Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum
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 quantificatio...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Published: |
Walter de Gruyter
2018
|
| Subjects: | |
| Online Access: | https://eprints.nottingham.ac.uk/52150/ |
| _version_ | 1848798659472261120 |
|---|---|
| author | Anand-Ivell, Ravinder Juul, Anders Albrethsen, Jacob |
| author_facet | Anand-Ivell, Ravinder Juul, Anders Albrethsen, Jacob |
| author_sort | Anand-Ivell, Ravinder |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T20:23:17Z |
| format | Article |
| id | nottingham-52150 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:23:17Z |
| publishDate | 2018 |
| publisher | Walter de Gruyter |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-521502024-08-15T15:29:21Z https://eprints.nottingham.ac.uk/52150/ Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum Anand-Ivell, Ravinder Juul, Anders Albrethsen, Jacob 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. Walter de Gruyter 2018-05-30 Article PeerReviewed 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 assay development; insulin-like factor 3 (INSL3); mass spectrometry https://www.degruyter.com/view/j/cclm.2018.56.issue-11/cclm-2018-0171/cclm-2018-0171.xml doi:10.1515/cclm-2018-0171 doi:10.1515/cclm-2018-0171 |
| spellingShingle | assay development; insulin-like factor 3 (INSL3); mass spectrometry Anand-Ivell, Ravinder Juul, Anders Albrethsen, Jacob Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum |
| title | Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum |
| title_full | Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum |
| title_fullStr | Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum |
| title_full_unstemmed | Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum |
| title_short | Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum |
| title_sort | development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum |
| topic | assay development; insulin-like factor 3 (INSL3); mass spectrometry |
| url | https://eprints.nottingham.ac.uk/52150/ https://eprints.nottingham.ac.uk/52150/ https://eprints.nottingham.ac.uk/52150/ |