Basic HTML Version
Bioscience Methods
BM 2011, Vo.2, No.1
http://bm.sophiapublisher.com
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Table 1 Data of the standard curve for stock solution
Concentration (μg/mL)
Peak
area
719
360
180
89.9
44.9
1
st
946 349.5
466 900.1
217 347.9
72 387.9
23 442.9
2
nd
1 051 213.0
461 082.5
186 301.4
70 680.6
19 649.2
Average
998 781.3
463 991.3
201 824.7
71 534.3
21 546.1
Figure 4 The standard curve for stock solution
Note: The equation reveals the mathematical expression of
standard curve and the correlation coefficient of standard curve
is 0.999 82
1.2.2 Standard curve for product
Using the external standard method as well, we
prepared gradient NGF concentration of products
based on the production formula by adding human
serum albumin and mannitol into NGF standard (keep
the same concentration of human serum albumin 1%
V and mannitol 5% V as selling products) to
manufacture the standard curve. Detected by
SEC-HPLC, we got chromatograms and the data of
retention time, peak height, peak area and other
parameters in every 90 μL injection. Compiling the
data of two parallel experiments in average, the stan-
dard curve was got in the linear range of 399.4 μg/mL
to 79.9 μg/mL (has manufactured the standard curve
in three different times, and reveals high repeatability
that RSD <1.42%) (Figure 5; Figure 6; Table 2).
1.3 Detection of NGF stock solution
The NGF stock solution of production batch I010 and
H015 were detected by SEC-HPLC based on the
chromatographic conditions established above (three
parallel experiments in average). The results were
calculated through the standard curve and compared
with that of BCA method (Table 3). There are tiny
Figure 5 The chromatograms of manufacturing the standard
curve for product
Note: The peaks around 15 minutes represent NGF and other
peaks for human serum albumin. Different color peaks reveal
the NGF in different concentrations
Figure 6 The standard curve for product
Note: The equation reveals the mathematical expression of
standard curve and the correlation coefficient of standard curve
is 0.999 97
differences of results between the two methods, that
proving of the reliability of SEC-HPLC quantitative
method in detecting NGF stock solution.