Method Validation (Week 3) Flashcards
Why we need method validation?
To show methods are fit for purpose
Its a requirement of accreditation to ISO 17025 and 15189
Provides confidence to customers that it can generate accurate and reliable data
Difference between validation and verification?
Validation - carried out on new methods or on incorporate changes
Verification - undertaken by a lab thats adopting a method that has been previously validated, or when a change has been made
Method Validation process
1) Define analytical requirement - what tests are you looking to develope
2) Develop/identify candidate method - look at other previous work
3) Plan validation experiments
4) Carry out experiments
5) use data to assess fitness for purpose
6) Analytical requirements met? Yes = Validation report, No = back to 2)
Defining analytical requirements
Customer requirements
Typical Tests:
- Qualitative - identify something
- Quantitative - how much is there
- Limit - could be purities, need to identify limit
What will happen to results? - high standards
What decisions made based on results?
Parameters to consider?
Validation of a HPLC analysis (case scene 1)
- A laboratory is looking to become
accredited for the analysis of caffeine,
saccharin, aspartame and benzoic acid in
fruit juices, squashes and other soft
drinks by HPLC. - A candidate method has already identified
and developed to provide the appropriate
chromatographic conditions.
Specificity (selectivity)
How discriminating is method? - can your method see what you’re trying to test for
Effect of other components on the analysis of the component of interest - make sure nothing else is effecting results - other compounds
Determined by adding materials that may be encountered in samples - CRMs
Sensitivity
How much response to an analyte changes when conc changes - peak area
Shown by slope - sensitive analyses have steep slope for graph
For sensitive methods - big changes
Question?
Ready-to-drink samples are going to be analysed undiluted
except for the addition of an internal standard.
Dilute-to-taste samples are going to be diluted by five times,
including the addition of an internal standard.
What other sample preparation may be needed?
May need to filter drinks - remove solid bits - will clog hplc columns
Degas the drinks
Limit of detection (LoD) or DL
The smallest amount of analyte that can be detected and
identified with an acceptable degree of uncertainty, but not
necessarily quantified.
We are looking for concentration which gives a response that is 3 × the response
for blank values.
LoD - graph
Baseline noise - squiggly line
Red line - LoD - 3x bigger the baseline peak
Blue line - LOQ - 10x bigger peak
Determining loD and LoQ
Analyse a series of blanks (10 x analyses).
Calculate standard deviation of response (spectroscopic
techniques) or mean noise level (chromatographic techniques).
Determine detection/quantitation limit response as below
Determining LoD and LoQ - 2
To determine response of standard, analyse a standard with a
concentration that will give a response close to the calculated
detection/quantitation limit responses.
Calculate LoD and LoQ using these equations:
Quantitative analysis - analytical range
Determines how much of compound we actually have
The specified calibration range for an analysis depends on the
intended use of the method.
Typically ±20% of the expected test concentration(s).
Major component: covers 80-120% of test concentration.
Impurity: typically reporting level to 120% of test concentration.
Quantitative analysis – linear range
The ability of a method to obtain results which are directly proportional to concentration. Determine with linear regression for line of best fit. Confirm linearity using residuals.
Calibration curve - straight line
R2 - closer to 1 is better
Quantitative analysis – linear range 2
Different methods have different ranges over which they are linear. ICP has a large dynamic range. LC-MS has a very small dynamic range.
Slightly s shaped graph - linear range in the middle
Quantitative analysis – working range
Linear range – has an acceptable level of uncertainty. The working range typically goes from the LoQ to a little beyond the linear range, up to where the sensitivity decreases.
Working range starts just after LoQ
Accuracy?
Accuracy is the closeness of agreement between the value found
for the analyte and the true or accepted value
Trueness?
Trueness is the closeness of agreement between a large number
of analyte values and the true value or accepted value.
Bias?
Bias is a measure of the total systematic error in a method.
Suggest appropriate concentrations for each additive standard for
the preparation of five point calibration graphs? Question should be before accuracy trueness cards
S - 80, C - 100, B -160
20% added - should be 120% above specification
S - 96
C - 120
B - 192
4 dilutions
S -
C -
B -
Determining truness and bias
Trueness/bias can be determined in two ways:
Repeat analysis of a certified reference material (CRM).
In general, at least 7 replicates should be analysed.
Determination of recoveries from spiked samples:
- Analyse samples before and after spiking (adding) a known amount of
standard.
- For a method with no bias, the difference in the results is the amount of standard that was added (100% recovery)
To check how well the analysis worked on actual samples, orange
juice was spiked with known amounts of additive standards.
2 cm3 of each of the additive spiked solutions was added to a
50 cm3 volumetric flask, plus an internal standard, and the
solutions made up to the mark with orange juice.
The recovery is calculated using the formula:
Recovery =
measured concentration × 100
/ actual concentration
Answer in book
Precision?
The closeness of agreement between a series of independent
measurements obtained from multiple sampling of the same
homogeneous sample under the prescribed conditions.
Precision reflects random errors which occur in the method.
Precision is calculated by determining the standard deviation of
test results from repeat measurements.
Expressing precision
An absolute measure of
precision = standard deviation (s or s.d)
or
variance (s2) A relative measure of precision = coefficient of variation (CV) or relative standard deviation (RSD)
Precision
Repeatability – same analyst & equipment; limited time span.
Assess by: minimum of 6 replicates at 100% of test concentration, or 3
replicates at each of 3 concentrations (9 replicates in all).
Intermediate Precision – within-laboratory variations: different
days, analysts and equipment.
Reproducibility – different laboratories, analysts, samples and
equipment; same method; extended time span.
The repeatability (r) of the method was checked by six injections
of appropriate additive standards. The concentrations measured
will be given in the next slide.
Determine the repeatability using the formula:
r = 3 x s
(where s is the standard deviation.)
Uncertainty of measurement?
Uncertainty of measurement is a quantitative measure of the
limits within which the true value of the analyte is expected to
lie, to a specified level of confidence.
Values should typical lie within 2 × standard deviations of the
true value, i.e. 95% confidence level.
ISO 17025 and ISO 15189 requires that uncertainty is stated
with test results.
Uncertainty not only considers repeatability and reproducibility,
it takes into account many factors that could affect the result,
such as systematic effects which may cause bias.
Uncertainty of measurement
Takes into account all random and systematic errors.
e.g. a glass pipette:
- The pipette will have a tolerance, i.e. ± 0.03 ml – this is a systematic error (it
will always be the same).
- Every time the operator uses the pipette, the position of the meniscus on the
line will vary slightly – random error.
- Therefore, there will be small variations in each volume of liquid dispensed.
The combination of these two errors will lead to the overall uncertainty in the
volume measured.
