 Principle.

› Production operations must follow clearly defined

procedures in accordance with manufacturing and
marketing authorizations, with the objective of obtaining
products of the requisite quality.
› All handling of materials and products, such as
 receipt cleaning
 quarantine, processing,
 sampling, packaging and labelling
 storage, distribution,
 dispensing
should be done in accordance with written procedures
or instructions and, where necessary, recorded.
› as far as possible no deviation from instructions or
procedures should be avoided. Any If deviations occur,
they should be done in accordance with an approved
procedure.
Routine Commercial Manufacturing
 Monitor critical operating and performance parameters
› Utilize appropriate tools, e.g., Statistical Process
Control
 Monitor product characteristics (e.g., stability, product
specifications)
 Monitor state of personnel training and material,
facility/equipment and SOP changes
 Investigate OOS for root cause and implement
corrective action.

Validated Manufacturing Process

 A validated manufacturing process has a high level of
scientific assurance that it will reliably produce
acceptable product
 Measurement
 Appropriate systems, variables and attributes

 Data Analysis
 Periodic analysis to allow for trending on a periodic
basis to detect problems as early as possible and
implement corrective action
 Statistical Process Control - preventative actions
 Selection of Critical Quality Attributes
 Meaningful specifications/ limits

 The objective of an Analytical Work is:

The achievement of reliable and dependable
analytical information
 For conducting nonclinical laboratory studies that
support or intended to support applications for research
or marketing permits for products regulated by the
National Food and Drug Administration, Good
Laboratory Practices should be followed in a strict sense
 Compliance with this part is intended to assure the
quality and integrity of the safety data
 Therefore: analytical method validation is required
Method validation provides an assurance of reliability
during normal use, and is sometime referred to as "the
process of providing documented evidence that the
method does what it is intended to do."
 Validation – the process of demonstrating that a laboratory
procedure is robust, reliable, and reproducible in the hands of
the personnel performing the test in that Laboratory
 Method validation is the process of establishing the
performance characteristics and limitations of a method and
the identification of the influences which may change these
characteristics and to what extent. It is also the process of
verifying that a method is fit for purpose, i.e., for use for solving
a particular analytical problem.
 ISO 17025:
 Validation is the confirmation by examination and the
provision of objective evidence that the particular
requirements for a specific intended use are fulfilled
 Validation is part of overall quality assurance program
in a laboratory to have:
 the correct answer when collecting data…
 to have confidence that the sample contains no un-
safe compound rather than there might have been
something wrong with the detection method
Costs/Benefits of Quality Assurance
Costs Benefits
– Test materials • More efficient outputs
– Standards
– Quality assurance • Fewer replicates for
equipment same
– Analysis of QA/QC reliability
samples • Fewer do-overs
– Quality assurance • Greater confidence of:
official – Staff
– Committee Work – Laboratory
– Interlab Studies – Customers
– Travel to meetings
 It is an important element of quality control.
 Validation helps provide assurance that a
measurement will be reliable.
 In some fields, validation of methods is a regulatory
requirement.
 The validation of methods is good science.

When is Validation needed?

 Before introduction of a new method into routine use
 Whenever the conditions change for which a method
has been validated, e.g., instrument with different
characteristics
 Whenever the method is changed, and the change is
outside the original scope of the method
Can laboratories validate methods on their own,  in-
house validation
The in-house methods should demonstrate that
a. the required tolerances of all measurements
undertaken within the method (volume, temperatures,
masses etc.);
b. the forms of the determinand measured, including
speciation;
c. the effect of interferences has been widely
investigated and quantified;
d. significant sources of error have been identified and
adequate means of controlling them have been
identified.”
questions
 Will methods validated in this way be recognized by
other laboratories?
 What sort of recognition can be expected for in-house
methods used in a regulatory environment?
  Gross error
Real Population   Errors which are so serious
that there is no real
alternative to abandoning
Accurate the experiment and making
Precise a complete start
Error: Systematic  Random error
 Error that cause the
Random individual result to fall on
both side of the average
value  affect the
Estimate of precision of an experiment
 Systematic error
Sample XS  Error that cause all the
result to be in error in the
same sense  affect the
accuracy, i.e. proximity to
Reliable method – the obtained the true value
results are accurate and
correctly reflect the sample
being tested
 Steps to have an Analytical Information:
› Sampling and sample storage
› Preparation of Samples
› Measurement
 Equipment and Equipment parameters
 Reagents
 System Suitability testing Analytical
Method
 Preparation of Standards Analytical
Procedure
 Procedure
› Evaluation Analytical Principle
 Calculations
 Reporting of results
› Documentation
 Assure that:
› What is reported there is really there
› The level is exactly as what you report
› There is no addition through contamination
› If reported as negative, it is really not there
 Appropriate for intended use
 Preferable standard method
› Published in international, regional national
standard, technical journals
 Non Standard methods  required
validation
› Journal article
› In-house method
For a large number of replicates the results approach
GAUSSIAN or NORMAL DISTRIBUTION CURVE

The mean value – x

gives the center of
the distribution

The standard deviation –

s measures the width of
the distribution

Experimental results are

commonly expressed in
the form:
mean  standard
deviation _
x  s Fac
Sri Noegrohati,
of Pharmacy
17 GMU
  Curve is symmetrical and
centred at m.
 The greater the value of σ,
the greater the spread of
the curve.
 Whatever values of µ and
y

σ,
› 68.27% of observations
0 20 40
x
60 80 100
are within µ  σ
› 95.45% of observations

y

exp  x    / 2 2
2
 are within µ  2 σ
› 99.97% of observations
 2 are within µ  3 σ

Sri Noegrohati, UGM 18

 Arithmetic mean: average of all observations
n If the sample is random then the

xi arithmetic mean is the best
x  i 1 estimate of the population (true)
n mean, m
 Variance: measures the extent to which the data differs
in relation to itself. Variance of population is the mean
squared deviation from the population mean, denoted
σ2, while the variance of the sample data is denoted s2.
n n

 ix  x 2

 ix  x 2

2  i 1
s i 1
n n 1
Sri Noegrohati, UGM 19
 “The closeness of agreement between a test result
and the accepted reference value.”
 “Accuracy of a measuring instrument is the ability
of a measuring instrument to give responses close
to a true value.”

 EURACHEM Guide (1998) The Fitness for Purpose of Analytical Methods: A

Laboratory Guide to Method Validation and Related Topics, pp. 39, 41;
available at http://www.eurachem.ul.pt/guides/valid.pdf
SYSTEMATIC ERROR
 Reproducible under the same conditions in the
same experiment
 Can be detected and corrected for
 It is always positive or always negative

To detect a systematic error:

 Use Standard Reference Materials
 Run a blank sample
 Use different analytical methods
 Participate in “round robin” experiments
(different labs and people running the same
analysis)
Sri Noegrohati,
Fac of Pharmacy
21
GMU
 “The closeness of agreement between independent
test results obtained under stipulated conditions.”
 “Precision depends only on the distribution of random
errors and does not relate to the true value or specified
value. The measure of precision is usually expressed in
terms of imprecision and computed as a standard
deviation of the test results.”
 • “A measure for the reproducibility of measurements
within a set, that is, of the scatter or dispersion of a set
about its central value.”

 EURACHEM Guide (1998) The Fitness for Purpose of Analytical Methods:

A Laboratory Guide to Method Validation and Related Topics, p. 45;
available at ttp://www.eurachem.ul.pt/guides/valid.pdf
RANDOM ERROR
 Uncontrolled variables in the measurement
 Can be positive or negative
 Cannot be corrected for
 Random errors are independent of each
other

Random errors can be reduced by:

 Better experiments (equipment, methodology,
training of analyst)
 Large number of replicate samples

Sri Noegrohati,
Fac of Pharmacy
23
GMU
 "Student's t-test deals with the problems associated with
inference based on "small" samples: the calculated mean
(Xavg) and standard deviation (σ) may by chance deviate
from the "real" mean and standard deviation (i.e., what you'd
measure if you had many more data items: a "large" sample).
 For example, it it likely that the true mean size of maple
leaves is "close" to the mean calculated from a sample of N
randomly collected leaves.
 At 95% of the time, the actual mean would be in the range:
› If N=5, Xavg± 2.776 σ/N1/2;
› if N=10: Xavg± 2.262 σ/N1/2 ;
› if N=20: Xavg± 2.093 σ/N1/2 ;
› if N=40; Xavg± 2.023 σ/N1/2 ;
› and for "large" N: Xavg± 1.960 σ/N1/2 .
 (These "small-sample" corrections are included in the
descriptive statics report of the 95% confidence interval.)
 Student" (real name: W. S. Gossett [1876-1937])
CONFIDENCE INTERVALS
The confidence interval is the expression stating that
the true mean, µ, is likely to lie within a certain
distance from the measured mean, x.
– Student’s t test

