126 / 129
Abbott Nutrition Division
Simultaneous Determination of Total Vitamin B
6,
B
2,
B
3
and B
1
in Infant
Formula Products by LC-MS/MS Using Enzymatic Digestion
8.
Calibration
a)
Calibration curves must have a correlation coefficient, r
2
, of >0.990.
i)
The calibration curve for a compound may be shortened to as few as 5 points, i.e. analyte
concentrations. This can occur due to an error in working standard preparation or
compromised instrument performance especially for lower standards (e.g. WS1 or WS2). Care
must be taken when doing so to ensure that the response factor for each product in the sample
set is within the response factor range of the remaining working standards. For example, if
WS1 for Riboflavin is outside the acceptance criteria, the WS1 level (all injections) may be
dropped for Riboflavin and the samples calculated using a calibration curve from WS2 to
WS6.
ii)
If individual standards are to be dropped due to atypical instrument response or injection error,
etc, care must be taken not to exclude too many working standard calibration points. As a
general rule, no more than 25% of the total number of working standard data points should be
excluded from a calibration curve while ensuring that the response factor for each product in
the sample set is within the response factor range of the remaining working standards.
iii)
If working standards are removed method performance criteria should be re-evaluated (Control
value, standard curve correlation, residuals, etc) to ensure each is met prior to approving and
reporting data.
iv)
Method deviations involving the removal of working standards from the final calibration curve
should be noted on the Method Worksheet and are to be reviewed and approved by the method
supervisor, system administrator or designee prior to the approval and reporting of data.
v)
Atypical instrument response should be noted on the Method Worksheet and reviewed by the
method supervisor and system administrator (or designees) to determine whether maintenance
is indicated.
b)
Calibration curve residuals (relative error) must be < 15% for WS1 and <10% for WS2-WS6.
i)
If the measured residual exceeds this limit, check for proper peak integration. If correction is
required, correct and recalculate.
ii)
A standard injection with an unacceptable residual can be excluded within guidelines discussion
above.
G.
MISCELLANEOUS - TIPS AND TROUBLESHOOTING
1.
System Operation
a)
Prior to starting operation ensure the Weak Needle Wash, Strong Needle Wash and Seal Wash
solvent reservoirs have been filled with the appropriate solvents. The system will shutdown should
any of these solvents run out during the course of an analysis.
b)
Communication between the computer and the UPLC and mass spectrometer is performed over an
Ethernet. After starting a sample set, monitor the UPLC to ensure that the computer and UPLC are
communicating. If the UPLC does not start within 2 to 3 minutes, the communication has failed.
To restore communication between the PC and the UPLC, delete the acquisition queue from the
42
BVIT-02
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE