36 / 258
L
in
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
1, 2017
147
(f)
50% Ethanol
.—Use a graduated cylinder to transfer
250 mL ethanol to a 500 mL volumetric flask. Dilute to
volume with laboratory water. Expiration: 1 month at room
temperature.
(g)
50% Methanol
.—Use a graduated cylinder to transfer
500 mL of methanol into a 1 L volumetric flask. Dilute to
volume with laboratory water. Expiration: 1 month at room
temperature.
(h)
Postcolumn reagent
.—Use a volumetric pipet to transfer
0.8 mL streptavidin fluorescein into a 1 L volumetric flask and
dilute to volume with biotin mobile phase. Expiration: 1 week
at room temperature.
(i)
Biotin stock standard solution (100 μg/mL)
.—
Weigh 0.05000 (±0.0050) g USP biotin reference standard.
Quantitatively transfer the standard to a 500 mL volumetric
flask using 50% ethanol. Mix well. Transfer to a 500 mL amber
bottle. Store refrigerated. Expiration: 3 months.
(j)
Biotin intermediate solution (1000 ng/mL)
.—Use
a volumetric pipet to transfer 1 mL biotin stock standard
solution into a 100 mL volumetric flask. Dilute to volume
with laboratory water. Mix well. Expiration: make fresh on
day of use.
(k)
Biotin working standards (5, 10, 20, 40, 60, 80, and
100 ng/mL)
.—For the 100 ng/mL working standard, use a
volumetric pipet to transfer 5mLbiotin intermediate solution into
a 50 mL volumetric flask. Dilute to volume with biotin mobile
phase and mix well. For the 5, 10, 20, 40, 60, and 80 ng/mL
working standards, use volumetric pipets to respectively transfer
0.5, 1, 2, 4, 6, and 8 mL of the 100 ng/mL working standard into
10 mL volumetric flasks. Dilute the volumetric flasks to volume
with biotin mobile phase and mix well. Expiration: make fresh
on day of use.
E. Sample Preparation
Note:
Because biotin is light-sensitive, all samples must
be prepared, handled, and stored in the dark or under yellow-
shielded lighting (
see
B
), unless otherwise stated.
(a)
Accurately weigh up to 4 g powder product or up to
20 g liquid product in a 50 mL volumetric flask. The final
concentration of biotin in the 50 mL solution should be in
the range of 8–60 ng/mL. Add 20 mL water to reconstitute
powder samples or add an appropriate amount of water to
dilute to a total volume of 24 mL for liquid products. Swirl
to mix well.
(b)
Add 10 mL 50% methanol to each sample. Swirl to mix
well.
(c)
Add 7.5 mL 2% metaphosphoric acid to each sample.
Immediately swirl to mix well.
(d)
Dilute the volumetric flasks to volume with laboratory
water. Invert the flask to mix well.
(e)
Transfer approximately 2 mL of each sample into a
microcentrifuge tube. Centrifuge at 10000 ×
g
for 10 min.
(f)
Filter the supernatant of each sample through syringe
filter into an HPLC autosampler vial.
F. Instrumentation
(a)
Instrumental operating conditions
.— (
1
)
FD wavelength
parameters
.—
λ
ex
= 495 nm,
λ
em
= 518 nm.
(
2
)
Run time
.—15 min.
(
3
)
Injection volume
.—20 μL.
(
4
)
Column temperature
.—20°C.
(
5
)
Mobile phase flow rate
.—For each injection run, the
mobile phase flow rate starts at 0.4 mL/min and the column
Figure 2016.11A. HPLC system configuration with a column switch.
36