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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 USE ONLY

DO NOT DISTRIBUTE