Page 83 - CRFTApplication Compendium_090613

5 Thermo Scienti c Poster Note • PN ASMS13_M503_ENiederko er_E 07/13S
Conclusion
The targeted workflow succes
variant extraction, LC separati
pan insulin Ab was sensitive f
 The automated sample
enrichment/extraction st
 Detection and quantitati
 The MSIA D.A.R.T. tips
for singly or multiply spi
 Decoupling data used f
reprocessing for potenti
 The Pinpoint 1.3 softwar
quantification for all insu
References
1. Thevis, M, Thomas, A.,
50
2. Nelson, R. W., Krone, J.
1153-58
FIGURE 8. LC-MS data analysis of sample processing of four insulin variants
spiked into plasma. The four samples are Lantus and Glulisine spiked at 0.48
nM, Humulin S (0.06 nM), and porcine as the internal standard. The resulting
full scan spectrum was averaged across the three co-eluting variants. Lantus
elutes 0.5 minutes prior to the three displayed insulin variants.
FIGURE 6. Normalized quantitative curve for Humulin S in plasma. The measured
porcine response was used to normalize each level.
FIGURE 9. Comparative qua
spiked into plasma at differe
into each sample at a const
insulin. All AUC values wer
iant extraction using a common
ion, MS data acquisition, and data
iking 0.24 nM of each variant in
d in each figure as well as the
rocessing method included
ntus Bovine
ov Nov Hum Lan Bov Nov
Porcine
Hum Lan Bov Nov
:100:39
.79e7
0.998
93 ppm
91:100:39
5.51e6
0.981
0.94 ppm
3c
75:100:28
3.87e6
0.995
0.81 ppm
mulin S. The measured AUC values
ss three charge states.
ftware to evaluate 5a) precursor
or each spiked Humulin S levels in
0.0150.030.060.12 0.240.480.96 Amount Spiked into Plasma
Mono
A+5
A+4
A+3
A+2
A+1
Porcine
+4
+5
+6
FIGURE 7. Normalized quantitative curves for bovine and Lantus insulin variants.
Each variant was spiked into the plasma separately with a constant amount of
porcine in all samples. The relative curves are reflective of the measured response
shown in Figure 3 .
0
20
40
60
80
100
20
40
60
80
100
Relative Abundance
20
40
60
80
100
6.2 7.0 7.8 8.6 9.4Time (min)
7.97
8.01
7.92
5.5E4
Humulin S
0.06 nM
Porcine
0.05 nM
4.8E4
Glulisine
0.48 nM
2.2E5
800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
802.5158z=1
846.5408z=1 1012.6253z=1
1056.6504z=1 1165.3358z=5 1456.4173z=4 935.5010z=1 996.6330z=1 1100.6767z=1
1156 1158 1160 1162 1164 1166 1168 1170 1172 1174m/z
1165.3358z=5
1162.3342z=5
1156.3319z=5
1172.9234z=5
1170.1277z=5 1160.7735
Glulisine
Porcine
Humulin S
A secondary test was perform
spiked at different levels on tar
resulting full scan MS to demo
insulin variants. The mass spe
formed predominantly singly c
which are ca. 2-5% of the total
extraction. Figure 9 shows th
over same dynamic range as
constant level. Porcine insulin
difference in measured signal,
resulting linear regression was
could be determined based on
the y value of 1.1649 from Fig
amount was 0.078 nM compar
Acknowledge
The authors would like to than
the donation of the insulin vari
For research use only. Not f
Humulin® is a registered trademark of E
Fisher Scientific and its subsidiaries.
This information is not intended to enco
intellectual property rights of others.
R2 = 0.998
y = 13.211x + 0.12833