Page 84 - CRFTApplication Compendium_090613

6 Integrated Targeted Quantitation Method for Insulin and its Therapeutic Analogs
Conclusion
The targeted workflow successfully demonstrated selective and sensitive insulin
variant extraction, LC separation, and quantitation using HR/AM MS for research. The
pan insulin Ab was sensitive for all six insulin variants used in the study.
 The automated sample extraction utilized one step as opposed to multiple
enrichment/extraction steps.
 Detection and quantitation ranges reached were at 0.015 nM in 0.5 L of plasma.
 The MSIA D.A.R.T. tips showed equivalent extraction and quantitative efficiency
for singly or multiply spiked insulin variants at different concentration ranges.
 Decoupling data used for quantitative and qualitative analysis facilitates
reprocessing for potential unknown insulin variants.
 The Pinpoint 1.3 software provides automated data extraction, verification, and
quantification for all insulin variants.
References
1. Thevis, M, Thomas, A., Schänzer, W. Mass Spectom. Reviews 2008, 27(1), 35-
50
2. Nelson, R. W., Krone, J., R., Bieber, A. L., Williams, P. Anal. Chem. 1995, 67,
1153-58
cessing of four insulin variants
tus and Glulisine spiked at 0.48
ternal standard. The resulting
ree co-eluting variants. Lantus
insulin variants.
umulin S in plasma. The measured
level.
FIGURE 9. Comparative quantitation curves for Lantus and Glulisine that were
spiked into plasma at different levels as well as Humulin S which was spiked
into each sample at a constant amount of 0.06 nM to replicate endogenous
insulin. All AUC values were normalized to the porcine AUC response.
bovine and Lantus insulin variants.
ately with a constant amount of
reflective of the measured response
1100 1150 1200 1250 1300 1350 1400 1450
m/z
.6504 1165.3358z=5 1456.4173z=4 1100.6767z=1
1162 1164 1166 1168 1170 1172 1174m/z
1165.3358z=5
1162.3342z=5
1172.9234z=5
1170.1277z=5
Glulisine
ulin S
A secondary test was performed to evaluate the effects of multiple insulin variants
spiked at different levels on targeted extraction and detection. Figure 8 shows the
resulting full scan MS to demonstrate the Q Exactive data acquisition of the different
insulin variants. The mass spectrum shows matrix from the MSIA elution solvents that
formed predominantly singly charged ions compared to the targeted insulin variants at
which are ca. 2-5% of the total signal and the resolution-facilitated peak detection and
extraction. Figure 9 shows the quantitation for two different insulin variants spiked
over same dynamic range as well as the porcine and Humulin S variants spiked at a
constant level. Porcine insulin was used as the internal standard. Despite the
difference in measured signal, each variant was detected at the lower levels and the
resulting linear regression was 0.99 or better. In addition, the amount of Humulin S
could be determined based on the individual quan curve presented in Figure 6. Using
the y value of 1.1649 from Figure 9 and the linear equation in Figure 6, the calculated
amount was 0.078 nM compared to the predicted amount of 0.06 nM.
Acknowledgements
The authors would like to thank Dr. Stephan Morely from the Sheffield Hospital, UK for
the donation of the insulin variants used in the study.
For research use only. Not for use in diagnostic procedures.
Humulin® is a registered trademark of Eli Lilly and Company. All other trademarks are the property of Thermo
Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.