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:

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

• Quantitative :Total Plate CountMPN (Most Probable Number)Assay of antibiotic potencySterility TestFenol coeffiscient test (desinfectant and antiseptic)Preservative Effectivity Test

Identification of Microorganism

Escherichia coli Salmonella thypi

Validation PreparationBefore validation is done, the laboratory should prepare some important matters :1. Microorganism of target or reference (see SR-02 :

persyaratan tambahan untuk akreditasilaboratorium, Pengujian Kimia dan BiologiSNI 17025, DP.01.16, Januari 2004)

2. Equipment and Instrument which regularly calibrated 3. Competent Personnels4. 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 100H = 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 Theory Assay Method Std Method

I 125 126 127II 125 120 119III 125 120

118IV 125 125

128V 125 132 131

Average 125 125 125

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

2 paiai and bi : assay result (1,2,3,and bi : assay result (1,2,3,……n)n)xi = average = 1/n (log xi = average = 1/n (log aiai + log bi) + log bi) P = number of sample testedP = number of sample testedCV = CV = CoeffCoeff of variation= 100 RSDof variation= 100 RSD

Example of Intermediate Precision on TPCNo. 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 0,000303

2 34 30 1,5315 1,4771 1,5043 0,0544 0,0013063 98 73 1,9912 1,8633 1,9273 0,1279 0,0044044 89 83 1,9494 1,9191 1,9342 0,0303 0,0002465 116 104 2,0645 2,0170 2,0407 0,0475 0,0005406 168 156 2,2253 2,1931 2,2092 0,0322 0,0002127 62 56 1,7924 1,7482 1,7703 0,0442

0,0006238 38 28 1,5798 1,4472 1,5135 0,1326 0,0076799 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 %

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 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

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-ŷ)2/n-2]1/2

where y = analite response ŷ = calculated from regression line

Linearity

• Coefficient of variation : regressionfunction

Vx0 = S y/x . 100% , (Vx0 ≤ 2%)b . x

• Coefficient of correlation ( r ≥ 0,999)

How to determine the conc of sample

Diameter of inhib

Log Ci

xx

xx

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 blank

log Cs = (Ds – a)/(Db – a) . log Cb

where :

Cb = conc. analit in ref/std

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 deteksiMedium : 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 Diagnostickit

• Step I : initial Selection, including evaluation to standard curve , any data reduction, evaluationof 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, stimulationor 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