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VALIDATION OF

MICROBIOLOGICAL ASSAY
METHOD
Slamet Ibrahim, Marlia Singgih
School of Pharmacy ITB

INTRODUCTION
(Good Analytical Practices) :
a. Test is done to fulfill a specific objective
or user need
b. Test is done using validated method,
procedure and instruments to guarantee
the result that suitable with the objective
c. Test should be carried out by qualified
personnel with highly competent skill

d. The result of test should be consistent,


not influenced by place, personnel or
instruments
e. Laboratory for testing should guarantee
that the procedures and quality control
system has be applied properly
f. The Laboratory Quality must be evaluated
and checked at regular basis by an
Authorized Government Council

Validation of Analytical Method


Definition :
Validation of Analytical Method is a process of proofing
or confirmation of a test in Laboratory and stated that
the method is fulfill the requirement , as stated in the
References
The objective of validation of an analytical procedure is
to demonstrate that it is suitable for its intended purpose.
A summary of the characteristics applicable to
identification, control of impurities and assay procedures
is included

Continued.

Objective :
Evaluate the method performances :
sensitivity, selectivity, accuracy, precision,
etc., also to evaluate the weaknesses, and
limitation of the method
Check factors that influence the
performances of method and how it
influence the results
Verify or Proof the analytical method which
is used in the laboratory

Requirement of Validation
for Analytical Method
Using Calibrated instruments and tools
Prepared by competent personnel

2 Method Validation
Primary Validation for new method, or
modified of standard method.
Secondary Validation for verification
process, if the lab adopted a validated
method

Parameter of Validation in
microbiology assay

Acurracy
Precision (repeatability, reproducibility
and intermediate precision)
Sensitivity
Selectivity and Specificity
Linearity
Acceptable range (upper and lower limit)
Robustness of method

References for Validation

The United States Pharmacopeia (USP) 30,


2008 or new edition
International Conference on Harmonization,
1996
Method Validation of Microbiological
Methods, guidance note : C & B and ENV
002, Singapore Accreditation Council, July
2002.
Feldsine, et.al., AOAC International method
Committee Guidelines for Validation of
Qualitative and Quantitative Food
Microbiological Official Method of Analysis,
Journal of AOAC International Vol.85, No.5,
2002

Water quality Guidance on Validation of


Microbiological Methods, Technical Report,
ISO/TR 13843 : 2000.
Procedure for The Estimation and
Expression of Measurement Uncertainty in
Chemical Analysis, Nordic Committee on
Food Analysis, NMKL, No.5, 1997.
APHA Standard methods for the
Examination of Water and Wastewater,
20th ed., 1998.

Microbiological Assay
1. QUALITATIVE
2. QUANTITATIVE

Qualitative Method
In qualitative method, its response is
presence or absence of parameter
detected, either direct or indirect to
samples

Quantitative Method
In Quantitative Method, its response is in
the form of analite amount, that can be
measured directly (i.e. enumeration of
microbes) or indirectly (i.e : Absorbance
value, Intensity, Impedancy, etc.)

Methods of Microbiological Analysis

Qualitative:

9
9
9
9

Direct identification
Morphological analysis :macroskopic and microskopic
Alternative method (Dye-reduction Test, Electrical Methods, ATP
Determination)
Rapid method (immunochemical, molecular techniques)

Quantitative :

9
9
9
9
9
9

Total Plate Count


MPN (Most Probable Number)
Assay of antibiotic potency
Sterility Test
Fenol coeffiscient test (desinfectant and antiseptic)
Preservative Effectivity Test

Identification of Microorganism

Escherichia coli

Salmonella thypi

Validation Preparation
Before validation is done, the laboratory should
prepare some important matters :
1. Microorganism of target or reference (see SR-02
:
persyaratan tambahan untuk akreditasi
laboratorium, Pengujian Kimia dan Biologi
SNI 17025, DP.01.16, Januari 2004)
2. Equipment and Instrument which regularly
calibrated
3. Competent Personnels
4. Statistic Program for calculation, evaluation and
result interpretation

Parameter of Validation

Accuracy (kecermatan) in
microbiological assay
Definition :
The accuracy of an analytical procedure
expresses the closeness of agreement
between the value which is accepted
either as a conventional true value or an
accepted reference value and the value
found

Accuracy
Recovery = in % = H/A x 100
Relative Deviation : (H A)/A x 100
H = result of assay method
A = result of real assay from microorganism
target
Relative Recovery : H/B x 100
H = result of assay method
B = result of standard method

Accuracy
Method of assay :
Spiked-placebo Recovery Method
Standard Addition Method
Use 9 times measurement ( 3 level
concentration with 3 replication)

Example of accuracy measurement


Total Plate Count
Strain
I
II
III
118
IV
128
V
Average

Theory
125
125

125
125

Assay Method Std Method


126
120
125

127
119
120

125

125
132
125

131
125

Calculation
% Recovery = Result of assay method/theory x
100%
= 125/125 x 100% = 100%
% Relative Recovery = Assay Method/std method
x
100% = 125/125 x 100% =
100%

