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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

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 USE ONLY

DO NOT DISTRIBUTE