Integrated Targeted Quantitation Method for Insulin and its Therapeutic Analogs

Program: Late-Breaking Abstracts
Session: MON-LB-Late-Breaking Poster Session 3
Bench to Bedside
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-LB-08
Eric E Niederkofler*1, Tara Schroeder2, Urban A Kiernan1, David A Phillips2, Kemmons A Tubbs2, Scott Peterman2, Bryan Krastins2, Amol Prakash2, Mary Lopez2 and Dobrin Nedelkov2
1Thermo Fisher Scientific, Tempe, AZ, 2Thermo Fisher Scientific
Novel Aspect
Incorporation of pan-Ab to capture human insulin and 5 commercially available analogs for global LC-SRM detection and quantitation assay.

The need to detect and quantify insulin and its analogs has become paramount for both medical and sports doping applications.  Insulin levels are typically present at sub ng/mL levels and are generally in the presence of low molecular weight background material requiring extraction/enrichment to increase the concentration prior to detection/quantitation.  In addition, slight sequence variations are used to change the bioavailability further complicating high-throughput quantitation. To date, researchers have utilized generic enrichment methods such as SPE to decrease background matrix effects.  We have developed a pan-insulin antibody capture method coupled to LC-SRM for high-throughput quantification for human insulin and variants.

A series of samples were prepared neat and in serum using human insulin and five additional variants.  The different insulin analogs were prepared independently and mixed at different levels to test the selectivity and sensitivity of the enrichment method employed.  Target enrichment was performed using custom MSIA D.A.R.T.’S derivatized with a pan-anti insulin antibody.  All detection and quantitation experiments were performed using LC-SRM on a newly released triple quadrupole mass spectrometer.  SRM transitions unique to each analog were optimized for the intact insulin molecules as well as for the corresponding beta chains.  Both sets were tested using a 15 minute experimental methods. 

Preliminary Data
The primary limitations to routine, high-throughput targeted quantitation of insulin and its various analogs have been limited by inefficient extraction/enrichment protocols.  The incorporation of custom MSIA D.A.R.T.’S loaded with the pan-insulin Ab facilitated capture for all variants from the samples while significantly decreasing the background matrix.  The increased capture efficiency permitted the development of an 8 minute method.  The pan-Ab has been shown to recognize a common epitope region in the beta chain that is conserved across all variants.  A unique set of SRM transitions were developed for each variant, intact as well as the beta chains and all transitions were included in a single, multiplexed method to identify presence/absence of each variant and relative/absolute quantitative determination.  The initial results demonstrated LOQ values <200 pg/mL for the intact variants and <40 pg/mL for the beta chains in serum.  In addition, the method was shown to detect the presence of multiple variants at spiked ratios in excess of 200-fold. 

Disclosure: EEN: Employee, Thermo Fisher Sceintific. TS: Employee, Thermo Fisher Sceintific. UAK: Employee, Thermo Fisher Sceintific. DAP: Employee, Thermo Fisher Sceintific. KAT: Employee, Thermo Fisher Sceintific. SP: Employee, Thermo Fisher Sceintific. BK: Employee, Thermo Fisher Sceintific. AP: Employee, Thermo Fisher Sceintific. ML: Employee, Thermo Fisher Sceintific. DN: Employee, Thermo Fisher Sceintific.

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