VALIDATION OF MICROBIOLOGY METHOD.pdf

8/10/2019 VALIDATION OF MICROBIOLOGY METHOD.pdf

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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

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