Precision (keseksamaan)
Definition
The precision of an analytical procedure
expresses the closeness of agreement (degree
of scatter) between a series of measurements
obtained from multiple sampling of the same
homogeneous sample under the prescribed
conditions
Parameter of Precision : repeatability,
reproducibility, intermediate precision

Precision
Relative Standard Deviation (RSD) for
intermediate precision :

[(log ai log bi )/xi] 2


2p

ai and bi : assay result (1,2,3,n)


xi = average = 1/n (log ai + log bi)
P = number of sample tested
CV = Coeff of variation= 100 RSD

Example of Intermediate Precision on TPC


No. TPC (cfu/mL)
a
b
1
2
3
4
5
6
7
8
9
10

logaritm
a
b

(II/I)2
avgr (I)

diff (II)

93
86
1,9685
1,9345 1,9515 0,0340
0,000303
34
30
1,5315 1,4771 1,5043 0,0544
0,001306
98
73
1,9912 1,8633 1,9273 0,1279
0,004404
89
83
1,9494 1,9191 1,9342 0,0303
0,000246
116
104
2,0645 2,0170 2,0407 0,0475
0,000540
168
156 2,2253 2,1931 2,2092 0,0322
0,000212
62
56
1,7924 1,7482 1,7703 0,0442
0,000623
38
28
1,5798 1,4472
1,5135 0,1326
0,007679
330
300
2,5185 2,4771
2,4978 0,0414
0,000275
2300 2040
3,3617 3,3096
3,3357 0,00521 0,000244
= 0,015832
RSD = 0,015832/2 x 10 = 0,0281
CV = 100 x RSD = 2,81 %

Range of Test Result


The range of an analytical procedure is the
interval between the upper and lower
concentration (amounts) of analyte in the
sample (including these concentrations) for
which it has been demonstrated that the
analytical procedure has a suitable level of
precision, accuracy and linearity.
Lower limit
The lowest test result is indicated by deviation of
analysis 20.0% of average (min measurement :
3 times)

Upper limit
The highest test result which indicated by
deviation of analysis 15.0% from average (min
measurement 3times)
2.LC HC-1

1,96

[ 2.LC HC ]1/2

Range of test result (plate agar)


Sample of food and drugs : 25-250 cfu/plate
Sample of water : 30-300 cfu/plate
Fungi (Aspergillus niger) : 8-80 colony/plate

Linearity
(for antibiotic potency assay)
Linearity : samples concentration and the assay
response are proportional , directly or by math
equation
Reference curve is made from at least 3
concentration between 50 150% of actual conc
(FDA)
To determine concentration in the sample : use
80, 100 and 120% of target concentration

Parameter of Linearity
Reference curve
Analysis sensitivity : F = y/ x
Residual deviation /residual regression
line
Sy/x = [(y-)2/n-2]1/2
where y = analite response
= calculated from regression line

Linearity
Coefficient of variation : regression
function
, (Vx0 2%)
Vx0 = S y/x . 100%
b.x
Coefficient of correlation ( r 0,999)

How to determine the conc of


sample
a. Use equation :

D = b log C + a
log Cs = Ds a/ b, where :
Diameter
of inhib

Cs = conc. Analit in sample

x
x
x

Ds = Diameter of inhibition
b. Use 1 ref.soln and blank
log Cs = (Ds a)/(Db a) . log Cb

where :
Cb = conc. analit in ref/std
Log Ci

Requirement of sample for


validation process
For qualitative assay, 3 samples used : no
contaminated , positive contaminated by a
certain amount near limit of detection (Low
Contamination Level), sample with High
Contamination Level
Low contamination level : 1-5 cfu/25 g sample
and high contamination level : 10-50 cfu/25 g
sample

For quantitative, sample devided by 4 :


one sample without inoculation, 3 samples
with low , medium dan high contamination
level.
Low : pada batas limit deteksi
Medium : 1 log more
High : equivalent to 2 log more

Validation of Reagent Kit


Kemudahan penanganan sampel dan
pereaksi yang digunakan,
How to store and stabilize the reagent
Toxicity
Quality hasil reaksi yang diberikan oleh kit
tersebut.

Process of evaluation of Diagnostic


kit
Step I : initial Selection, including evaluation to

standard curve , any data reduction, evaluation


of sensitivity of reagent
Step II : Understand procedure of kit usage , to
evaluate the influence of matrix efect, recovery,
cross-reaction , interferences, handling of
sample and reagent
Step III : Development of validation data,
including evaluation of normal range of test
result, test under abnormal condition, stimulation
or supression.

Robustness of Method
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.

Robustness of method
Should be done during the development of
method and depends on factors that
influence the assay
If the assay is very sensitive to
environment, the assay should be
controlled carefully

Robustness of method
Many conditions should be controlled :
Stability of sample
Temperature of incubation
Time of incubation
Aerobic or anaerobic
media (nutrition), etc

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