Selective Determination of Human and Synthetic Insulins in Plasma Using LC-MS – How Does it Help the Clinician? Jean-Christophe Prost (Presenter) Bern University Hospital (Inselspital)

Authors: Jean-Christophe Prost (1), Lia Bally (2), Cédric Bovet (1), Christoph Stettler (2)
(1) University Institute of Clinical Chemistry, Inselspital, Bern University Hospital, University of Bern, Switzerland; (2) Department of Diabetes, Endocrinology, Clinical Nutrition & Metabolism, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Quantification of insulin in blood is of high importance for diagnostic, therapeutic, forensic and research purposes. Although commercially available immunoassays are very sensitive, but they lack selectivity between insulin and insulin analogues. The recent introduction of immunoaffinity workup coupled to ultra high-performance liquid chromatography mass spectrometry offers a promising approach for the simultaneous quantification of endogenous and synthetic insulins. The assay developed on our triple quadrupole mass spectrometer demonstrated good sensitivity and selectivity for endogen and exogen insulins, and could help clinician along avoiding several immunoassay analysis to monitor selectively multiple insulins by insulin-treated diabetes patients.

Introduction

Quantification of insulin levels in blood is of high importance for diagnostic, therapeutic, forensic and research purposes. In insulin-treated patients, the measurement of endogenous insulin secretion may help to monitor residual pancreatic beta cell function and assist clinical decisions on optimal glucose-lowering treatment. Additionally, insulin analysis is used in combination with glucose, C-peptide, beta-hydroxybutyrate, and proinsulin determination for the investigation of adult hypoglycemia. Furthermore, simultaneous detection of human and synthetic insulins including their ratio profile is a promising approach for identifying the misuse of insulin for doping purposes.

Popular automated immunoassays for insulin have distinctly heterogeneous performance in detecting synthetic insulins with large range of cross-reactivity (0% - >100% from the ratio of the measured and nominal concentrations) [1]. The ability to detect synthetic insulins is assay-specific and varies between different insulin analogues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) offers a means to circumvent these analytical issues. As such, the recent introduction of an immunoaffinity workup coupled to ultra high-performance liquid chromatography mass spectrometry (UHPLC-MS) [2] enables simultaneous quantification of endogenous and synthetic insulins.

Methods

With the help of immunoaffinity micropipette tips (InsuQuant Mass Spectrometric Kit, Thermo Scientific) we established a UHPLC-MS/MS method for the quantification of insulin and five recombinant human insulins (lispro, degludec, aspart, determir, glargine) in plasma between 7.5 and 960 pM. The preparation of the samples require 500 µL of plasma and is automated with a liquid handling system thanks to the 96-well plate format of the immunoaffinity tips.

Results

The assay developed on our triple quadrupole mass spectrometer (Xevo TQ-S, Waters) demonstrated good sensitivity for all insulins (limit of quantification 7.5 - 15 pM) and selectivity (peak area of the insulin analogs in blank matrix < 20 % LOQ). In general, low carryover was measured (< 15 %). We will illustrate the applicability of the method for the simultaneous analysis of endogenous and exogenous insulins in metabolic studies focussing on individuals with insulin-treated diabetes.

Conclusions & Discussion

The combination of immunoaffinity micropipette tips with UHPLC-MS/MS allowed the quantification of insulin and insulin analogs in blood. Our method can be used as a new diagnositic tool for clinicians.

[1] Slim, S.; Griffiths, M. J.; Gama, R. Annals of clinical biochemistry 2007, 44, 196-8.

[2] Nedelkov, D.; Niederkofler, E. E.; Oran, P. E.; Peterman, S.; Nelson, R. W. Journal of proteomics 2018, 175, 27-33.