Analytical Test Method Validation - Quantitation and Detection Limit

2
with known concentrations of analyte and by establishing the minimum level at which the analyte
can be quantified with acceptable accuracy and precision. The quantitation limit must not be
greater than 50% of the specification, where technically feasible.
· Based on Signal-to-Noise Approach
This approach can only be applied to analytical procedures that exhibit baseline noise.
Determination of the signal-to-noise ratio is performed by comparing measured signals from
samples with known low concentrations of analyte with those of blank samples, and by
establishing the minimum concentration at which the analyte can be reliably quantified. A typical
signal-to-noise ratio is 10:1.
· Based on Capability of the Instrument
In some cases the instrument itself is the limiting factor for the analysis regardless of the sample.
An example of this is an LOD test using an analytical balance. In this case a discussion of the
quantitation limit may be constructed in the validation documentation based on the calibration
tolerance of the equipment rather than analysis of actual samples. The actual limit of quantitation
would still be presented in numerical terms relevant to the assay method based on the discussion.
Another example of this may be for KF titration assays where the ability of the instrument to
deliver a minimum amount of titrant would be the limiting factor. It is recommended that
experiments to determine this minimum amount of sample should be conducted for the specific
instrument model if this approach is taken. The experiment(s) could then be referred to in any
validation that utilizes the same model of equipment.
· Based on the Standard Deviation of the Response and the Slope
The quantitation limit (QL) may be expressed as: QL = 10 􀄱/ S where, 􀄱= the deviation of the
response; S = the slope of the calibration curve. The slope S may be estimated from the
calibration curve of the analyte. The estimate of 􀄱 is carried out in a variety of ways including:
o Based on the Standard Deviation of the Blank:
Analyzing an appropriate number of blank samples and calculating the standard
deviation of these responses and perform measurement of the magnitude of
analytical background response.
o Based on the Calibration Curve:
A specific calibration curve should be studied using samples containing an
analyte in the range of the QL. The residual standard deviation of a regression
line or the standard deviation of y-intercepts of regression lines may be used as
the standard deviation.
In all cases, the quantitation limit can be subsequently validated by the analysis
of a suitable number of samples known to be near or prepared at the quantitation
limit or reporting level.
Two possible approaches include:
A)
Three replicate preparations of a spiked sample are prepared at the quantitation level or
reporting level and analyzed. Calculate the % recovery. Calculate the average of the
replicates and % RSD.