Validation

Question 1

Selectivity

Answer 1

Method is able to differentiate analyte from endogenous compounds (matrix, metabolites, co-admin drugs, back-conversion).
> 6 individual sources blank matrix –> evaluated + analysed for interference
Response <20% LLOQ / 5% IS

Question 2

Carry-over

Answer 2

Run blank after high concentration

<20% LLOQ / <5% IS

Question 3

Lower limit of quantification

Answer 3

Lowerst concentration of analyte that can be reliably quantified + lowest calibration standard
S/N > 5

Question 4

Highest limit of quantification

Answer 4

Highest concetration of analyte that can be reliably quantified + highest calibration standard

Question 5

Accuracy

Answer 5

Closeness of the mean test results to the actual value
Within-run: 5x4 standards (LLOQ, 3xLLOQ, medium, >75% HLQ)
Between-run: 3 runs on two days
A = (Av / nominal value) * 100%
Within 99-95% (quality control) or 85-80% (bioanalysis) of the nominal value

Question 6

Calibration curve

Answer 6

Test results are directly proportional to the concentration of analyte in the sample
6 cali concentrations (LLOQ - HLQ)
Concentration of the back calculation is within 15% nominal value
LLOQ within 20% nominal value
Weighted regression!

Question 7

Precision

Answer 7

Closeness of repeated measurements
Within-run: 5x4 standards (LLOQ, 3xLLOQ, medium, >75% HLQ)
Between-run: 3 runs on two days
CV = (SD/Av) * 100%
CV < 15%
LLOQ < 20%

Question 8

Dilution integrity

Answer 8

Dilutions should not affect accuracy + precision
Spiked sample, dilute, measure concentration for each dilution 5x
Accuracy + precision within 15%

Question 9

Limit of detection

Answer 9

Lowest amount of sample which can be detected, but not quantified
S/N > 3

Question 10

Matrix effect

Answer 10

Needs to be investigated when using MS methods (ion suppression)
6 blank matrices, determine MF
MF = (peak area analyte + matrix) : (peak area - matrix)a
CV MF = < 15%

Question 11

Stability

Answer 11

Ensure that every step during sample preparation + sample analysis + storage does not affect the concentration of the analyte
3 x LLOQ + 3 x HLQ analysed immediately after prep + after applied storage conditions (against fresh cali curve)
Mean concentration within 15% of nominal concentration

Question 12

Range

Answer 12

Interval between upper and lower concentration of the analyte for which analytical procedure has suitable level of precision, accuracy and linearity (LLOQ - HLQ)

Question 13

Robustness

Answer 13

Capacity of analytical procedure to remain unaffected by small variations in method parameters + indication of reliability during normal usage