Proteomics, 2014 Jun:  12(14):  1445-56

An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HRAM)

Peterman, S. M.1, Niederkofler, E. E.2, Phillips, D. A.2, Krastins, B.1, Kiernan, U. A.2, Tubbs, K. A.2, Nedelkov, D.2, Prakash, A.1, Vogelsang, M. S.1,  Schroeder, T.3, Couchman, L.4, Taylor, D. R.4, Moniz, C. F.4, Vadali, G.1, Byram, G.1, and Lopez, M. F.1

1Thermo Fisher Scientific, BRIMS, Cambridge, MA, USA

2Thermo Fisher Scientific, Tempe, AZ, USA

3Thermo Fisher Scientific, Somerset, NJ, USA

4Department of Clinical Biochemistry, King’s College Hospital NHS Foundation Trust, London, UK



The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensics applications.  Synthetic variants contain slight sequence variations to affect bioavailability.  To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS.  A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report.  The assay is sensitive, selective, semi-automated, and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.

PMID: 24668948; Pmic:  201300300



The role of insulin on metabolic pathways has led to tremendous amounts of research for many decades.  With increased understanding of insulin and its role in signaling, biotherapeutic variants have been created to specifically perturb metabolic rates based on specific sequence variants.  As a result, drug development, medical, sports doping, and forensic applications desire the ability to attribute total activity to particular compounds or their metabolites. Due to different cross-reactivities of various immunoassays, the ability to ascertain this information has been lacking.  Therefore, methods to enable this have been of interest.

The focus of the study was to develop a routine, automated mass spectrometric immunoassay (MSIA™) to confirm the presence of and quantify endogenous insulin and any known variants present in plasma/serum.  To achieve the stated goals, the resulting assay must be sensitive enough to detect and robustly quantify targeted analytes at sub ng/ml levels while maintaining a wide dynamic range (3 orders of magnitude).   Selectivity afforded by the assay becomes critical for confidently differentiating target analyte spectral signal from the background matrix.  The resulting method must be universally applicable for endogenous insulin as well as administered insulin variants individually or simultaneously without the need to drastically change the components of the assay.


f1Figure 1.  Targeted extraction process using covalently bound pan-insulin Ab to MSIA D.A.R.T.’S.  All samples were processed using the same protocols


The most critical aspect of the assay is related to its efficiency and selectivity to simultaneously extract each of the various insulin variants from the plasma/serum matrix.  Incorporation of the Thermo Scientific™ insulin MSIA™ Disposable Automated Research Tips (D.A.R.T.’S), devices integrating affinity micro-columns coated with pan insulin antibody into a functional pipette tip, facilitates automated extraction of insulin and insulin variants from plasma through the use of an automated liquid handler.  (Figure 1)  Therefore, all insulin variants are extracted simultaneously, conserving time, buffers, and plates which directly feed into the standardized liquid chromatography mass spectrometry (LC-MS) analysis. Utilizing a pan-insulin Ab enabled incorporation of an internal standard used to determine targeted insulin variant amounts as well as enable quality control/system suitability determination.  Additionally, the samples post-extraction of insulin remain relatively unchanged, allowing for further processing of the samples for the analyses of other proteins using MSIA D.A.R.T.’S incorporating other affinity reagents.

MSIA incorporation significantly reduce the biological matrix, thereby extending the detection and quantitation range for the insulin analysis.  Figure 2 shows the full scan MS for a sample spiked with three insulin variants at 50 pM for porcine insulin and 24 pM for Humulin S and Glulisine.  The matrix for the initial studies consisted of BSA/PBS.



Figure 2.  High resolution full scan MS spectrum showing the co-isolation and detection of three insulin variants spiked into plasma.  The inset shows the narrow mass region coving the +5 precursor charge state for Porcine insulin spiked as an internal standard, Humulin S, and Glulicine.


Performing the LC-MS experiments using the Q Exactive mass spectrometer facilitates high resolution/accurate mass analysis of the targeted analytes.  Detection using HR/AM MS strategies efficiently separate the insulin variant ion signal from the background matrix.  Automated data processing was performed to determine presence/absence and area under the curve (AUC) values to determine relative/absolute amounts of all insulin variant.  The Pinpoint software was used for all data processing.  A single workbook contained all of the targeted insulin variants and corresponding m/z values representing precursor and product ions.  By depleting the background matrix, the resulting data extraction was performed using ±5 ppm tolerance.  The top six isotopic m/z values per charge state was used for determination of presence/absence, verification, and quantification. A total of 18 extracted ion chromatograms (XICs) per analyte were used for processing. We utilized porcine insulin as an internal standard and spiked it into each sample prior to MSIA extraction.  Verification was done based on isotopic distribution overlap between the relative AUC values per isotope as compared to the theoretical isotopic distribution.  A calculated correlation coefficient 0.95 was used the acceptance criteria for all analytes.

The benefit of our method is that the entire routine can be applied to all known variants.  A single plate was prepared containing all samples spiked into the BSA/PBS and plasma matrix.  A total of eight levels were used to determine limits of detection and quantitation (LOD/LOQ) per insulin variant, and evaluate the putative background interference effects.   All samples were analyzed and processed in less than 15 hrs.  Figure 3 shows the cumulative results from spiking different insulin variants into human plasma.  The displayed range covers the lowest six levels (excluding 240 and 960 pM to demonstrate low-end linearity.



Figure 3.  Quantification curve for each of the variants as well as multiple variants spiked in the same sample.  Porcine was spiked into each sample as ISTD at 50 pM.


The results show linearity for each of the insulin variants across the lower range and all levels had measured RSD values less than 10% except the 1.5 pM level for Novolog.    In addition to extracting and accurately measuring the spiked insulin variant levels, the method also extracted endogenous human insulin.  The MSIA extraction demonstrated excellent capacity by simultaneously extracting two variants spiked at 960 pM as well as the porcine and endogenous human insulin.  The measured human insulin response was shown to be consistent across all samples.


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