Lecture 9: Quantitation

Question 1

purpose of bioanalytical method validation (BMV)

Answer 1

provide results that are reliable and reproducible as studies involving determination and quantitation require a valid interpretation

For work in which a high level of confidence is required (e.g. pharmaceutical/clinical analysis, environmental monitoring, forensic analysis, etc.) and provides a quality assurance of data acquired with the validated protocol.
- Forms part of GLP criteria

Question 2

when is validation required

Answer 2

1. Prior to commencing studies involving routine testing
2. When a protocol is transferred to another lab
3. If any part of the protocol is changed, that could affect the figures of merit measured in the validation
   o i.e. alternative instrument/sample matrix

Question 3

phases of BMV

Answer 3

pre study validation
in study validation

Question 4

pre-study validation

Answer 4

involving analytical method development

Question 5

in-study validation

Answer 5

involve the application of the method to biological/chemical matrices for a measure of parameters such as pharmacokinetics, bioavailability, etc.; if carried out at different sites, the method will require validation at each site to establish inter-laboratory reliability.

Question 6

types of validation

Answer 6

full
partial
cross

Question 7

full validation use

Answer 7

for testing new method and target substance

Question 8

partial validation use

Answer 8

– i.e. transferring an existing fully validated method to alternative lab/instrument/sample processing procedures, etc.

Question 9

cross validation use

Answer 9

i.e. comparison of two methods during the same study

Question 10

1. Accuracy

Answer 10

the degree of closeness of the determined value to the nominal or known true value under prescribed conditions

Question 11

2. Precision

Answer 11

is the closeness of agreement among a series of measurements.

Question 12

3. Selectivity

Answer 12

is the extent to which the method can determine a particular compound in the analyzed matrices without interference from matrix components.

Question 13

4. Sensitivity

Answer 13

defined as the lowest analyte concentration in the matrix that can be measured with acceptable accuracy and precision (i.e., LLOQ).

Question 14

5. Reproducibility

Answer 14

represents the precision between two laboratories. It also represents the precision of the method under the same operating conditions over a short period of time.

Question 15

6. Limit of Quantification

Answer 15

is the lowest amount of an analyte that can be quantitatively
determined with acceptable precision and accuracy.

Question 16

7. Stability

Answer 16

is a measure of the intactness an analyte (lack of degradation) in a given matrix under specific storage and use conditions

Question 17

8. Dynamic range

Answer 17

range of linearity of calibration/standard line

Question 18

9. Recovery

Answer 18

refers to the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the method.

Question 19

Internal standard (IS) method

Answer 19

Most reliable for trace level analysis – accounts for analyte recovery in preparation of complex samples & ionisation suppression

 Add a constant & known amount of isotopically labelled version of target analyte as a certified/standard reference material (surrogate IS) to both unknown sample & calibration standards.
 Measure instrument response of target substance to internal standard,
generating a relative response factor (RRF) for both the calibration
standards & unknown