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Licensed Unlicensed Requires Authentication Published by De Gruyter May 30, 2018

Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum

  • Jakob Albrethsen ORCID logo EMAIL logo , Hanne Frederiksen , Anna-Maria Andersson , Ravinder Anand-Ivell , Loa Nordkap , Anne Kirstine Bang , Niels Jørgensen and Anders Juul



The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantification of INSL3 by mass spectrometry.


We developed an assay in which the INSL3 A-chain is released from the INSL3 A-B heterodimer by chemical reduction and alkylation. The alkylated INSL3 A-chain is quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as substitute for serum INSL3. The method was compared to a validated and sensitive in-house serum INSL3 immunoassay using 97 serum samples from 12 healthy boys during pubertal transition. Adult levels were determined based on sera from 72 adult healthy males aged 18–40 years.


An LC-MS/MS assay with limit of detection and limit of quantification (LOQ) of 0.06 and 0.15 ng/mL, respectively, and intra-assay CVs <9% in the relevant ranges was obtained. The LC-MS/MS compared well with the in-house immunoassay (Deming regression slope: 1.28; Pearson correlation: R=0.86). INSL3 concentrations increased with pubertal maturation in healthy boys. INSL3 concentrations were above the LOQ in all samples from the adult men. The mean (±2 SD range)for serum INSL3 concentrations in the adult men was 2.2 (0.5–3.9) ng/mL.


We have developed a robust and sensitive method suitable for quantitation of serum INSL3 in a clinical setting using LC-MS/MS instrumentation available in modern clinical laboratories. The method paves the way for future studies into the clinical role of serum INSL3 measurements.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: Innovation Fund Denmark 14-2013-4; International Center for Research, and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC); ReproUnion.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.


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Supplementary Material:

The online version of this article offers supplementary material (

Received: 2018-02-14
Accepted: 2018-05-03
Published Online: 2018-05-30
Published in Print: 2018-10-25

©2018 Walter de Gruyter GmbH, Berlin/Boston

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