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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.

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