Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies

North Foulon; Elisha Goonatilleke; Michael J MacCoss; Michelle A Emrick; Andrew N Hoofnagle

doi:10.1016/j.jmsacl.2022.06.003

Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies

J Mass Spectrom Adv Clin Lab. 2022 Jun 10:25:19-26. doi: 10.1016/j.jmsacl.2022.06.003. eCollection 2022 Aug.

Authors

North Foulon¹, Elisha Goonatilleke¹, Michael J MacCoss², Michelle A Emrick¹, Andrew N Hoofnagle^{1

3}

Affiliations

¹ Department of Laboratory Medicine & Pathology, University of Washington, Seattle, WA, USA.
² Department of Genome Sciences, University of Washington, Seattle, WA, USA.
³ Department of Medicine, University of Washington, Seattle, WA, USA.

Abstract

Introduction: The measurement of insulin and C-peptide provides a valuable tool for the clinical evaluation of hypoglycemia. In research, these biomarkers are used together to better understand hyperinsulinemia, hepatic insulin clearance, and beta cell function. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an attractive approach for the analysis of insulin and C-peptide because the platform is specific, can avoid certain limitations of immunoassays, and can be multiplexed. Previously described LC-MS/MS methods for the simultaneous quantification of insulin and C-peptide measure the intact analytes and most have relied on immunoaffinity enrichment. These approaches can be limited in terms of sensitivity and interference from auto-antibodies, respectively. We have developed a novel method that does not require antibodies and uses proteolytic digestion to yield readily ionizable proteotypic peptides that enables the sensitive, specific, and simultaneous quantitation of insulin and C-peptide.

Methods: Serum samples were precipitated with acetonitrile. Analytes were enriched using solid phase extraction and then digested with endoproteinase Glu-C. Surrogate peptides for insulin and C-peptide were analyzed using targeted LC-MS/MS.

Results: Inter-day imprecision was below 20 %CV and linearity was observed down to the lower limit of quantitation for both analytes (insulin = 0.09 ng/mL, C-peptide = 0.06 ng/mL). Comparison to a commercially available insulin immunoassay (Beckman Coulter UniCel DxI 600 Access) revealed a 30% bias between methods.

Conclusion: A novel LC-MS/MS method for the simultaneous analysis of insulin and C-peptide using Glu-C digestion was developed and evaluated. A detailed standard operating procedure is provided to help facilitate implementation in other laboratories.

Keywords: C-peptide; Insulin; LC-MS/MS; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LLOQ, lower limit of quantitation; Liquid chromatography-tandem mass spectrometry; Plasma; Serum; TaMADOR, Targeted Mass Spectrometry Assays for Diabetes and Obesity Research.

Grants and funding

U01 DK121289/DK/NIDDK NIH HHS/United States