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VALIDATION OFMICROBIOLOGICAL ASSAY
METHODSlamet Ibrahim, Marlia Singgih
School of Pharmacy ITB
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INTRODUCTION
(Good Analytical Practices) :
a. Test is done to fulfill a specific objective
or user needb. Test is done using validated method,
procedure and instruments to guaranteethe result that suitable with the objective
c. Test should be carried out by qualified
personnel with highly competent skill
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d. The result of test should be consistent,not influenced by place, personnel or
instrumentse. 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 anAuthorized Government Council
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Validation of Analytical Method
Definition :
Validation of Analytical Method is a process of proofingor 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 toidentification, control of impurities and assay procedures
is included
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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
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Requirement of Validation
for Analytical Method
Using Calibrated instruments and tools
Prepared by competent personnel
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2 Method Validation
Primary Validation for new method, or
modified of standard method.
Secondary Validation for verification
process, if the lab adopted a validatedmethod
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Parameter of Validation in
microbiology assay Acurracy
Precision (repeatability, reproducibilityand intermediate precision)
Sensitivity
Selectivity and Specificity Linearity
Acceptable range (upper and lower limit) Robustness of method
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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 ENV002, Singapore Accreditation Council, July2002.
Feldsine, et.al., AOAC International methodCommittee Guidelines for Validation ofQualitative and Quantitative Food
Microbiological Official Method of Analysis,Journal of AOAC International Vol.85, No.5,
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Microbiological Assay
1. QUALITATIVE
2. QUANTITATIVE
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Qualitative Method
In qualitative method, its response is
presence or absence of parameterdetected, either direct or indirect to
samples
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Quantitative Method
In Quantitative Method, its response is in
the form of analite amount, that can bemeasured directly (i.e. enumeration of
microbes) or indirectly (i.e : Absorbance
value, Intensity, Impedancy, etc.)
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Methods of Microbiological Analysis
Qualitative: Direct identification
Morphological analysis :macroskopic and microskopic
Alternative method (Dye-reduction Test, Electrical Methods, ATPDetermination)
Rapid method (immunochemical, molecular techniques)
Quantitative :
Total Plate Count MPN (Most Probable Number)
Assay of antibiotic potency
Sterility Test
Fenol coeffiscient test (desinfectant and antiseptic)
Preservative Effectivity Test
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Identification of Microorganism
Escherichia coli Salmonella thypi
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Validation Preparation
Before validation is done, the laboratory shouldprepare some important matters :
1. Microorganism of target or reference (see SR-02:
persyaratan tambahan untuk akreditasi
laboratorium, Pengujian Kimia dan BiologiSNI 17025, DP.01.16, Januari 2004)
2. Equipment and Instrument which regularly
calibrated3. Competent Personnels
4. Statistic Program for calculation, evaluation and
result interpretation
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Parameter of Validation
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Accuracy
Recovery = in % = H/A x 100
Relative Deviation : (H A)/A x 100H = result of assay method
A = result of real assay from microorganism
target
Relative Recovery : H/B x 100
H = result of assay methodB = result of standard method
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Accuracy
Method of assay :
Spiked-placebo Recovery Method Standard Addition Method
Use 9 times measurement ( 3 level
concentration with 3 replication)
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Example of accuracy measurement
Total Plate Count
Strain Theory Assay Method Std Method
I 125 126 127
II 125 120 119
III 125 120
118
IV 125 125
128
V 125 132 131
Average 125 125 125
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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%
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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 samehomogeneous sample under the prescribed
conditions
Parameter of Precision : repeatability,reproducibility, intermediate precision
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Precision
Relative Standard Deviation (RSD) for
intermediate precision :
[(log ai log bi )/xi] 2
2 p aiai and bi : assay result (1,2,3,and bi : assay result (1,2,3,n)n)xi = average = 1/n (logxi = average = 1/n (log aiai + log bi)+ log bi)
P = number of sample testedP = number of sample tested
CV =CV = CoeffCoeffof variation= 100 RSDof variation= 100 RSD
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Example of Intermediate Precision on TPC
No. TPC (cfu/mL) logaritm (II/I)2
a b a b avgr (I) diff (II)
1 93 86 1,9685 1,9345 1,9515 0,0340
0,0003032 34 30 1,5315 1,4771 1,5043 0,0544 0,001306
3 98 73 1,9912 1,8633 1,9273 0,1279 0,004404
4 89 83 1,9494 1,9191 1,9342 0,0303 0,000246
5 116 104 2,0645 2,0170 2,0407 0,0475 0,000540
6 168 156 2,2253 2,1931 2,2092 0,0322 0,000212
7 62 56 1,7924 1,7482 1,7703 0,04420,000623
8 38 28 1,5798 1,4472 1,5135 0,1326 0,007679
9 330 300 2,5185 2,4771 2,4978 0,0414 0,00027510 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 %
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Range of Test Result
The range of an analytical procedure is the
interval between the upper and lowerconcentration (amounts) of analyte in the
sample (including these concentrations) for
which it has been demonstrated that theanalytical 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)
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Upper limitThe 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
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Linearity
(for antibiotic potency assay)
Linearity : samples concentration and the assayresponse are proportional , directly or by mathequation
Reference curve is made from at least 3concentration between 50 150% of actual conc(FDA)
To determine concentration in the sample : use80, 100 and 120% of target concentration
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Parameter of Linearity Reference curve
Analysis sensitivity : F = y/ x
Residual deviation /residual regressionline
Sy/x = [(y-)2
/n-2]1/2
where y = analite response
= calculated from regression line
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Linearity
Coefficient of variation : regression
functionVx0 = S y/x . 100% , (Vx0 2%)
b . x Coefficient of correlation ( r 0,999)
How to determine the conc of
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How to determine the conc of
sample
Diameter
of inhib
Log Ci
x
x
x
x
x
a. Use equation :
D = b log C + a
log Cs = Ds a/ b, where :
Cs = conc. Analit in sample
Ds = Diameter of inhibition
b. Use 1 ref.soln and blanklog Cs = (Ds a)/(Db a) . log Cb
where :
Cb = conc. analit in ref/std
Requirement of sample for
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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
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For quantitative, sample devided by 4 :
one sample without inoculation, 3 sampleswith low , medium dan high contamination
level.
Low : pada batas limit deteksi
Medium : 1 log more
High : equivalent to 2 log more
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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 kittersebut.
Process of evaluation of Diagnostic
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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 ofsample and reagent
Step III : Development of validation data,
including evaluation of normal range of testresult, test under abnormal condition, stimulationor supression.
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Robustness of Method
The robustness of an analytical procedure
is a measure of its capacity to remainunaffected by small, but deliberate
variations in method parameters and
provides an indication of its reliabilityduring normal usage.
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Robustness of method
Should be done during the development of
method and depends on factors thatinfluence the assay
If the assay is very sensitive to
environment, the assay should be
controlled carefully
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Robustness of method
Many conditions should be controlled :
Stability of sample Temperature of incubation
Time of incubation
Aerobic or anaerobic
media (nutrition), etc