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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
MassLynx main page. Shutdown the Acquity control panel, the inlet editor, the tune page and
finally the MassLynx main window. Run the program “Stop Acquity Process” from the PC
desktop. Once the program completes launch MassLynx and allow the software to fully load. If
upon startup MassLynx does not require a security login or comes up with a sample queue running
the PC will need to be rebooted to clear the error. From the main window of MassLynx, launch
the Tune and Inlet editors. From the desktop, launch the Acquity Console. Once all four process
windows are loaded verify that the tune page and Acquity console show the system ready for
analysis. The sample list can then be restarted.
2.
Chromatography
a)
Column Stability
i)
Previous experience has indicated that the BEH C-18 column is stable for approximately 2000
injections.
ii)
The column should be replaced when peak shape (e.g. tailing, fronting, of broadening) or
retention times indicate deteriorating performance.
b)
Pyridoxine
i)
Pyridoxine normally elutes at approximately 1.47 minutes. The retention of Pyridoxine is readily
affected by changes in mobile phase composition. If the acid concentration in mobile phase A
is incorrect the retention time of Pyridoxine will shift significantly.
ii)
Pyridoxine can also be used as a good indicator of column performance. Past experience has
demonstrated that as the column begins to fail the retention of Pyridoxine shifts earlier from ~
1.5 minutes to ~1.2 minutes.
3.
Mass Spectrometry
a)
Sensitivity
Data review should include monitoring of the signal to noise (S/N) of the low working standard
of each compound. Pyridoxamine and Pyridoxal have the weakest signal of the compounds being
analyzed. The S/N for both compounds should be greater than 20. If routine cleaning of the
source fails to restore system performance contact the system administrator or Waters service.
b)
Signal Saturation
Waters triple quadrupole mass spectrometers have a maximum signal intensity of 1.34e
8
in SRM
analysis. A signal that approaches this level may be saturating the detector and not producing a
true response for the injected concentration. Evaluation of the high standards and samples should
be conducted for samples with intensities greater than 1.0e
8
. These injections should be evaluated
for peak shape (flat topped) and residuals of standards to ensure that the detector is not at
saturation.
4.
Quantitation
a)
Response Ratio Evaluation
i)
Care should be taken to review the response ratio for all compounds for the sample to ensure that
the analyte concentration for the analytical sample is within the concentration range of the
43
BVIT-02
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE