Lecture 5

Dr. Miranda
PHARMACEUTICAL
Fawzy
QUALITY CONTROL Lecturer of Pharmaceutical
PPS513 Analytical Chemistry
Faculty of Pharmacy
Damanhour University
 Analytical Quality Control
PHR 525

Analytical validation characteristics

1. Accuracy
2. Precision
A. Repeatability
B. Intermediate precision
C. Reproducibility (Ruggedness)
3. Specificity
4. Detection limit (DL), Limit of
Detection (LOD).
5. Quantitation limit (QL), Limit of
Quantitation (LOQ).
6. Linearity
7. Range
8. Ruggedness *
9. Robustness *
* These 2 are not included in the ICH
documents.
 5. The Range of Analytical
Procedure
› The range is the interval between the lower &
upper concentrations of the procedure that can
be determined with acceptable precision,
accuracy and linearity.
› The range must include both the upper
and lower levels.
› The range should have the same unit as
the test results.
Minimum Specified Range of
Procedure
 Analytical Quality Control
PHR 525

Data Elements Required for Assay Validation

› ICH characteristics for validation:
Analytical
a) Impurities
characteristics b) Degradation products
Quantitative test
(Performance Identification test
a) Drug substance
Characteristics) b) Drug product Drug substance
(Validation c) Dissolution Quantitative
characteristics) Limit test
test

1. Accuracy + + - -
2. Precision + + - -
3. Specificity + + + +
4. DL - - + +
5. QL + + - -
6. Linearity + + - -
7. Range + + - -
8. Ruggedness + + - -
9. Robustness + + - -
 6. Detection Limit (D.L.)
Limit of Detection (L.O.D)
› D.L. is the minimum concentration that
can be reliably detected.
› D.L. is the point at which the measured
value is just above the uncertainty of the
procedure.

HOW?!
 1. Visual Method
› The Visual method can be used for instrumental & non-
instrumental procedures
› D.L. is assessed by analyzing a number of samples
containing increasing, low and known amounts of analyte.
› The minimum concentration of analyte that can be reliably
detected represents D.L.
 2. Signal-to-Noise Ratio Method
› Applied to analytical procedures which
exhibit base-line noise. e.g. HPLC &
spectrophotometric methods.
› D.L. is the concentration of the sample that
gives

› Measure the signals of a series of samples of

increasing, low, known, concentrations.
› Measure the noise of a number of blank
experiments.
› The concentration that gives (signal of
analyte) / (noise of blank) equals 2/1 or 3/1
represents D.L.
 3. Standard Deviation of
Response & Slope Method:
Recommended Data for
Registration Application:
› The Value of D.L. (e.g 0.221µg / 100 mL)
› The Method used (Visual ,……..etc)
› The Chromatograms
› Experimental verification of D.L.
› The D.L. should be verified experimentally
by analyzing samples known to contain
amounts near D.L. (0.05 – 1.0 % of test
concentration).
ICH Guidelines:
› ICH Guidelines describes D.L. as:

𝐷.𝐿.=​𝟑.𝟑(𝑠𝑡𝑑.𝑒𝑟𝑟𝑜𝑟 𝑜𝑓 𝑦−𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡)/𝑆
› D.L. & Q.L. can be deter mined
simultaneously with linearity.
› The target value of D.L. or Q.L. is 0.05-1.0
% of test concentration.
› The parameter obtained should be
verified experimentally.
 7. Quantitation limit (Q.L.)
Limit Quantitation (L.O.Q.)
› Q.L. is the lowest concentration that can be
quantitatively determined with acceptable
accuracy &precision.
› Q.L is the parameter used for the quantitative assay
of low levels of drugs in sample matrix (0.05 – 1.0 %
of target concentration).
› Q.L. is the parameter used for the quantitative assay
of:
› Impurities
› Degradation products
› Trace substances
 Analytical Quality Control
PHR 525

Data Elements Required for Assay Validation

› ICH characteristics for validation:
Analytical
a) Impurities
characteristics b) Degradation products
Quantitative test
(Performance Identification test
a) Drug substance
Characteristics) b) Drug product Drug substance
(Validation c) Dissolution Quantitative
characteristics) Limit test
test

1. Accuracy + + - -
2. Precision + + - -
3. Specificity + + + +
4. DL - - + +
5. QL + + - -
6. Linearity + + - -
7. Range + + - -
8. Ruggedness + + - -
9. Robustness + + - -
Assessment (Evaluation) of Q.L.
1. Visual Method:
› The same as under D.L.
› Establish the minimum concentration of
analyte that can be reliably quantitated
with acceptable accuracy &precision.
2. Signal / Noise Ratio Method:
Q.L.=​signal of analyte/noise of blank =​10/1
› Applied to analytical procedures which
show base-line noise. e.g. HPLC.
› Measure signals of response of samples of
increasing known, low concentrations.
› Measure noise of response of a number of
blank samples.
› Calculate the ratio ​ signal of analyte/noise of
blank .
› Select the minimum concentration of
analyte that gives ​ signal of analyte/noise of
blank =​10/1 .
3. Standard Deviation of Response and
Slope Method:
› Example:
› Calculate Q.L. of drug substance
analyzed by HPLC. The least squares
analysis of data gave a slope of 2.09 and
S.D. of 0.14.
› Q.L. ?????
› Any concentration less than 0.67µ𝑔/100𝑚𝐿
can not/ can be quantified.
› Q.L. = 3.03 D.L.
Recommended Data For
Registration Application:
As under D.L.
› The value of Q.L.
› The method used.
› The chromatograms.
› Experimental verification of Q.L.
 8. Ruggedness
› Ruggedness of analytical procedure is the
reproducibility of the test results of the same
sample under a variety of conditions such as
different temperature & different days.
› The higher the reproducibility, the higher the
r u g g e d n e s s .
› Ruggedness is expressed as lack of influence on
test results of:
› Operational variables of the procedure. (lab.,
analyst, …)
› Environmental variables of the procedure
(temperature – humidity).
› The smaller the influence, the higher the
ruggedness.
› Ruggedness is a measure of reproducibility of test
results under variation normally expected from lab
to lab & from analyst to analyst.
 8. Ruggedness
› Assessment of Ruggedness:
› Assay under variables (different analyst /
labs/ equipment/ days).
› Assay under normal conditions (same
analyst / labs/ equipment).
› Compare results (S.D. + RSD% + Er%).
 9. Robustness
› Robustness of analytical procedure is the capacity
of the procedure to remain unaffected by small but
deliberate variation in method parameters.
› Robustness measures the reliability of a method
under deliberate variation in method parameters.
› Robustness Testing:
› Evaluate the extent to which procedure parameters
can be varied without affecting the performance of
the procedure.
› Examine the quantitative influence of variables on
test results. e.g.: In HPLC the following parameters
may be evaluated by measuring the quantitative
influence on test results caused by their variation.
› Parameters:
1. pH of mobile phase ± 0.2 pH
2. Flow rate ± 0.2 mL/min
3. Detector λ ± 2 nm
4. Injection volume ± 0.1 mL
5. HPLC manufacturer
6. Column supplier
7. Column lot
The range over which a parameter is
evaluated should be meaningful i.e. will the
method perform successfully if the specified
pH is adjusted ± 0.2 pH unit. (not 2 pH units)
› In HPLC, responses (resolution, peak
tailing) are tracked, while parameters are
adjusted.
› Robustness should be considered during
method development (ICH
recommendations).
› If the influence of a parameter variation
is within specified range, the variation is
said to be within method robustness
range.
› Data resulting from variation of
parameters will allow judgement if the
method needs revalidation.