The confidence interval is given by:

_
ts
μ  x
n
Where t is the value of student’s t taken from the
table

Sri Noegrohati,
26 UGM
 d.f. 90% 95% 99% 99.9%

2 2.920 4.303 9.925 31.596

3 2.353 3.182 5.841 12.941
4 2.132 2.776 4.604 8.610
5 2.015 2.571 4.032 6.869
10 1.812 2.228 3.169 4.587
20 1.725 2.086 2.845 3.850
30 1.697 2.042 2.750 3.646
Worked example:
Fluoride content of a sample determined potentiometrically in water is
(mg/l) 4.50, 3.80, 3.90, 4.20, 5.00 and 4.80 for separate analyses.
Mean = 4.37 Standard deviation = 0.48
90% confidence limits are: µ = 4.37  2.015 x (0.48/6) = 4.37  0.39
99% confidence limits are: = 4.37  4.032 x (0.48/6) = 4.37  0.79
Sri Noegrohati, UGM 27
 Sri
Noe
gro
hati
,
Fac
A ‘t’ test is used to compare sets of measurements.
of
Pha
Usually 95% probability is good enough. rma
cy
GM
28 U
APPLYING STUDENT’S T:
1) COMPARISON OF MEANS
Comparison of a measured result with a ‘known’
(standard) value

known value  x
t calc  n
s
tcalc > ttable at 95% confidence level
 results are considered to be different
 the difference is significant!

Statistical tests are giving only probabilities.

They do not relieve us of the responsibility of interpreting
our results!
29
 2) COMPARISON OF REPLICATE MEASUREMENTS
For 2 sets of data with number of measurements n1 , n2
and means x1, x2 :

x1  x2 n1n2
t calc 
spooled n1  n2
Where Spooled = pooled std dev. from both sets of data

s12 (n1  1)  s22 (n2  1)

spooled 
n1  n2  2

tcalc > ttable at 95% confidence level

 difference between results is significant.
Sri Noegrohati,
Degrees of freedom
30
= (n1 + n2 – 2) Fac of Pharmacy
GMU
Impact of Number of Experiments on Capturing Variability in a Population
of Data
Q TEST FOR BAD DATA
gap
Q calc 
range
The range is the total spread
of the data.
The gap is the difference
between the “bad” point
and the nearest value.
Example:
Gap Sri
Noe
12.2 12.4 12.5 12.6 12.9 gro
hati
Range ,
Fac
of
Pha
If Qcalc > Qtable  discarded rma
cy
questionable point GM
33 U
1. Analytical measurements should be made to satisfy an
agreed requirement.
2. Analytical measurements should be made using
methods and equipment that have been tested to
ensure they are fit for their purpose.
3. Staff making analytical measurements should be both
qualified and competent to undertake the task.
4. There should be a regular and independent assessment
of the technical performance of a laboratory.
5. Analytical measurements made in one location should
be consistent with those made elsewhere.
6. Organizations making analytical measurements should
have well defined quality control and quality assurance
procedures.
 Statement of Problem
 Definition of Objective
 Selection of Procedure
 Sampling, Sample Transport and Storage
 Sample Preparation
 Measurement/Determination
 Data Evaluation
 Conclusions and Report

Sri Noegrohati,
35
UGM
 The equipment on which the work is being done is
broadly suited to the application. It is clean, well
maintained and within calibration.
 The staff carrying out the validation are competent in
the type of work involved.
 There are no unusual fluctuations in laboratory
conditions and there is no work being carried out in the
immediate vicinity that is likely to cause interferences.
 The samples being used in the validation study are
known to be sufficiently stable.
 Fitness for purpose is the “degree to which data
produced by a measurement process enables a
user to make technically and administratively
correct decisions for a stated purpose.”
 Based on Lambert - Beer law  linear correlation between
absorbance and concentration  calibration curve

