Industrial Technology Institute

Test Method Validation & Verification

H.P.P.S.SomasiriPrincipal Research Scientist / SDD-QAD /QM

Industrial Technology Institute

SLAB2015/11/10, BMICH

VER 6

Industrial Technology Institute 2

Introduction

An analytical method is the series of procedures from receipt of a sample to the production of the final report.

Validation is the process of verifying that a method is fit for purpose.

Industrial Technology Institute 3

Basic Concepts in Method ValidationSpecificityAnalyte StabilityAnalytical RangeCalibration RangeRecovery (Accuracy)PrecisionLimit of Detection (LOD)Limit of Quantification (LOQ)Measurement uncertainty

Industrial Technology Institute 4

Specificity

Shows that the detected signal is due to the analyte, not another compounds/ s.

Use matrix blanks, reagent blanks for conformation of specificity

5

Example: Confirmation of Identity

Standard Non Polar Column

0

2

4

6

8

10

12

0 0.5 1 1.5 2 2.5 3

Time (minutes)

Resp

onse

Sample Non Polar Column

0

2

4

6

8

10

12

0 0.5 1 1.5 2 2.5 3

Time (minutes)

Resp

onse

Standard Polar Column

0

2

4

6

8

10

12

0 0.5 1 1.5 2 2.5 3

Time (minutes)

Resp

onse

Sample Polar Column

0

2

4

6

8

10

12

0 0.5 1 1.5 2 2.5 3

Time (minutes)

Resp

onse

Industrial Technology Institute 6

Analyte StabilityAnalyte stability during sample storage, analyte

stability in sample processing, analyte stability in sample extract should be studied.

Industrial Technology Institute 7

Analytical RangeRange of the analyte that can be analysed with the

proposed selected method.

Industrial Technology Institute 8

Calibration Range If a linear calibration is used, the range in which the

response for the anayte shows a linear correlation with the analyte’s concentration should be determined.

This is known as linear dynamic range.

Industrial Technology Institute 9

Linear Dynamic RangeColour Vs time

y = 0.7518x2 + 0.9696x + 9.2679R2 = 0.9891

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6

Time

Col

our

Coconut oil

Palm Oil

Expon. (Coconut oil)

Poly. (Palm Oil)

Industrial Technology Institute 10

Recovery% Recover of the anlalyte on extraction, clean-up,

derivatization and measurement.Can be done with

- Certified Reference Material (CRM,s)- Standard Reference materials (SRM,s)- Interlaboratory comparisons- Spiking (@ 20 %, 50 % and 80 %)

Industrial Technology Institute 11

Accuracy (Trueness)

How close the result to the expected (assigned) value

12

Example: AccuracySample Blank value

(mg/L)RM value (mg/L)

1 0.001 1.462 0.018 1.803 0.001 1.174 0.018 1.205 0.002 1.226 0.002 0.747 0.002 1.168 0.014 1.239 0.001 1.2010 0.002 1.29Mean value 0.004 1.25

RM assigned value: 1.22 ± 0.08 mg/L; Laboratory value for RM: 1.246 mg/L

Mean laboratory Value fromRM value certificate1.246 mg/L 1.22 mg/L

Bias

value

13

Accuracy versus Precision

Accuracy (Trueness)

PrecisionAccuracy &

Precision

Industrial Technology Institute 14

Precision Is measured as “repeatability “ or reproducibility

Repeatability Measurement is repeated with a minimum of variations (same person, same laboratory, same equipment, over a short period of time.ReproducibilityMeasurement is repeated with as much variation as possible. (Different analyst, different laboratory, different equipment, over a long period of time).

Industrial Technology Institute 15

Limit of Detection (LOD) Point where the variability of the measurement is such that it is

impossible to distinguish between random fluctuation in the value measured and actual signal.

– LOD = YB + 3XSB

• YB = Mean value measured from the blank

• SB = Standard Deviation of the value measured from the blank

16

Replicate (n) Instrument response

Calculated Concentration

(ppb)1 29550 0.1002 28653 0.0973 29401 0.0994 28532 0.0975 29712 0.1016 29983 0.1017 28167 0.095

     Mean   0.099StdDev   0.0023 Times StdDev   0.007Detection Limit   0.106

TBT in Biota

Limit of Detection

Industrial Technology Institute 17

Limit of Quantification (LOQ) Point where the measured value could be quantified reliably

LOQ = YB + 10XSB

• YB = Mean value measured from the blank

• SB = Standard Deviation of the value measured from the blank

18

Example:TBT in BiotaReplicate (n) Instrument

responseCalculated

Concentration (ppb)

1 29550 0.1002 28653 0.0973 29401 0.0994 28532 0.0975 29712 0.1016 29983 0.1017 28167 0.095

     Mean   0.099StdDev   0.0023 Times StdDev   0.007Detection Limit   0.106

Limit of Quantitation

Blk + 5s 0.110 Blk + 6s 0.113Blk + 10s 0.122

Limit of Quantification (LOQ)

19

Sensitivity- Cadmium Analysis by AAS

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.00 0.20 0.40 0.60 0.80 1.00 1.20

Cd Concentration (mg/L)

Abs

orba

nce

slope = 0.241 ± 0.011

intercept = 0.009 ± 0.003

r-square = 0.99442 XX

xx

xx

Sensitivity

20

Measurement Uncertainty

Uncertainty estimates should take account of:Overall long-term precisionBias and its uncertainty, including the

statistical uncertainty involved in the bias measurements, and the reference material or method uncertainty

Calibration uncertaintiesAny significant effects operating in addition to

those listed above

21

Test Method Verification

Any standard method shall be verified before being used in the laboratory

Verify the RecoveryAccuracyPrecision (Repeatability and Reproducibility)LOD and LOQ

Industrial Technology Institute 22