MSIA Insulin Workflow for the Comprehensive Identification and Analysis of Isobaric Insulins: A Method for Insulin Mixture Quantitation Using HR/AM and Multiplexed LC Lewis Couchman, Senior Clinical Scientist, David R. Taylor, Senior Clinical Scientist, Caje F. Moniz, Clinical Lead and Head of Department, Department of Clinical Biochemistry, King’s College Hospital, London, UK.

Eric E. Niederkofler, R&D Manager, Amanda Ribar, Research Technician, Kwasi Antwi, Application Scientist and Urban A. Kiernan, Business Development Manager, Thermo Fisher Scientific, Tempe, Arizona.

Bryan Krastins, Senior Applications Scientist, Scott Peterman, Senior Applications Scientist, BRIMS Center, Thermo Fisher Scientific, Cambridge, Massachusetts.

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GoalTo provide a high-throughput mass spectrometric immunoassay (MSIA) Insulin workflow, with the inclusion of rich peak identification MS data, for the analysis of composite mixtures of isobaric insulins (insulin and insulin lispro) that may be present in human plasma.

IntroductionThe analysis of insulin is important (i) in the study of and development of treatments for metabolic diseases; (ii) in forensic toxicology, including post-mortem investigations, and (iii) in sports doping. Insulin physiology is complex and the range of therapeutic insulins continues to expand. Methods that are routinely used for insulin analysis, including traditional immunoassays, are unable to detect subtle structural differences among these insulin variants. As a result, there is a growing movement towards the analysis of insulins using LC-MS to provide additional assay specificity and allow simultaneous quantification of insulin species. However, currently developed methods are not well-suited to high-throughput applications and automation, and may fail to meet assay performance specifications that are being demanded by the industry.

Insulin lispro (Humalog®, Eli Lilly) is an insulin analog which is isobaric with human insulin (in insulin lispro, the lysine and proline residues at positions B28 and B29 are reversed – Figure 1). Differentiation of these two species has been reported by Thevis et al. and others due to a unique product ions in their MS2 spectra resulting from the reversal of residues B28 and B29. Herein, a rapid separation of these species using normal-flow LC is described. By using high-resolution, accurate mass detection (HR/AM) with chromatographic separation,

additional qualitative peak identification data is obtained. Furthermore, multiplexed LC maintains assay throughput.

MaterialsEquipment:• ThermoScientific™ Versette™ Automated

Liquid Handler

• ThermoScientific™ Finnpipette™ F1 Adjustable-volume pipettes

• ThermoScientific™ Q Exactive™ Mass Spectrometer

• ThermoScientific™ TLX-II Transcend™ LC system

• ThermoScientific™ Pinpoint™ Software (Version 1.4)

• ThermoScientific™ XCalibur™ Software (Version 2.2)

Laboratory consumables:• ThermoScientific™ MSIA™ Insulin D.A.R.T.’S

• ThermoScientific™ Dionex™ ProSwift RP-4H monolithic column (50 x 1 mm)

• ThermoScientific™ 96-well polypropylene microplates

• ThermoScientific™ 96-well robotic PCR plate

Reagents:• Insulinanalogs:Insulinlispro(Humalog®, Eli Lilly, 100

IU/mL), recombinant human insulin (Humulin S®, Eli Lilly, 100 IU/mL), 4[D10]-Leu insulin internal reference standard (Peptides International, Louisville, KY)

• ThermoScientific™ Pierce BupH phosphate buffered saline (PBS) - Wash Buffer

• ThermoScientific™ N-Octyl-ß-D-Glucoside (NOG)

• FisherChemical™ Optima LC/MS water (H2O), acetonitrile (MeCN), formic acid (FA), and trifluoroacetic acid (TFA)

• Insulin-depletedserum(BBIsolutions,Madison,WI)

• ACTH1-24carrierpeptide(Bachem,Torrance,CA)

G Q E

V I

T S I S L Y Q L

E N Y N

F V N Q H L G S H L V E A L Y V L G

E R G

F F

Y T

T C C C

C

C C

A Chain

B Chain

Insulin lispro (Humalog®) P K P K

K P K

P Human insulin (as Humulin S ®)

Figure 1. Structures and amino acid sequences for human insulin (as Humulin S®) and insulin lispro (Humalog®)

2 MethodIn the described experiments, a mixture of human insulin (500 mIU/L) and insulin lispro (500 mIU/L) were prepared in Elution buffer (15 mg/L ACTH 1-24 in 33% MeCN/0.4% trifluoroacetic acid in water). Aliquots of this solution (100 µL) were diluted with deionized water (30 µL) and used as a standard to optimize chromatographic separation.

The purification of insulins from individual samples was achieved using MSIA Insulin D.A.R.T.’S (Disposable Automation Research Tips), following the protocol provided in MSIA Insulin D.A.R.T.’S Technical Manual. Insulin and insulin LisPro were prepared in plasma both at 100 mIU/L. MSIA Insulin D.A.R.T.’S embed functionalized microcolumns that are coated with an immobilized anti-insulin antibody. The MSIA Insulin D.A.R.T’S are exposed to biological samples and the sample solution is pipetted up and down through the microcolumn to purify insulins from sample matrix. As a result of this analytical affinity purification, insulin analogs are enriched from plasma in a consistent and highly reproducible fashion. In the developed protocol, immunoenrichment of insulin analogs took approximately 60 minutes.

