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ABBOTT NUTRITION DIVISION

TITLE

PAGE NO.

Determination of Biotin in Infant, Pediatric and Adult

Nutritionals by HPLC and Fluorescence Detection

7 of 11

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)

FOR ERP USE ONLY

DO NOT DISTRIBUTE