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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
5.
UPLC ANALYSIS
a)
Upon verifying equilibration of the UPLC system, inject the working standards (WS1 to WS6)
followed by a mobile phase blank, control sample and sample extracts.
b)
Sample extracts are injected in single.
c)
Samples should be bracketed between two sets of working standards. Vitamin content in product
samples is then determined by comparison of the response ratio for the sample to the calibration
curve.
d)
For larger sample sets, working standards should be injected every 6 hours or approximately every
50 sample extracts.
e)
Calibration curves must have a correlation coefficient, r
2
, of >0.990 as defined in Section H
(Quality Control).
f)
Calibration curve residuals (relative error) must be < 15% for WS1 and <10% for WS2-WS6 as
defined in Section H (Quality Control).
g)
The Method utilizes calibration curves for each nutrient that cover approximately 2 orders of
magnitude in analyte concentration.
h)
The calibration curve for a compound may be shortened to as few as 4 points, i.e. analyte
concentrations. This can occur due to an error in working standard preparation or compromised
instrument performance especially for lower standards (WS1, WS2 or WS3). Care must be taken
when doing so to ensure that the response factor for each sample extract in the sample set is within
the response ratio range of the remaining working standards. For example, if WS1 for Pyridoxal is
outside the acceptance criteria, the WS1 level (all injections) may be dropped for Pyridoxal and the
samples calculated using a calibration curve from WS2 to WS6.
i)
If individual standards are to be dropped due to atypical instrument response or injection error, etc,
care must be taken to not 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.
30
BVIT-02
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE