 3
concentrations/3 replicates each. (ICH)
- a minimum of 6 determinations at 100% of the
test concentration (ICH)

PERFORMANCE CHARACTERISTICS:
PRECISION
 Example: Expected precision (repeatability) as a
function of analyte concentration (from AOAC)

16
PERFORMANCE CHARACTERISTICS:
PRECISION
 Example: Predicted relative standard deviation of
reproducibility (from AOAC)

PERFORMANCE CHARACTERISTICS:
RUGGEDNESS/ROBUSTNESS
 Ruggedness/robustness:
- A measure of its capacity to remain unaffected by
small, but deliberate variations in method
parameters.
- Provides an indication of the method’s reliability
during normal usage.
- The evaluation of robustness should be
considered during the development phase and
depends on the type of procedure under study.

PERFORMANCE CHARACTERISTICS:
RUGGEDNESS/ROBUSTNESS
 Ruggedness/robustness:
- Tested by deliberately introducing small changes
in the procedure and examining the effects in the
results
- Examples of typical variations: concentration of
reagent; pH of solution, temperature of reaction,
extraction time, flow rate, different columns.

17
PERFORMANCE CHARACTERISTICS:
RUGGEDNESS/ROBUSTNESS
 Evaluation of Ruggedness/robustness:
1. Using “F-test”: comparing 2 precision (SD) with
the hypothesis are not significantly different.
2. Using ANOVA (analysis of variance): analyze
the differences among group means in a sample.
 Ruggedness study is in most cases not necessary
at the single-laboratory level.

FITNESS FOR PURPOSE

 The extent to which the performance of the
method matches the criteria, agreed between the
analyst (or the laboratory) and the end-user of
the data.
 Conclusion of the validation study should state
that the method used is fit for the intended use.
 Measurement uncertainty: A parameter linked to
the measurement result and characterising the
spread of the values that can reasonably be
added to the measurement magnitude.

PERFORM METHOD VALIDATION

 Get a copy of the method to be validated and
study all the requirements.
 Define the application, scope and purpose of the
validation.
 Define performance parameters/characteristics
and acceptance criteria.
 Perform pre-validation test (where appropriate)

 Perform full validation or partial validation.

 Evaluate results and prepare validation report

 Develop SOP for executing the method in routine

analysis

18
REFERENCES / REGULATION AGENCIES /
INDUSTRIAL COMMITTEES

1. AOAC
2. United states Pharmacopeia
3. US FDA
4. ICH (International Conference on
Harmonization)
5. US EPA
6. Codex Alimentarius
7. Eurachem Method Validation Guide
8. IUPAC

QUALITY ASSURANCE
AND
QUALITY CONTROL
IN
CHEMICAL
LABORATORY

OBJECTIVES

 To understand the importance of quality

assurance and quality control in the
laboratory.
 To be able to apply the quality control
tools in the daily lab activities.

19
COURSE OUTLINE

I. Introduction to quality assurance and quality

control
II. QA programs
III. QC procedures
IV. Evaluation of QC data

QUALITY CONCEPT
 What is Quality?
 Conformance to requirements / specifications
 Fitness to purpose
 Getting it right, first time, every time
 Customer Satisfaction

QUALITY CONCEPT

20
QUALITY ASSURANCE VS QUALITY CONTROL

Quality assurance Quality control

 Part of quality  Part of quality

management focused
on providing
confidence that
quality requirements
will be fulfilled.
management focused
on fulfilling quality
requirements

QUALITY ASSURANCE VS QUALITY CONTROL

Quality assurance Quality control

 All the planned and  The operational

systematic activities techniques and
implemented within the activities used to
quality system that can fulfill requirements
be demonstrated to
for quality."
provide confidence that
a product or service will
fulfill requirements for
quality."

QUALITY ASSURANCE VS QUALITY CONTROL

IN THE LABORATORY

Quality assurance Quality control

 Plans and programs  Processes used to

in order to continually monitor and evaluate
monitor the reliability the validity of the test
of the reported test results.
results.

21
QUALITY ASSURANCE PROGRAMS
 Should be able to accomplish
the following:
 Establish Standard Operating
Procedures (SOP) for each step
of the laboratory testing process
starting from sample handling
to reporting of results.
 Define administrative
requirements: mandatory
recordkeeping, data evaluation,
and internal audits to monitor
adherence to SOPs.

QUALITY ASSURANCE PROGRAMS

 Specify corrective actions,

documentation, and the
persons responsible for
carrying out corrective actions
when problems are identified.
 Sustain high quality
performance.

