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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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6.
The mobile phase (20% methanol in 0.02M phosphate buffer at pH 7.0) is pumped at 0.4 mL/min. The
post column reagent (0.8
P
g/mL streptavidin fluorescein in biotin mobile phase) is pumped at 0.2
mL/min. The biotin after eluting from the column binds with the streptavidin fluorescein to become a
fluorescent conjugate and is detected by the fluorescence detector (FLD).
7.
This method uses a column switch. The HPLC run takes 15 min for each injection when a column
switch is used. The column switch allows the elution of riboflavin at 11 min with a high flow rate of 1.5
mL/min. Without the column switch, the run takes 30 min for each injection with the elution of
riboflavin at 24 min.
C.
APPARATUS
1. Chromatography Instrumentation:
a)
Analytical column: Agilent Poroshell 120 EC-C18, 4.6 x 50 mm, 2.7
P
m or equivalent
b)
Agilent Autosampler G1329B or equivalent for 20 uL injection
c)
Agilent Fluorescence Detector G1321B or equivalent
d)
Agilent Quaternary Pump 1311B or equivalent isocratic pump for mobile phase
e)
Agilent Isocratic Pump 1310B or equivalent for post column reagent solution
f)
Agilent Column Compartment G1316A with column switch or equivalent
g)
Agilent OpenLAB Software or equivalent for data collection
h)
Post column knitted reactor coil
: 15 meter Teflon tubing (0.25 mm ID, 1/16” OD), total volume
0.75 mL, protected from light, Aura Industries, USA or equivalent
i)
Compatible VALCO nuts and grooved Ferrule 1/16" or equivalent for connection of knitted
reactor coil
j)
VALCO stainless steel T-connector w/0.25mm bore or equivalent for post column reaction
k)
HPLC autosampler vials, clear or amber glass vials with Teflon/silicon/Teflon caps
2. General Laboratory Equipment/Facilities
a)
Analytical balance, Mettler Toledo Microbalance XP 56 or equivalent for standard weighing
b)
Analytical balance, Mettler Toledo Balance AB304S or equivalent for sample weighing
c)
Cover adequate to keep post-column reaction coil from light
d)
Disposable syringe: 1 mL or equivalent
e)
Syringe filter: nylon 0.45
P
m or equivalent
f)
Repeating pipettes: 5-20 mL capacity or equivalent
g)
Filter assembly with nylon filter membrane, 0.45
P
m or equivalent
h)
Graduated cylinders: assorted sizes
i)
Lab wipes
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