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et al
.:
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ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
1, 2017
proteins to produce a filtrate, which is subjected to subsequent
HPLC analysis. The mobile phase (20% methanol in 0.02 M
phosphate buffer at pH 7.0) is pumped at 0.4 mL/min. The
postcolumn reagent (0.8 μg/mL streptavidin fluorescein in
biotin mobile phase) is pumped at 0.2 mL/min. The biotin, after
eluting from the column, binds with streptavidin fluorescein to
become a fluorescent conjugate and is detected by an FD. 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.
B. Apparatus
Common laboratory equipment and, in particular, the
following:
(a)
HPLC system
.—Two isocratic pumps; autosampler
capable of injecting 20 μL; FD; high-pressure mixing tee;
Aura’s postcolumn knitted reactor coil: 15 m Teflon tubing
(0.25 mm id, 1/16 in. od), 0.75 mL total volume, Cat. No. KRC
15-25, or equivalent; and reactor coil–compatible VALCO
nuts and grooved ferrule, or equivalent. The system should
be configured as shown in Figure
2016.11A
when the column
switch is used as an option.
(b)
Cover
.—Adequate to keep the postcolumn reaction coil
from light.
(c)
Analytical column
.—Agilent Poroshell 120 EC-C18,
4.6 × 50 mm, 2.7 μm, or equivalent.
(d)
Analytical balance
.—Capable of weighing to the nearest
0.00001 g.
(e)
pH Meter
.
(f)
Microcentrifuge
.—Capable of centrifuging at 10 000 ×
g
.
(g)
Microcentrifuge tubes
.—2 mL.
(h)
Disposable syringe
.
(i)
Syringe filter
.—Nylon 0.45 μm or equivalent.
(j)
Filter assembly for filtering mobile phase
.
(k)
Nylon filter membrane for filtering mobile phase
.—0.45 μm
or equivalent.
(l)
Graduated cylinders
.—Assorted sizes.
(m)
Disposable transfer pipets
.
(n)
Volumetric flasks
.—Class A, assorted sizes.
(o)
Volumetric pipets
.—Class A, assorted sizes.
(p)
Yellow lights or yellow shields with a cutoff of 385 nm
.
(q)
Magnetic stirring plate
.
(r)
Magnetic stirring bars
.
C. Chemicals and Reagents
(a)
Biotin standard
.—U.S. Pharmacopeia (USP) reference,
official lot. Store per label instructions.
(b)
Ethanol (reagent alcohol)
.—American Chemical Society
(ACS) or equivalent.
(c)
Streptavidin fluorescein conjugate
.—1mg/mL, GeneTex
Streptavidin–fluorescein isothiocyanate (Streptavidin-FITC;
Cat. No. GTX30950) or AnaSpec Streptavidin-FITC (Cat. No.
AS-60659-FITC), or equivalent. Store per label instructions.
(d)
Laboratory water
.
(e)
Methanol
.—HPLC grade or equivalent.
(f)
pH Meter buffer solutions for pH meter standardization
.
(g)
Metaphosphoric acid
.—ACS, 33.5–36.5%, or equivalent.
(h)
Sodium phosphate monobasic (SPM)
.—ACS or equivalent.
(i)
Sodium phosphate dibasic (SPD)
.—ACS or equivalent.
D. Standard and Solution Preparation
Note:
Because biotin is light sensitive, all standards must
be prepared, handled, and stored in the dark or under yellow-
shielded lighting (
see
B
), unless otherwise stated.
(a)
0.2 M SPM
.—Weigh 27.800 (±2.780) g SPM into a 1 L
volumetric flask. Add about 800 mL laboratory water and mix
until all solids dissolve. Dilute to volume with laboratory water.
Mix well. Expiration: 1 month at room temperature.
(b)
0.2 M SPD
.—Weigh 28.390 (±2.840) g SPD into a 1 L
volumetric flask. Add about 800 mL laboratory water and mix
until all solids dissolve. Dilute to volume with laboratory water.
Expiration: 1 month at room temperature.
(c)
0.02 M phosphate buffer (pH 7)
.—Place a 400 mL beaker
on a stir plate. Using graduated cylinders, transfer 78 mL 0.2 M
SPM and 168 mL 0.2 M SPD into the beaker. Adjust pH of the
solution in the beaker to 7.00 (±0.05) using SPM and SPD. This
solution is 0.2 M phosphate buffer (pH 7) and is made fresh for
mobile phase preparation. Using a graduated cylinder, transfer
200 mL 0.2 M phosphate buffer (pH 7) into a 2 L volumetric
flask. Dilute to volume with laboratory water and mix well. This
solution is 0.02 M phosphate buffer (pH 7). Expiration: 1 week
at room temperature.
(d)
Biotin mobile phase
.—Use a graduated cylinder
to transfer 400 mL methanol into a 2 L volumetric flask.
Dilute to volume with 0.02 M phosphate buffer (pH 7). Mix
well, filter, and degas in a sonicator for 5 min. Transfer to a
suitable mobile phase container. Expiration: 1 week at room
temperature.
(e)
2% Metaphosphoric acid
.—Weigh 60 (±6) g of
metaphosphoric acid (33.5–36.5%) into a 1 L beaker. Add
about 800 mL laboratory water and mix until all solids dissolve.
Quantitatively transfer the solution to a 1 L volumetric flask,
dilute to volume with laboratory water, and mix well. Store
refrigerated. Expiration: 1 week.
Figure 1. Chemical structures of biotin and biocytin.
35