QUALITY ASSURANCE PROGRAMS

 Planning lab activities
 Good Laboratory Practices (GLP)
 6 “S” Concept
 LEAN Techniques
 Audit and inspection

22
GOOD LABORATORY PRACTICES (GLP)
 GLP should not only be confused
with standards for laboratory safety
- appropriate gloves, glasses &
clothing to handle lab materials
safely.
 GLP is a quality system concerned
with the organizational process and
the conditions under which
laboratory study / test / experiments
are planned, performed, monitored,
recorded, archived and reported.

GOOD LABORATORY PRACTICES (GLP)

 Why do we need GLP?
 To ensure the generation of high quality and
reliable test data related to the safety of
industrial chemical substances and
preparations in the framework of harmonising
testing procedures for the Mutual Acceptance
of Data (MAD).
 To limit waste in doing the laboratory testing

GOOD LABORATORY PRACTICES (GLP)

 Fundamental points:
 Resources - personnel, facilities & equipment
 Characterization - test item and test system
 Rules for performing lab activities – study plan
or protocol, standard operating procedures
(SOPs)
 Results - raw data and data collection, final
report and archiving
 Quality Assurance Unit – monitoring the
process

23
5 “S” CONCEPT
 5 “S”: a process for creating and maintaining
an organized, clean, and high performance
work place, which serves as a foundation for
continuous improvement activities
1. Sort Out (Seiri) - housekeep
2. Set in Order (Seiton) - organize

3. Shine (Seiso) – clean up

4. Standardize (Seiketsu) – keep clean

5. Sustain (Shitsuke) - discipline

Plus Safety: throughout the entire process

safety is number 1.

LEAN TECHNIQUES
 LEAN: a manufacturing/production system
best characterized as relentlessly eliminating
waste from all of its activities and operations.
 Principles of LEAN:
 Identify the value

 Map the value stream

 Create the flow

 Establish pull
 Seek perfection

AUDIT AND INSPECTION

 Conduct of internal audit
- Acknowledge findings (deviations), conduct
thorough root cause analysis and appropriate
corrective actions.
- Identify risk and opportunities that may arise
from the internal audit.

24
AUDIT AND INSPECTION
 Conduct of inspection
- Inspection: an examination of a
product, process, service, or
installation or their design and
determination of its conformity with
specific requirements or, on the basis
of professional judgment, with general
requirements (ISO/IEC 17020
definition).
- Regular inspection of the facility and
all the resources related to laboratory
activities.
- Use of checklist as a good tool when
conducting inspection.

QUALITY CONTROL PROCEDURES

 Homogeneity of test item

 Purity of reagents
 Type of glassware
 Preparation of reagents and standard solutions
 Equipment / instrument selection and
calibration
 Intermediate checks

QUALITY CONTROL PROCEDURES

 Precision and accuracy checks

 Use of control charts
 Participation to Proficiency testing or
interlaboratory comparison tests
 Intralaboratory comparison tests
 Use of retained test items and testing of blind
samples

25
HOMOGENEITY OF THE TEST ITEM
 The laboratory sample is the material received by
the laboratory and usually must be reduced in
bulk and fineness to an analytical sample from
which the test portions are removed for analysis.
 Conduct homogeneity test as necessary.

 Ensure that the selected test portion is the best

representative of the whole batch.

PURITY OF THE REAGENTS

 Water purity
 defined in many different ways, but the generally
accepted definition states that high-purity water
is water that has been distilled or deionized, or
both, so that it will have a specific resistance of
500,000Ω or greater (or a conductivity less than
2.0mho/cm).
 refer to “Standard for the Examination of Water
and Wastewater” on the requirement for water
purity.

PURITY OF THE REAGENTS

 Water purity
Degree of Maximum Approximate
purity conductivity, concentration
(umho/cm) of electrolyte,
mg/L
Pure 10 2–5
Very pure 1 0.2 – 0.5
Ultrapure 0.1 0.01 - 0.02
Theoretically .055 0
pure

26
PURITY OF THE REAGENTS
 Grades of Chemicals:
- Reagent grade A.C.S.
- Guaranteed Reagent (GR)
- AR
- Primary standard
- USP grade
- Technical grade

TYPE OF GLASSWARE
 Laboratory vessels serve three functions: storage
of reagents, measurement of solution volumes,
and confinement of reactions.
 Depending on the manufacturer, various trade
names are used for specific brands possessing
special properties such as resistance to heat,
shock, and alkalies. Example: Kimax- or Pyrex-
brand glass is a relatively inert all-purpose
borosilicate glass

TYPE OF GLASSWARE
 Volumetric glassware: accurately calibrated
glassware for precise measurements of volume.
 This group includes volumetric flasks, volumetric
pipets, and accurately calibrated burets.
 Volumetric apparatus is calibrated to contain or
to deliver a definite volume of liquid.
 Solutions must be measured at the temperature
at which the apparatus was calibrated.
 Consider frequency of calibration.

 Read calibration certificate and note for any

corrections and MU.

