Page 82 - CRFTApplication Compendium_090613

4 Integrated Targeted Quantitation Method for Insulin and its Therapeutic Analogs
n of insulin and different insulin
be effective. The sensitivity and
t reach biological levels as well as
ful internal standard was included
reparation, LC-MS analysis, and
tive for most insulin variants to
biological levels, facilitate a low-
ate the workflow to expedite
ect is based on effective targeted
shows the automated steps to first
pounds, and elution into a new
e was then prepped for LC-MS
viously reported while increasing
xactive mass spectrometer.
FIGURE 8. LC-MS data analysi
spiked into plasma. The four
nM, Humulin S (0.06 nM), and
full scan spectrum was avera
elutes 0.5 minutes prior to the
FIGURE 6. Normalized quantit
porcine response was used to
valently bounded pan-insulin Ab
ed using the same protocol.
lution, the samples were dried
olvent composition.
the Pinpoint 1.3 software based
ulin variant was extracted
sor charge states. Integrated
generate the reported values.
to score each insulin variant
top and stop, apex, and tailing
lap.
FIGURE 3. Comparative analysis of insulin variant extraction using a common
workflow, including the same tips, LC separation, MS data acquisition, and data
processing. Each sample was prepared by spiking 0.24 nM of each variant in
different wells. The measured results are listed in each figure as well as the
results for porcine (Figure 3e). The Pinpoint processing method included
precursor m/z values for each variant.
Hum Lan Bov Nov
Humulin S Lantus Bovine
Hum Lan Bov Nov Hum Lan Bov Nov
Novorapid Porcine
Hum Lan Bov Nov Hum Lan Bov Nov
69:100:28
3.87e7
0.998
0.88 ppm
91:100:39
3.79e7
0.998
0.93 ppm
91:100:39
5.51e6
0.981
0.94 ppm
63:100:31
4.70e6
0.991
0.73 ppm
3b 3a 3c
3d 3e
75:100:28
3.87e6
0.995
0.81 ppm
R2 = 0.989
y = 74399239x – 110936.40712
FIGURE 4. Targeted quantitation curve for Humulin S. The measured AUC values
were summed from 18 isotopic m/z value across three charge states.
data enabled sufficient selectivity to
nal using multiple precursor charge
as shown in Figure 2 demonstrates
profiles. Data dependent MS/MS
ermination as well. (data not shown)
sequence confirmation (MS/MS)
riants, but to perform significant post-
wn, provided the b-chain epitope
parative extraction and detection
n variants. FIGURE 5. Qualitative output from Pinpoint software to evaluate 5a) precursor
charge state and 5b) +5 isotopic distribution for each spiked Humulin S levels in
plasma.
Mono
A+5
A+4
A+3
A+2
A+1
0.0150.03 0.060.12 0.240.48 0.96 Amount Spiked into Plasma 0.0150.03 0.060.12 0.24 0.480.96 Amount Spiked into Plasma
Mono
A+5
A+4
A+3
A+2
A+1
Humulin S Porcine
+4 +4
+5 +5
+6 +7
FIGURE 7. Normalized quantit
Each variant was spiked into t
porcine in all samples. The re
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 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
P
R2 = 0.998
y = 13.211x + 0.