Once the insulins were purified, they were eluted from the MSIA Insulin D.A.R.T.’S and diluted for MS detection and analysis. This begins with sample injection into the LC-MS. The chromatographic separation of insulin lispro and human insulin was achieved using a simple binary gradientelution(Table1,EluentA:0.1%(v/v)formicacidinOptimaLC/MSwater,EluentB:0.1%(v/v)formicacidin Optima LC/MS MeCN), and a Dionex ProSwift RP-4H monolithic capillary column (50 x 1 mm). Total analysis time was 15 minutes, eluent was diverted to waste for the first 3 minutes following each injection, and the data window was set to acquire MS data from 5-9 minutes for multiplexing.

In this MSIA Insulin HR/AM workflow, the insulins were detected on a Thermo Scientific Q Exactive mass spectrometer, operating in positive ionization full-scan mode (70,000 FWHM, 700-1,800 m/z) with alternating targeted MS2 scans (35,000 FWHM, NCE 35) based on a pre-determined inclusion list. All HR/AM full-scan MS data generated were extracted (±5 ppm) and processed

using Pinpoint 1.4 for insulin quantification. Ion ratio data (the top 6 most intense isotopes for each of the +4, +5 and +6 charge states) were used for sequence verification and the measured AUC values for 4[D10]-Leu insulin were used as an internal standard for all samples.

Results and Discussion

The MSIA Insulin Workflow has been developed with the ability to quantify multiple forms of insulin that may be present in human plasma samples. An extended chromatographic analysis was performed to resolve the isobaric lispro analog from human insulin. The retention times for insulin lispro and insulin were 6.75 and 7.15 minutes, respectively. This was in line with the hydrophobicity factors predicted in silico by Pinpoint for insulin lispro and insulin, which were 59.15 and 61.17, respectively. Figure 2 shows a typical MS data generated using the MSIA Insulin Workflow for the sample prepared containing both insulin (Humulin S) and insulin lispro (Humalog). Also shown is the theoretical vs observed ion ratio plot for the human insulin peak, and confirmatory MS2 data, based upon previously reported unique MS2 product ions. The insulin peak retention time was confirmed by co-elution with the internal reference standard (4[D10]-Leu insulin retention time 7.10 minutes; data not shown). The MSIA Insulin Workflow using enhanced chromatographic separation in tandem with HR/AM MS and MS2 detection offers a solution to the analysis of insulin lispro.

ConclusionsThe described MSIA Insulin Workflow with a modified chromatographic separation protocol provides the ability to routinely resolve human insulin from the isobaric insulin analog, insulin lispro. Both forms of insulin are simultaneously purified from samples in a standardized and reproducible fashion. Combined with the HR/AM method described, it is therefore possible to acquire full-scan MS and MS2 data for both analytes indepen-dently, allowing (i) robust peak identification criteria (e.g. isotope ratios, product ions and retention time) to be applied for both analytes, and (ii) fully quantitative analysis of both compounds using the internal reference standard. This robust methodology for the differential analysis of these isobaric forms of insulin significantly impacts insulin analytics. The development of the MSIA Insulin Workflow provides the foundation for standard-ization of insulin analytical measurements for applications in clinical research, therapeutic development, forensics and sports doping applications.

Furthermore, the additional chromatographic analysis time can be overcome, and MS acquisition time maximized by using the Thermo Scientific Transcend multiplexing technology in which two, or four, LC channels are used with a single MS detector.

For more information on the MSIA workflow,

www.thermoscientific.com/msia

2

Step Start Sec Flow Grad %A %B

1 0 30 0.250 Step 83 17

2 0.50 570 0.250 Ramp 75.6 24.4

3 10.00 30 0.250 Ramp 0 100

4 10.50 120 0.250 Ramp 0 100

5 12.50 30 0.250 Ramp 83 17

6 13.00 120 0.400 Ramp 83 17

7 15.00 3 0.250 Ramp 83 17

Table 1. Chromatographic gradient conditions. Eluent A: 0.1 % (v/v) formic acid in Optima LC/MS Water, Eluent B: 0.1 % (v/v) formic acid in Optima LC/MS MeCN. The column was maintained at 55 °C.

thermoscientific.com/msia © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Humulin S® is a registered trademark of Eli Lilly and CompanyHumalog® is registered trademark of Eli Lilly and CompanySpecifications, terms and pricing are subject to change. Not all products are available in all countries. For Research Use Only. Not for use in diagnostic procedures.

For more info: e-mail msia.info@thermofisher.com

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Thevis M, Thomas A, Delahaut P, Bosseloir A, Schänzer W. Qualitative determination of synthetic analogues of insulin in human plasma by immunoaffinity purification and liquid chromatography-tandem mass spectrometry for doping control purposes. Anal Chem. 2005 Jun 1;77(11):3579-85

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Esposito CL, Berube M, Espourteille F. Simultaneous analysis of opiates and benzodiazepines in urine in under three minutes per sample using LC-MS/MS. Application Note 486b, AN63173_E12/09S; Thermo Fisher Scientific; San Jose, CA, 2010.

Figure 2. Typical data content generated using the described MSIA Insulin Workflow. Shown are: theoretical (purple) vs measured (green) isotopic distribution patterns for the +5 charge state of insulin lispro (a), and insulin (b); composite isotope chromatograms for insulin lispro (c), and insulin (d); MS2 product ion scans for insulin lispro (e), and insulin (f); extracted ion chromatograms (g) for the specific product ions for insulin lispro (m/z 217.1182) and insulin (m/z 226.1548)

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