37 / 258
148
L
in
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
1, 2017
switch valve position starts at 1
→
2. At 5 min, after biotin is
eluted and detected by the FD, the valve position is switched to
1
→
6. The flow rate then ramps to 1.5 mL/min over 1 min and
keeps until 12 min, when riboflavin is eluted and detected by the
FD. Lastly, the flow rate decreases to 0.4 mL/min over 1 min
before the valve position is switched back to 1
→
2. The flow rate
keeps at 0.4 mL/min until 15 min.
(
6
)
Postcolumn pump flow rate
.—0.2 mL/min.
(b)
Instrument configuration with an optional column
switch
.—The system should be configured as shown in
Figure
2016.11A
.
(c)
System pressure
.—Column pump head pressure
maximum at 600 bar or per column manufacturer’s instructions;
postcolumn reaction coil head pressure maximum at 40 bar or
per manufacturer’s instructions.
(d)
System equilibration
.—(
1
) Turn on the FD at least 1 h
before start of analysis.
(
2
) Inject the most concentrated standard (approximately
100 ng/mL) onto the column and observe the response on
the FD. If necessary, adjust the detector gain and sensitivity
settings so that the standard curve is within the range of the
detector. After the detector settings have been determined,
inject the most concentrated standard three to four 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 is >2%,
then 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. After the system
has reached equilibrium and the RSD is ≤2%, inject a set of
standards, unknown samples, and another set of standards.
Every set of unknown samples must be bracketed by standards.
(e)
Column and system maintenance
.—The column,
postcolumn reaction coil, and system may be cleaned by using
50% methanol at an appropriate pressure, referring to
F(c)
.
G. Calculations
Quantification is obtained by using a seven-level external
standard consisting of the following concentrations: 5,
10, 20, 40, 60, 80, and 100 ng/mL. The calibration of the
standards is determined by using a polynomial regression
curve (cubic-fit).
(a)
Calculation of stock standard concentration:
SS = S
w
× P ÷ 500 × D
1
where SS = the stock standard concentration (μg/mL); S
w
=
the standard weight (g); P = the purity of the standard (g/g);
500 = the volume of the stock solution (mL); and D
1
= the unit
conversion factor: D
1
= 1000000 μg/g.
(b)
Calculation of intermediate standard (IS) concentration:
IS = (volume of SS used) × (SS concentration) ÷ dilution volume
(c)
Calculation of working standards concentration level
7 (WS7):
WS7 = (volume of IS used in mL) × (concentration of IS in
µ
g/mL) ÷ (WS7 volume in mL) × 1000 ng/
µ
g
(d)
Calculation of working standards concentration level
1–6 (WS1–6):
WS1–6 = (volume of WS7 used in mL) × (concentration of
WS7 in ng/mL) ÷ (WS1–6 volume in mL)
(e)
Calculation of the biotin concentration in the injected
product samples (C
i
) is from its biotin peak area and the standard
curve generated from the standards.
(f)
Calculation of original product concentration (C
p
) is,
therefore, based on the dilution scheme used for the sample:
C
p
= C
i
× D
0
÷ ss × D
1
× D
2
where C
p
= the original product concentration (μg/100g); C
i
= the
injected sample’s biotin concentration, from standard curve
(ng/mL); D
0
= the dilution of original product before filtration:
D
0
= 50 mL; ss = the sample size (g); D
1
= the unit conversion
(from per g to per 100 g sample and from ng to μg of biotin):
D
1
= (100 g/100 g)(1 μg/1000 ng) = 1/10 g·μg/ng/100 g; and
D
2
= the conversion from sample to reconstituted finished product:
for powder, D
2
= 25 g powder ÷ (25 g powder + 200 g water) =
25 g ÷ 225 g = 1/9; for liquid (2× dilution by weight), D
2
= 2; and
for ready-to-feed (RTF; as is), D
2
= 1.
Results and Discussion
Method Validation
This method has undergone a thorough single-laboratory
validation (SLV) usingAOAC INTERNATIONAL guidelines to
probe its linearity, LOQ, specificity, accuracy, and ruggedness.
The analytical range for SPIFAN biotin-fortified matrixes
was found to be between 1.7 and 142 μg/g reconstituted final
product or RTF.
Calibration fit
.
—
During each analytical run, seven standards
with biotin concentrations ranging from 5 to 100 ng/mL were
injected before and after each sample set. Calibration curves were
constructed from these standards using a polynomial regression
curve (cubic-fit) and used to back-calculate the concentration of
each working standard in order to calculate calibration error at
each level. The method demonstrated good polynomial regression
(cubic) fit, over a standard range of 5–100 ng/mL biotin, with
r
2
>0.9990. The calibration errors for the lowest two levels (near
the LOQ level) are around 25% and 10%; the calibration errors
for the remaining levels were <8% (Table 1; Figure 2).
Suitable calibration curve range
.
—
Due to the characteristics
of the postcolumn protein binding reaction, saturated
Table 1. Representative calibration standard data
Level
Concentration,
ng/mL
HPLC peak
area
Amount, ng/mL Error, %
1
5
0.2019
6.21
24
2
10
0.6600
9.00
10
3
20
2.2932
18.61
7
4
40
6.1743
41.03
3
5
60
9.2305
60.45
1
6
80
11.353
77.66
3
7
100
12.863
103.9
4
37