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ABBOTT NUTRITION DIVISION
TITLE
PAGE NO.
Determination of Biotin in Infant, Pediatric and Adult
Nutritionals by HPLC and Fluorescence Detection
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e)
Mobile phase flow rate: for each injection run, the mobile phase flow rate starts at 0.4 mL/min and
the column switch valve position starts at 1
Æ
2. At 5 min, after biotin is eluted and detected by FLD,
the valve position is switched to 1
Æ
6. The flow rate then ramps to 1.5 mL/min over 1 min time and
keeps till 12 min when riboflavin is eluted and detected by FLD; finally the flow rate decreases to
0.4 mL/min over 1 min time before the valve position is switched back to 1
Æ
2. The flow rate keeps
at 0.4 mL/min till 15 min.
f)
Post column pump flow rate: flow rate for the post column reagent is 0.2 mL/min.
g)
Column switch setup.
h)
System pressure: column pump head pressure maximum at 600 bar; post column reactor pressure
maximum at 40 bar.
2.
System Equilibration
a)
Fluorescence detector: turn on the detector at least one hour prior to start of analysis.
b)
Inject the most concentrated standard (approximately 100 ng/mL) onto the column and observe the
response on the fluorescence detector. If necessary, adjust the detector gain and sensitivity settings
so that the standard curve is within the range of the detector. Once the detector settings have been
determined, inject the most concentrated standard 3
–
4 times and note the peak areas. If the system
is equilibrated, the RSD of the standard peak areas should be < 2% and the peak areas should not
steadily increase or decrease by more than 4% from the first injection to the third or fourth injection.
If the RSD >2%, locate the source of the imprecision and correct it before beginning the sample
analysis. If peak areas steadily increase or decrease by more than 4%, the system is not equilibrated
and must be allowed to equilibrate longer. Once the system has reached equilibrium and the RSD is
≤2%, inject a set of standards, the control sample
(SRM 1849a), unknown samples, and another set
of standards. Every set of unknown samples must be bracketed by standards and the first sample in
each set must be the control sample.
Bio-03 (February 2016)
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