Method validation in practice

WP 2

Saša Šorgić (ESV)

Method validationMethod validation is process of demonstrating or confirming that a method is suitable for its intended purpose.

Validation includes demonstrating performance caracteritic such is:

• Linearity

• Range

• Limit of detection

• Limit of quantitation

• Accuracy

• Precision

• Sensitivity

• Selectivity

Validation in ESV

• Method

• Plan of validation

• Linearity

• Limit of detection

• Accuracy and precision

• Measurement uncertainty

• Interlaboratory comparison

• Quality control

Linearity

Linearity was investigated using 6 different concentration levels in two repeats

Parameters of interest:

• Coefficient of correlation

• Standard uncertainty of intercept on the y-axis

• F-test

Example: Linearity analysis of copper by ICP-OES method for detection of heavy metals in water

Matrix matched calibrations

ICP-OES – 1%HNO3

GC/FID

• Methanol in vine - 5% Ethanol

• Volatile components in alcoholic drinks – 40% Ethanol

GC/MS

• Calibrations in representative matrix (apple, strawberry, pepper...)

Limit of detection

Different approaches:

• Blank approach

• Matched LoD approach

Example: Determination of IDL and MDL for copper in water by ICP-OES technique according to US EPA 200.7 method

Example: Determination of LoD and LoQ for PCB 101 in chocolate by GC/MS

LoD = X+3SDLoD = (1 + 0,3) µg/kg = 1,3 µg/kg ≈ 1µg/kg

LoQ = X+10SDLoQ = (1 + 0,65) µg/kg = 1,65 µg/kg ≈ 2µg/kg

Example: Confirmation of matched LoD of pyrimethanil in commercial tea sample, used for validation of pesticide’s content in food by GC/MS

Precision and accuracy• RM or CRM

• Spike of representative matrix on two concentration levels

Representative matrix

• Apple – Fruit

• Pepper – Vegetable

• Strawberry – Pigment

• Tea – Coffeine

• Chocolate – Fat and Oil

• Water

• Soil

Example: Palmitic acid in CRM-Olive oil, used for validation of FAMEs content in fats and oils by GC/FID

Example: Spike of sodium in commercial water on two concentration levels:

• 1st level added 5mg/l of sodium standard

• 2nd level added 20mg/l sodium standard

Example: Spike of pyrimethanil in commercial tea sample, used for validation of pesticide’s content in food by GC/MS

Interlaboratory comparison

Quality Control

Measurement's uncertainty

Includes uncertainty of:

• Equipment• Standard solutions• Calibration equation• Precision• Control charts• Interlaboratory comparison

Keeping method under control

• Interlaboratory comparison in period less then four years

• Internal control of method

• Internal and external control of equipment

• Control charts

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