27
PREPARATION OF REAGENTS AND
STANDARD SOLUTIONS

 Sourcing (COA, lot numbers, expiry date)

 Storage
 Interferences and potential problems
 Records

 Proper labeling

INSTRUMENT / EQUIPMENT SELECTION

 Understanding of instrument design

 Degree of accuracy
 Performance checks
 IQ (instrument qualification); OQ (operational
qualification); PQ (performance qualification)

INTERMEDIATE CHECKS

 Perform intermediate checks to maintain

confidence in the calibration status of measuring
and test equipment.
 Not a substitute for calibration but may provide
justification for the extension of calibration
intervals, where results are favorable.
 Maintain records and consider the uncertainty of
measurement when confirming if the calibration
status continues to satisfy the requirements for
the test or measurement.

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VALIDITY CHECKING
 Calibration check
- For calibration curve, standard solutions should
be analysed within the required range of
concentration.
- The ideal calibration curve is linear within its
most useful range, with a regression coefficient of
0.99 or greater.
- The standard curve is then verified by the
analysis of a midpoint standard concentration.

VALIDITY CHECKING
 Use of blank
- Method blank: a sample containing all
components except analyte, and it is taken
through all steps of the analytical procedure.
Processed simultaneously with and under the
same conditions as samples containing an
analyte of interest through all steps of the
analytical procedure.
- Run in order to evaluate the system for high
biased readings of background contamination.

VALIDITY CHECKING
 Use of blank
- Field blank: reagent water that has been bottled
in the laboratory, shipped with sample bottles to
the sampling site, processed and preserved as a
routine sample and returned with the routine
samples to the laboratory for analysis.
Purpose: assess contamination from field
conditions during sampling.

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VALIDITY CHECKING
 Use of blank
- Trip blank: a clean sample of a matrix that is
taken from the laboratory to the sampling site
and transported back to the laboratory without
having been exposed to sampling procedures.
Typically, analyze only for volatile compounds.
Purpose: assess contamination introduced during
shipping and field handling procedures.

VALIDITY CHECKING
 Recovery check
- A sample spiked with a known amount of the
variable should be tested in each batch and the
closeness of fit to the expected value calculated.
- In some cases this procedure also provides a
check on accuracy but, in assays where a variable
is extracted from the original matrix (such as in
many sample clean-up procedure), it can be used
to monitor the extraction step.

PRECISION AND ACCURACY CHECKS

 Replicate testing
- Tool to check precision.
- Since the samples are analysed using the same
method, equipment and reagents, the same bias
should affect all results.
- Evaluation of results varies and may depend on
test method/reference.

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PRECISION AND ACCURACY CHECKS

 Precision control using pooled reference material

- The reference material is normally prepared by
taking previously analysed samples with known
concentrations of the analyte under test, mixing
them and aliquoting the resultant pool.
- The aliquots are then stored in readiness for
analysis. A small sample of the aliquots is
analysed to determine the mean concentration of
the analyte, and the standard deviation and the
coefficient of variance at that concentration level.

PRECISION AND ACCURACY CHECKS

 Precision control using pooled reference material

- This has the advantage of providing some
monitoring of accuracy but a viable control only if
the material to be used will be stable in storage
for a sufficiently long period of time.
- This requires that the original pool materials
must have been prepared during method
validation, and that new materials must be
prepared before the old ones are finished.

PRECISION AND ACCURACY CHECKS

 Accuracy control using certified reference

material
- Certified reference materials (CRMs) are matrix-
matched materials with assigned target values
and assigned ranges for each variable, reliably
determined from data obtained by repeated
analysis.
- Since CRMs are prepared and checked under
carefully controlled conditions, they are costly to
produce and expensive to purchase.
- More cost effective to use CRM for the periodic
checking of accuracy, in combination with a
rigorous IQC programme.

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USE OF CONTROL CHARTS
 Control charts: monitors variance in a process
over time and alerts the business to unexpected
variance which may cause defects.
 Principle of control charts is that internal quality
control data can be graphically plotted so that
they can be readily compared and interpreted
 Criteria for evaluation: references; test method;
Westgard rule

USE OF CONTROL CHARTS

 Levey-Jennings chart:
- graph that quality control data is plotted on to
give a visual indication whether a laboratory test
is working.
- a mark is made indicating how far away the
actual result was from the mean (which is the
expected value for the control).
- lines run across the graph at the mean, as well as
one, two and three standard deviations to either
side of the mean.

USE OF CONTROL CHARTS

 Levey-Jennings chart:
(a) select appropriate control materials.
(b) analyze those materials to characterize method
performance by collecting a minimum of 20
measurements over at least 10 days.
(c) calculate the mean and standard deviation of
those data.
(d) Select the number of control measurements to
be used per run and
(e) select the control rules to be applied.

