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

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Anand-Ivell, Ravinder, Juul, Anders and Albrethsen, Jacob (2018) Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum. Clinical Chemistry and Laboratory Medicine, 56 (11). ISSN 1437-4331

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Abstract

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

Item Type: Article
Keywords: assay development; insulin-like factor 3 (INSL3); mass spectrometry
Schools/Departments: University of Nottingham, UK > Faculty of Science > School of Biosciences > Division of Animal Sciences
Identification Number: https://doi.org/10.1515/cclm-2018-0171
Depositing User: Anand-Ivell, Dr Ravinder
Date Deposited: 01 Jun 2018 11:08
Last Modified: 30 May 2019 04:30
URI: https://eprints.nottingham.ac.uk/id/eprint/52150

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