Finding the “Best”

Straight Line:
a line that
minimises the
deviations in the y-
direction using the
sum of the square
of these
deviations)-
method of “least
squares”.
Sri Noegrohati, UGM 39
 Linearity “defines the ability of the method to obtain
test results proportional to the concentration of
analyte.”
 “The Linear Range is by inference the range of analyte
concentrations over which the method gives test results
proportional to the concentration of the analyte.”
 Working range is a “set of values of measurands for
which the error of a measuring instrument is intended to
lie within specified limits.”
Assumption:
 There is a linear relationship
 Errors in the y-values (measured values) are greater than the
errors in the x-values minimise only the vertical deviations.
 Uncertainties for all y-values are the same

 minimise only the vertical deviations

Equation of a straight line: y = mx + c

where m = slope and c = y-intercept

n xi yi    xi  yi
c
x  y   x y  x
2
m
  n x    x 
i i i i i
n xi2   xi 2 i
2
i
2

Sri Noegrohati,
41
UGM
 The vertical deviation can be calculated as follows:
di = yi – (mxi + c)
 Some deviations are positive (point lies above the
curve) and some are negative (point lies below the
curve)  square the values so that the sign does not
play a role
di2 = (yi – mxi - c)2
 Estimate the standard deviation for all y values

sy   i 
d 2

n2
Sri Noegrohati, UGM 42
  Standard deviation for the slope
(m): 2
sy n
    x 
2
sm 
n xi 2
i
2

 Standard deviation for the intercept (c):

s y2  xi2  
    x 
2
sc 
n xi 2
i
2

 Detectability
Is the Analytical Signal distinguishable from the Blank? 
need to know the uncertainty of the measurements.sc
Y detection limit = c+3sc  LOD = (c+3sc)-c/m
Sri Noegrohati,
43
UGM
Sensitivity:
 calibration sensitivity = slope (m) of calibration curve.= ability
to discriminate between small differences in analyte
concentration
 analytical sensitivity (g) = slope (m)/standard deviation (Ss)

 lnear range, where

the response of the
analyte is linear with
concentration.
 The dynamic
range is the
concentration where
there is a
measureable
response to the
analyte, even if it is
not linear.
Sri Noegrohati,
44
UGM
Linear Dynamic Range: linear region of calibration curve where
the lower limit is ten times the standard deviation of the blank.
LOQ - limit of quantitation
LOL - limit of linearity

Concentration (mM) Sri Noegrohati,

45 UGM
Calibration of the fundamental Analytical Procedure
 Limit of detection (LOD) – “the concentraton that can
be measured with reasonable statistical certainty of
significantly different from blanc signal.”
 Limit of quantitative measurement (LOQ) – “the lowest
concentration of an analyte that can be determined
with acceptable precision (repeatability) and accuracy
under the stated conditions of the test using the chosen
analytical procedure.”
 Point of saturation for an instrument detector, so that higher
amounts of analyte do not produce a linear response in
signal
 Reproducible method – the same or very similar results
are obtained each time a sample is tested
 “Precision under reproducibility conditions, i.e.
conditions where test results are obtained with the same
method on identical test items in different laboratories
with different operators using different equipment.”
 Will you get the same result each time you test a
sample?
 Different from repeatability, which is the “precision
under repeatability conditions, i.e. conditions where
independent test results are obtained with the same
method on identical test items in the same laboratory
by the same operator using the same equipment within
short intervals of time.”
 Robust method – successful results are obtained a high
percentage of the time and few, if any, samples need
to be repeated
 “The robustness of an analytical procedure is a measure
of its capacity to remain unaffected by small, but
deliberate variations in method parameters and
provides an indication of its reliability during normal
usage.”
 You do not want the method to fail when you only have
enough material for a single try.
 “The ability of a method to measure only what it is
intended to measure.”
 “Specificity is the ability to assess unequivocally the
analyte in the presence of components which may
be expected to be present. Typically these might
include impurities, degradants, matrix, etc.”
 Will the method produce a result reliably over
time?
 Control chart i.e. Shewhart chart, are an effective
tool for monitoring stability and quality assurance
over time
 Funded by the
Company

Performed by
manufacturer

Performed by Lab