concentration. The calibration curve is a plot of how the instrument responds (the analytical signal) to changes in
the concentration of the analyte (the substance to be measured). The operator prepares a series of standards
across a range of concentrations near the expected concentration of the analyte in the unknown sample.
Calibration standards are used to build calibration curves. Incorrect readings or missing readings on some of the
calibration samples might indicate issues with the analytical run. Follow acceptable methods found in literature
and regulatory agency guidances to create a calibration curve. Examples of good practice in the preparation of
calibration curves include:
• Preparing calibration standards in blank matrix in which the analyte is to be measured.
• Generating a calibration curve for each analyte to be measured.
• Making sure of the coverage of the expected concentration range of the analyte, including typical and atypical
specimens.
• Using six to eight standards to generate the curve.
This is not a comprehensive list and other guidances should be used in determining the best practice in developing
a calibration curve for the laboratory.
Note:
In some analytical runs, single-point calibration standards are used. Single-point calibrations are performed
using a matrix blank sample and a single standard concentration. The relationship between instrument response
and analyte concentration is determined by the line created by these two points. Both the acquisition and
processing methods should be validated before being accepted for their intended use.
Regression Equations
This section describes the equations used to calculate the regression curves. In the following equations, x represents
the analyte concentration for
Standard
samples and y represents the corresponding peak area or height. The
exact variables used for the regression depend on whether an internal standard is being used and whether the
peak area or the peak height is used as shown in
.
Table 2-2 Regression Variables
y
x
Area Used?
Internal
Standard Used?
A
a
/ A
is
C
a
/ C
is
/ DF
Yes
Yes
H
a
/ H
is
C
a
/ C
is
/ DF
No
Yes
A
a
C
a
/ DF
Yes
No
H
a
C
a
/ DF
No
No
where:
SCIEX X500 QTOF System
System User Guide
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RUO-IDV-05-2334-E
Principles of Operation