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USE OF CONTROL CHARTS
 Chart analysis:
(a) Control limit—If one measurement exceeds a
CL, repeat the analysis immediately. If the
repeat measurement is within the CL, continue
analyses; if it exceeds the CL, discontinue
analyses and correct the problem.

USE OF CONTROL CHARTS

 Chart analysis:
b) Warning limit—If two out of three successive
points exceed a WL, analyze another sample. If the
next point is within the WL, continue analyses; if
the next point exceeds the WL, evaluate potential
bias and correct the problem.

USE OF CONTROL CHARTS

 Chart analysis:
c) Standard deviation—If four out of five successive
points exceed 1s, or are in decreasing or increasing
order, analyze another sample. If the next point is
less than 1s, or changes the order, continue
analyses; otherwise, discontinue analyses and
correct the problem.

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USE OF CONTROL CHARTS
 Chart analysis:
c) Trending—If seven successive samples are on
the same side of the central line, discontinue
analyses and correct the problem

PROFICIENCY TESTING (PT) INTERLABORATORY

COMPARISON TEST (ICT)

 PT: evaluation of participant performance

against pre-established criteria by means of
interlaboratory comparisons (ISO 17043).
 Determines the performance of individual
laboratories for specific tests or measurements.
 Commonly used to monitor laboratories’
continuing performance.

PROFICIENCY TESTING (PT) AND

INTERLABORATORY COMPARISON TEST (ICT)

 PT / interlab comparison test process

 A coordinating body sends a test item or artifact
to a reference laboratory for testing.
 Coordinating body sends the item to each
participating laboratory for subsequent testing.
 Each participant laboratory will independently
test the item, submit their results to the
coordinating body.
 Coordinating body will evaluate the all the test
results and issue a performance report to each
participating laboratory.

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PROFICIENCY TESTING (PT) AND
INTERLABORATORY COMPARISON TEST (ICT)

 ICT: the organization, performance, and

evaluation of measurements or tests on the same
or similar items by two or more laboratories or
inspection bodies in accordance with
predetermined conditions (ISO 17043).
 Participant laboratories are only comparing
performance amongst the group of participating
members.

PROFICIENCY TESTING (PT) AND

INTERLABORATORY COMPARISON TEST (ICT)

 Results are commonly evaluated using two

methods described in ISO/IEC 17043:
- Normalized Error: statistical evaluation used to
compare proficiency testing results between the
participant and the reference laboratory where
the uncertainty in the measurement result is
included.
- First evaluation used to determine conformance
or nonconformance (i.e. Satisfactory/
Unsatisfactory) in proficiency testing.
- ISO 13528: Statistical methods for use in
Proficiency testing

PROFICIENCY TESTING (PT) AND

INTERLABORATORY COMPARISON TEST (ICT)

 Normalized Error

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PROFICIENCY TESTING (PT) AND
INTERLABORATORY COMPARISON TEST (ICT)

 Normalized Error:
 When the value of |En| ≤ 1 (i.e. between -1 and
+1), the results are considered satisfactory.
 When the value of |En| > 1 (i.e. greater than +1
or less than -1), the results are considered
unsatisfactory.

PROFICIENCY TESTING (PT) AND

INTERLABORATORY COMPARISON TEST (ICT)

- Z-Score:
- statistical measurement of a score’s relationship
(i.e. how many standard deviations above or
below the population mean) to the mean in a set
of scores.
- used to review the results of all participants and
identify outliers and exclude their data from
proficiency testing results.

PROFICIENCY TESTING (PT) AND

INTERLABORATORY COMPARISON TEST (ICT)

 Z-Score:

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PROFICIENCY TESTING (PT) AND
COMPARISON TEST (ICT)

 Z-Score: the following rules are used

 When the value of Z <=2, the results are
considered satisfactory.
 When the value of Z >=3, the results are
considered unsatisfactory.
 When the value of Z >=2 and Z <=3, the results
are considered questionable.

INTRALABORATORY COMPARISON TEST

 Intralaboratory comparison test: comparison

study of 1 or more methods by multiple
analysts/technicians in the laboratory.
 Include comparison with another competent
laboratory where available.
 Baseline for satisfactory agreement shall be
included.

USE OF RETAINED TEST ITEMS

 Previously tested samples are submitted for

testing.
 Assign another analyst/technician to conduct the
test.
 Compare results and evaluate percentage
difference or standard deviation.

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USE OF BLIND SAMPLES

 Submit any sample with similar matrix.

 Can make use of retained samples and usually,
assign the same analyst to conduct the test.
 Compare results and evaluate percentage
difference or standard deviation.

EVALUATION OF QC DATA
 Review your data
 Frequency of review of the data
 What to do when results are not within the
defined criteria

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